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3D orbital tracking for super-resolving the dynamics of gene expression.
Novel Techniques in Microscopy. Vancouver, Canada. April 12-15, 2015.
OSA Technical Digest. 2015; (online), NM3C.1.Optical super resolution of fast dynamic processes can be achieved using the 3D orbital tracking technique and fluctuation spectroscopy analysis. Here we show application of the method to the dynamics of loci of gene expression.
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3D Nanoimaging and detection of molecular flow using the nSPIRO method.
Novel Techniques in Microscopy. Vancouver, Canada. April 12-15, 2015.
OSA Technical Digest. 2015; (online), NM4C.1.Detecting proteins dynamics within cells grown in 3D micro-environments is challenging. We developed a 3D nano-imaging technique to uniquely probes proteins in cells grown on collagen. Results show paxillin and actin diffusion rates are unique.
Ganesan G, Santoro Y, Warren RV, Leproux A, Gratton E, Malik S, Galassetti PR, Tromberg BJ.
Spectral changes in subcutaneous fat with weight loss measured by diffuse optical spectroscopy.
BiOS 2015. Part of SPIE Photonics West. San Francisco, California, February 7-12, 2015.
Proc SPIE. 2015; 9319, 9319-2.Adipose tissue (AT) is increasingly understood to contribute to dynamic changes in physiology and metabolic disease. We sought to use broadband diffuse optical spectroscopy (DOS) to measure changes in subcutaneous AT optical properties with weight loss in adults. We describe a method of self-referencing differential spectroscopy (SRDS) to assess spectral changes found, independent of absorption due to primary chromophores. Preliminary data indicate that weight loss is associated with unique spectral signatures in the wavelength ranges corresponding to deoxyhemoglobin and bulk lipid.
Stringari C, Donovan PJ, Gratton E.
Phasor FLIM metabolic mapping of stem cells and cancer cells in live tissues.
BiOS 2012. Part of SPIE Photonics West. San Francisco, California, January 21-26, 2012.
Proc SPIE. 2012; 8226, 82260D.We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers in conjunction with image segmentation and the concept of cell phasor for deriving metabolic maps of cells and living tissues in vivo. In issues we identify and separate intrinsic fluorophores such as collagen, retinol, retinoic acid, porphyrin, flavins, free and bound nicotinamide adenine dinucleotide (NADH). Metabolic signatures of tissues are obtained by calculating the phasor fingerprint of single cells and by mapping the relative concentration of metabolites. This method detects small changes in metabolic signatures and redox states of cells. Phasor fingerprints of stem cells cluster according to their differentiation state in a living tissue such as the C. elegans germ line and the crypt base of small intestine and colon. Phasor FLIM provides a label-free and fit-free sensitive method to identify metabolic states of cells and to classify stem cells, normal differentiated cells and cancer ... [truncated at 150 words]
Crosignani V, Dvornikov AS, Aguilar JS, Stringari C, Edwards R, Mantulin WW, Gratton E.
In vivo deep tissue fluorescence imaging of the murine small intestine and colon.
BiOS 2012. Part of SPIE Photonics West. San Francisco, California, January 21-26, 2012.
Proc SPIE. 2012; 8226, 822609.Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence ... [truncated at 150 words]
Rossow MJ, Gatto R, D'Amico E, Mantulin WW, Gratton E.
Blood flow measurements and clot detection with near-infrared spectroscopy.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME72, 2006. Detecting impeded blood flow and locating the clot causing it is a major challenge in neurosurgery. We propose an instrument that uses near-infrared spectroscopy to simultaneously detect clots and measure blood flow.
Tanner K, D'Amico E, Kukreti S, Matulin WW, Gratton E, Kaczmarowski A, Malpeli JG.
Spectrally resolved neurophotonics: the optical BOLD effect and vascular components in the mammalian brain.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME20, 2006. We developed a broad band spectral technique that is independent of the light transport modality to separate optical changes in scattering and absorption in the cat’s brain due to the hemodynamic signal following visual stimulation.
Gratton E, Tanner K, Beitel E, D'Amico E, Matulin WW.
Effects of vasodilation on intrinsic optical signals.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME29, 2006. Using a spectral technique, we showed that during visual stimulation of the cat brain there was an apparent change in water content. We developed a simple model to explain how vasodilation could cause this effect.
Simonson PD, D'Amico E, Gratton E.
Proposal and proof of principle for a novel optical biopsy needle: modulating an optical needle's refeclectivity alters the average photon path in scattering media.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME38, 2006. We show through Monte Carlo simulations and experiments that a novel optical needle can be used to probe different volumes of tissue in the needle’s vicinity by simply modulating the needle’s reflectivity.
Machado NG, Pearlman A, Gratton E.
Near-infrared spectroscopy and the swallowing event.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME46, 2006. In this work, we applied near infrared spectroscopy (NIRS) to observe brain hemodynamics in the frontal lobes during the swallowing event.
Gatto R, D'Amico E, Mantulin WW, Gratton E, Charbel FT.
Optical microprobe for blood clot detection.
OSA Biomedical Topical Meeting (BIO), Fort Lauderdale, Florida, March 18-23, 2006.
Biomedical Optics (Technical Digest). Optical Society of America, ME47, 2006. One of the mayor complications during vascular surgery is the formation of blood clot. We tested an animal model and assembled a prototype device that shows the blood clot signature spectrum and its temporary growth.
Behne MJ, Barry NP, Moll I, Gratton E, Mauro TM.
Fluorescence lifetime to image epidermal ionic concentrations.
Femtosecond Laser Applications in Biology (Proceedings of SPIE, Vol. 5463). By S Avrillier and JM Tualle (Editors). pp. 37-44, 2004. Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept ... [truncated at 150 words]
Toronov VY, D'Amico E, Hueber DM, Gratton E, Barbieri BB, Webb A.
Optimization of the phase and modulation depth signal-to-noise ratio for near-infrared spectroscopy of the biological tissue.
Saratov Fall Meeting 2003: Optical Technologies in Biophysics and Medicine V (Proceedings of SPIE, Vol. 5474). By VV Tuchin (Editors). pp. 281-284, 2004. Frequency-domain near-infrared spectroscopy and imaging offer significant advantages over the continuous-wave method in human brain applications. However, the drawback of existing instruments is a low signal-to-noise ratio for measured phase and modulation depth changes caused by cerebral activation. In this paper we show that in the case of the geometry specific for the activated area in the human brain, the SNR can be significantly improved by increasing the modulation frequency. We present the results of a study performed experimentally using a sub-nanosecond pulsed light source and a spherical absorbing object immersed in a highly scattering solution. We show that changes caused by the absorbing inhomogeneity in both phase and modulation depth increase with frequency and reach maximum values at frequencies between 400 and 1400 MHz, depending on the particular source-detector distance. We also show that for the semi-infinite geometry an increase of the modulation frequency from 100 to 500 MHz ... [truncated at 150 words]
Toronov VY, D'Amico E, Hueber DM, Gratton E, Webb A, Barbieri BB.
Optimization of the frequency-domain instrument for the near-infrared spectro-imaging of the human brain.
Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems XIV (Proceedings of SPIE, Vol. 5312). By KE Bartels, LS Bass, and others (Editors). pp. 378-383, 2004. Although many results on the near infrared spectroscopy and imaging of the human brain have been already published, the signal-to-noise ratio (SNR) of the instrumentation used in these studies has never been systematically optimized for the anatomy of the human head. Assuming a typical anatomical structure of the adult human head, we have studied the SNR of the frequency-domain instrument as a function of the modulation frequency and the source-detector distance. The study was performed both experimentally (using a pulsed laser system) and numerically (using the diffusion approximation and Monte Carlo simulations). For the specified geometry we have found that changes caused by non-homogeneities in the phase and in the ratio of the modulation amplitude to the mean intensity significantly increase with the modulation frequency and exhibit peaks between 400 and 1100 MHz, depending on the source-detector distance. Assuming the shot noise, we have found that the corresponding SNR peaks ... [truncated at 150 words]
Choi JH, Wolf M, Toronov VY, Michalos A, Gratton E.
Spatio-temporal analysis of the cerebral spontaneous oscillation.
Complex Dynamics, Fluctuations, Chaos, and Fractals in Biomedical Photonics (Proceedings of SPIE, Vol. 5330). By VV Tuchin (Editors). pp. 29-37, 2004. Cerebral vasomotion was studied on the human brain in vivo by use of multi-optode frequency domain near-infrared spectroscopy (NIRS). Vasomotion is a spontaneous oscillation with a frequency of 0.1 Hz in the arterial flow. We investigated (1) the fluctuations of cerebral hemodynamics on the dynamical characteristics of cerebral vasomotion and (2) the dynamical coupling between vasomotion in the skin and brain. We found that (1) vasomotion is temporal coherent at least for about 3 min; (2) vasomotion observed from NIRS is low-dimensional chaotic with its fractal dimension of about 4.5; (3) vasomotion is spatially coherent with coherence length of about 1 - 2 cm but cerebral vasomotion is dynamically independent from vasomotion in the skin.
Toronov VY, Franceschini MA, Fantini S, Webb A, Gratton E.
Study of complex hemodynamic fluctuations in the human brain by simultaneous near-infrared spectro-imaging and functional magnetic resonance imaging.
Complex Dynamics, Fluctuations, Chaos, and Fractals in Biomedical Photonics (Proceedings of SPIE, Vol. 5330). By VV Tuchin (Editors). pp. 22-28, 2004. In this paper we discuss temporal and spatial patterns of brain hemodynamics under rest and motor stimulation conditions obtained by functional magnetic resonance imaging and simultaneous fast multi-channel near-infrared spectro-imaging in the human motor cortex. Our data indicate that the main difference between the brain hemodynamics under the repetitive stimulation and the rest conditions is not in the appearance of hemoglobin concentration changes during the stimulations (since fluctuations occur at rest as well), but in their more regular, i.e. phase-synchronous with the stimulation behavior.
McKinney SA, Tan E, Wilson TJ, Nahas MK, Déclais AC, Clegg RM, Lilley DMJ, Ha T.
Single-molecule studies of DNA and RNA four-way junctions.
Nucleic Acids Chemistry and Biology: The 5th Cambridge Symposium, Queen's College, Cambridge, 31 August–3 September 2003.
Biochem Soc Trans. 2004; 32(1): 41-45.Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.
Levi V, Ruan Q, Kis-Petiková K, Gratton E.
Scanning FCS, a novel method for three-dimensional particle tracking.
Intermolecular Associations in 2D and 3D, University of Nottingham, UK, 19 - 20 June 2003.
Biochem Soc Trans. 2003; 31(Pt 5): 997-1000.We describe a novel method to track fluorescent particles in three dimensions with nanometre precision and millisecond time resolution. In this method, we use our two-photon excitation microscope. The galvomotor-driven x-y scanning mirrors allow the laser beam to move repetitively in a circular path with a radius of half the width of the point spread function of the laser. When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-y plane can be determined by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective was used to change the laser focus at two planes (half width of the point spread function apart). The difference of the fluorescence intensity in the two planes is used to calculate the z-position of the fluorescent particle. The laser beam is allowed to scan multiple circular orbits before it ... [truncated at 150 words]
Toronov VY, Webb A, Walker SA, Gupta R, Choi JH, Gratton E, Hueber DM.
Study of the fMRI blood oxygen level dependent effect by near-infrared spectroscopy.
Saratov Fall Meeting 2002: Optical Technologies in Biophysics and Medicine IV (Proceedings of SPIE, Vol. 5068). By VV Tuchin (Editors). pp. 222-226, 2003. In order to study the behavior of cerebral physiological parameters and to further the understanding of the fMRI blood-oxygen-level-dependent (BOLD) effect, we have recorded simultaneously multi-source frequency-domain near-infrared and BOLD fMRI signals during motor functional activation in humans. From the near-infrared data we obtained information on the changes in cerebral blood volume and oxygenation. In order to relate our observations to changes in cerebral blood flow we employed the "balloon" model of cerebral perfusion. Our data showed that the deoxyhemoglobin concentration is the major factor determining the time course of the BOLD signal.
Wolf M, Franceschini MA, Paunescu LA, Toronov VY, Michalos A, Wolf U, Gratton E, Fantini S.
Absolute frequency-domain pulse oximetry of the brain: methodology and measurements.
7th Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT). Hanover NH, USA.
Oxygen Transport to Tissue XXIV (Advances in Experimental Medicine and Biology, Vol. 530). By HM Swartz and J Dunn (Editors). Springer, pp. 61-73, 2003. ISBN: 9780306477744A new method to non-invasively measure the absolute tissue oxygen saturation (SO2) and arterial oxygen saturation (fdSaO2) by frequency-domain spectroscopy is described. This method is based on the quantitative measurement of the tissue absorption spectrum, which is used to determine global SO2. From the amplitude of absorption changes caused by arterial pulsation oscillations, in the range of 633-841 nm, the fdSaO2 can be calculated. During deoxygenation (air/N2 mixture) experiments, we measured the fdSaO2 and SO2 on the forehead of three healthy volunteers and compared them to the arterial oxygen saturation measured by conventional pulse oximetry (poSaO2) on the finger. fdSaO2 and poSaO2 agree very well (mean difference: -1.2 ± 2.6%). Changes in SO2 were systematically smaller than in fdSaO2 or poSaO2 probably due to autoregulation. The measurements with 4 and 8 wavelengths had comparable quality.
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Fluctuation correlation spectroscopy in cells. Determination of molecular aggregation.
European Conference on Lasers and Electro-Optics and the International Quantum Electronics Conference. 22-27 June 2003, Munich, Germany.
Lasers and Electro-Optics Europe. CLEO/Europe. pp. 707, 2003. The determination of molecular interactions in cells has become a field of great interest in the post-genomic era. Ideally, we will like to know which protein is interacting with which, the migration and the localization of the proteins in the cell. During the past years very powerful optical methods have been developed that have the sensitivity to detect single molecules and molecular interactions. Although most of these methods have been demonstrated in solutions or on molecules immobilized on a surface, we will like to extend this methodology to the cell environment. Fluctuation correlation spectroscopy has been proposed as a method to detect single molecules, determine some of the chemico-physical parameters such as local diffusion and molecular brightness. Finally, advances in the determination of spatio-temporal correlations in a cell will be discussed.
Wolf U, Wolf M, Choi JH, Paunescu LA, Safonova LP, Michalos A, Gratton E.
Mapping of hemodynamics on the human calf with near infrared spectroscopy and the influence of the adipose tissue thickness.
29th Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT), Philadelphia, Pennsylvania, August 11-15, 2001.
Oxygen Transport to Tissue XXIII: Oxygen Measurements in the 21st Century: Basic Techniques and Clinical Relevance (Advances in Experimental Medicine and Biology, Vol. 510). By DF Wilson, SM Evans, J Biaglow, and A Pastuszko (Editors). Springer, pp. 225-30, 2003. ISBN: 9780306473944We investigated the influence of the adipose tissue thickness (ATT) on near-infrared spectroscopy (NIRS) measurements of the absorption coefficient (mu a), the reduced scattering coefficient (mu s') and changes in concentrations of oxyhemoglobin ([O2Hb]) and deoxyhemoglobin ([HHb]). We used a frequency domain spectrometer and a special probe to generate maps of these parameters on the human calf during venous occlusion. For ATT below 6 mm mu a remained constant, whereas for ATT between 6 and 14 mm mu a decreased quickly and became almost constant again for ATT larger than 14 mm. Mu s' was not significantly altered by the ATT but the values showed a high variability between subjects. We found significantly different changes in both the [O2Hb] and the [HHb] between the proximal and distal locations of measurement. Although ATT influences the recovery of the optical properties of the underlying tissue, these differences depending on the location cannot ... [truncated at 150 words]
Wolf M, Wolf U, Choi JH, Gupta R, Safonova LP, Paunescu LA, Michalos A, Gratton E.
Detection of the fast neuronal signal on the motor cortex using functional frequency domain near infrared spectroscopy.
29th Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT), Philadelphia, Pennsylvania, August 11-15, 2001.
Oxygen Transport to Tissue XXIII: Oxygen Measurements in the 21st Century: Basic Techniques and Clinical Relevance (Advances in Experimental Medicine and Biology, Vol. 510). By DF Wilson, SM Evans, J Biaglow, and A Pastuszko (Editors). Springer, pp. 193-7, 2003. ISBN: 9780306473944Using non-invasive near infrared spectroscopy fast changes in the range of ms in the optical properties of neurons during brain activity have been described. Since the signal is small, the system to detect it has to be highly noise optimized. We used a frequency-domain tissue oximeter, whose laser diodes were modulated at 110 MHz and the amplitude (AC), mean intensity (DC) and phase (phi) of the modulated optical signal was measured at 96 Hz sample rate. In two volunteers, 36 and 37 years old, the probe consisting of 4 crossed source detector pairs was placed above the motor cortex (C3 position), contralateral to the hand performing the tapping exercise. The tapping frequency was set at 2.5 times the heart rate of the subject to avoid the influence of harmonics on the signal. An electronic device recorded the tapping movement. Control-data were obtained from a solid medium of approximately the same ... [truncated at 150 words]
Toronov VY, Webb A, Choi JH, Wolf M, Safonova LP, Wolf U, Gratton E.
Functional cerebral activation detected by frequency-domain near-infrared spectroscopy.
Saratov Fall Meeting 2001: Optical Technologies in Biophysics and Medicine III (Proceedings of SPIE, Vol. 4707). By VV Tuchin (Editors). pp. 92-96, 2002. The aim of our study was to explore the possibility of detecting haemodynamic changes in the brain using frequency- domain near-IR spectroscopy by exploiting the phase of the intensity modulated optical signal. To obtain optical signals with eh highest possible signal-to-noise ratio, we performed simultaneous NIRS-fMRI measurements, with subsequent correlation of the time courses of both measurements. The cognitive paradigm used arithmetic calculations, with optical signals acquired with sensors placed on the forehead. In three subjects we demonstrated correlation between the haemodynamic signals obtained using NIRS and BOLD fMRI.
Morren G, Wolf M, Lemmerling P, Wolf U, Choi JH, Gratton E, de Lathauwer L, van Huffel S.
Extraction of fast neuronal changes from multichannel functional near-infrared spectroscopy signals using independent component analysis.
Functional Monitoring and Drug-Tissue Interaction (Proceedings of SPIE, Vol. 4623). By MD Kessler and GJ Mueller (Editors). pp. 68-76, 2002. Fast changes in the range of milliseconds in the optical properties of cerebral tissue, which are associated with brain activity, can be detected using non-invasive near-infrared spectroscopy (NIRS). These changes in light scattering are due to an alteration in the refractive index at neuronal membranes. The aim of this study was to develop highly sensitive data analysis algorithms to detect this fast signal, which is small compared to other physiological signals. A frequency-domain tissue oximeter, whose laser diodes were modulated at 110MHz was used. The amplitude, mean intensity and phase of the modulated optical signal was measured at 96Hz sample rate. The probe consisting of 4 crossed source detector pairs was placed above the motor cortex, contralateral to the hand performing a tapping exercise consisting of alternating rest- and tapping periods of 20s each. The tapping frequency, which was set to 3.55Hz or 2.5 times the heart rate of the ... [truncated at 150 words]
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Near-infrared detection of correlated activity in the brain.
Conference on Lasers and Electro-Optics (CLEO'02) and the 10th Quantum Electronics and Laser Science Conference (QELS'02), Long Beach, CA, May 19-24, 2002.
Lasers and Electro-Optics, 2002. CLEO '02. Technical Digest. pp. 368, CWC3, 2002. Summary form only. Near-IR light can pass trough the skull and reach the surface of the brain. It is well established in exposed cortex experiments that brain activity changes the brain surface optical properties in the near-IR, due both to changes in blood flow and to scattering from the brain cells. Several researches have proposed optical methods and the near-IR spectral region to measure brain function non-invasively with high temporal resolution and good localization. Our research has shown that it is possible to increase by at least one order of magnitude the detection of the small changes associated with neuronal activity. Our technical developments and a new sensor could make this optical technique widely available and complementary to fMRI.
Wolf M, Wolf U, Choi JH, Safonova LP, Gupta R, Toronov VY, Michalos A, Paunescu LA, Gratton E.
Functional fast neuronal signals in the visual and motor cortex detected by frequency-domain near-infrared spectroscopy.
OSA Biomedical Topical Meeting (BIO), Miami Beach, Florida, April 7, 2002.
Biomedical Topical Meeting. Optical Society of America, SuF4, 2002. Choi JH, Wolf M, Wolf U, Polzonetti CM, Safonova LP, Gupta R, Michalos A, Mantulin WW, Gratton E, Toronov VY, Hueber DM.
Noninvasive determination of optical properties of adult brain with frequency-domain near-infrared spectroscopy.
OSA Biomedical Topical Meeting (BIO), Miami Beach, Florida, April 7, 2002.
Biomedical Topical Meeting. Optical Society of America, SuD33, 2002. Absolute optical values for the human adult brain are difficult to measure due to the
layered structure of head. We used long-range multi-distance frequency-domain near-infrared
spectroscopy (NIRS) to measure absolute optical values of each tissue layer in the human head.
We determined baseline optical values for the forehead in 30 adults (age range 21 to 50). We
found that tissue oxygenation was relatively narrowly distributed (STD~3%) within the subject
group, whereas hemoglobin concentrations and optical parameters had a relatively broader
distribution. In addition, the distance dependence of optical and physiological parameters showed
the layered structure of the human head. These measurements allowed determination of absolute
optical properties in each layer of the adult head.
Michalos A, Safonova LP, Hueber DM, Wolf U, Wolf M, Choi JH, Gupta R, Polzonetti CM, Barbieri BB, Mantulin WW, Gratton E.
Reduced cerebral hemodynamic response in sleep disorders: a NIRS frequency-domain study.
OSA Biomedical Topical Meeting (BIO), Miami Beach, Florida, April 7, 2002.
Biomedical Topical Meeting. Optical Society of America, MA1, 2002. We applied NIRS to investigate changes in cerebral oxygenation and hemodynamics in sleep-disordered breathing; namely, snoring and obstructive sleep apnea. A detected reduced brain hemodynamic response to hypoxia may be a predictor of cerebrovascular morbidity.
Safonova LP, Michalos A, Hueber DM, Wolf U, Wolf M, Choi JH, Gupta R, Polzonetti CM, Barbieri BB, Mantulin WW, Gratton E.
Age correlated changes in cerebral hemodynamics assessed by near-infrared spectroscopy.
OSA Biomedical Topical Meeting (BIO), Miami Beach, Florida, April 7, 2002.
Biomedical Topical Meeting. Optical Society of America, SuD35, 2002. Using near-infrared frequency-domain spectroscopy we observed cerebral hemodynamic changes in normal subjects during breath holding, which correlated with age. Snoring affected changes did not allow us to observe the age effect in a group of snorers.
Barry NP, Hanson KM, Gratton E, Clegg RM, Behne MJ, Mauro TM.
Applications of ultrafast lasers to two-photon fluorescence and lifetime imaging.
Commercial and Biomedical Applications of Ultrafast and Free-Electron Lasers (Proceedings of SPIE, Vol. 4633). By GS Edwards, J Neev, A Ostendorf, and JC Sutherland (Editors). pp. 50-61, 2002. Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focused into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two- photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (~500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. ... [truncated at 150 words]
Nayfeh MH, Therrien J, Belomoin G, Akcakir O, Barry NP, Gratton E.
Stimulated blue emission and second harmonic generation from films of ultrasmall Si nanoparticles.
2000 MRS Fall Meeting, Symposium F. November 27- 30, 2000. Boston, MA.
Microcrystalline and Nanocrystalline Semiconductors (MRS Proceedings, Vol. 638). By PM Fauchet, JM Buriak, LT Canham, N Koshida, and BE White Jr (Editors). pp. F9.5.1-12, 2001. ISBN: 9781558995482We describe a procedure for dispersion bulk Si into ultrasmall nanoparticles, much smaller than what is available today, with high throughput and excellent size definition and control. The quantum dots are ~ 1 nm in diameter and contain 29 Si atoms[1-2]. We show a most unexpected and totally surprising phenomenon. Unlike bulk Si, an optically inert indirect gap material, the particles are extremely active optically in the blue, exceeding the activity of fluorescein, such that single particles are readily detected and imaged, using two-photon nearinfrared femto second excitation. This comes nearly a decade after Canham first surprised the scientific community by reporting red luminescence from anodized Si[3]. In addition to being ultrabright, the particles have other useful properties that are unlike those of bulk including being a source of stimulated emission and collimated beam emission[4-5], and harmonic generation[6]. Single electron charging and the confinment energy spacing, both are much larger ... [truncated at 150 words]
Nayfeh MH, Mitas L, Gratton E, Brau P.
Si29 Nanoparticles and reconstituted films: a highly fluorescent new form of silicon.
6th International Symposium on Quantum Confinement: Nanostructures Materials and Quantum Devices. September 5-6, 2001. San Francisco, CA.
Quantum Confinement VI: Nanostructured Materials and Devices (Electrochemical Society Proceedings, Vol. 2001-19). By M Cahay, JP Leburton, DJ Lockwood, S Bandyopadhyay, and JS Harris (Editors). Electrochemical Society, Inc, pp. 157-160, 2001. ISBN: 9781566773522Toronov VY, Webb A, Choi JH, Wolf M, Gratton E, Hueber DM.
Simultaneous functional magnetic resonance and near-infrared imaging of adult human brain.
Optical Tomography and Spectroscopy of Tissue IV (Proceedings of SPIE, Vol. 4250). By B Chance, RR Alfano, BJ Tromberg, M Tamura, and EM Sevick-Muraca (Editors). pp. 380-382, 2001. The aim of this study was to compare functional cerebral hemodynamic signals obtained simultaneously by near infrared spectroscopy (NIRS) and by functional magnetic resonance imaging (fMRI). The contribution of superficial layers (skin and skull) to the NIRS signal was also assessed.
Huang H, Dong CY, Kwon HS, Sutin JDB, Kamm RD, So PTC.
Studying cellular mechanics using magnetic tweezers and two-photon microscopy.
Optical Diagnostics of Living Cells IV (Proceedings of SPIE, Vol. 4260). By DL Farkas and RC Leif (Editors). pp. 94-102, 2001. The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron ... [truncated at 150 words]
Paunescu LA, Michalos A, Choi JH, Wolf U, Wolf M, Gratton E.
In-vitro correlation between reduced scattering coefficient and hemoglobin concentration of human blood determined by near-infrared spectroscopy.
Optical Tomography and Spectroscopy of Tissue IV (Proceedings of SPIE, Vol. 4250). By B Chance, RR Alfano, BJ Tromberg, M Tamura, and EM Sevick-Muraca (Editors). pp. 319, 2001. We study the correlation between (mu) s' and THC obtained in vitro, in a highly scattering medium containing human blood. We used a frequency domain near infrared spectrometer (modulation frequency: 110 MHz, wavelengths: 758 and 830 nm) to measure in real time (acquisition time: 0.64 s) (mu) s' and THC. We used Liposyn suspension and red blood cells in saline buffer solution. After a couple of minutes of baseline acquisition, several consecutive increments of 3-5 ml blood were added to the solution yielding THC equals 15-100 (mu) M and (mu) a equals 0.03-0.3 1/cm. At the last amount of blood added, increments of glucose in the range of 0.5-20 g/L were added. For each step of blood and glucose added, data were acquired for a couple of minutes. This was repeated 6 times. Average of data was calculated for both (mu) s' and THC for each of the red blood ... [truncated at 150 words]
Toronov VY, Webb A, Choi JH, Wolf M, Gratton E.
Simultaneous assessment of human brain functional hemodynamics by magnetic resonance and near-infrared imaging.
Medical Imaging 2001: Physics of Medical Imaging (Proceedings of SPIE, Vol. 4320). By LE Antonuk and MJ Yaffe (Editors). pp. 861-867, 2001. Near-infrared spectroscopy is a relatively new imaging method, which can provide important information on concentrations of oxy- and deoxy-hemoglobin in cortical areas of the brain. We discuss the advantages of the integration of magnetic resonance and optical imaging techniques and present the results of our experimental study on the comparison of optical and fMRI signals obtained simultaneously on humans during functional activity and at rest. In all subjects we found a good collocation of the brain activity centers revealed by both methods. We also found a high temporal correlation between the BOLD signal (fMRI) and the deoxy-hemoglobin concentration (near-infrared spectroscopy) in the subjects who exhibited low fluctuations in superficial head tissues. The contamination of optical signals by superficial tissue layers urges applying algorithms of three-dimensional optical tomography.
Ragan TM, So PTC, Kwon HS.
3D particle tracking on the two-photon microscope.
Multiphoton Microscopy in the Biomedical Sciences (Proceedings of SPIE, Vol. 4262). By A Periasamy and PTC So (Editors). pp. 247-258, 2001. We have developed a 3D single-particle-tracking (SPT) system based around the two-photon laserscanning fluorescence microscope that can track particles in all three dimensions and at a high frequency response. We have implemented two different techniques to achieve this goal. The techniques employ feedback control in order to track the particle but differ in the approach they use to ascertain the particle's 3D position. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle position. The second technique scans only a single plane but utilizes optical aberrations which have been introduced into the optical system that break the axial symmetry of the point spread function and serves as an indicator ofthe particle's axial position. The current system has a frequency response at the video rate and an axial range of 100 tim. We tested the system by ... [truncated at 150 words]
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Growth of myelin figures made of water soluble surfactant.
1st Annual International Conference on Microtechnologies in Medicine and Biology, 2000.
1st IEEE Embs Special Topics Conference on Microtechnology in Medicine & Biology. pp. 345-348, Poster 126, 2000. ISBN: 0780366034The swelling and emulsification dynamics of a soluble lamellar phase of an ionic surfactant is investigated. Upon mixing this surfactant with brine the authors observe a myelinic instabilities at the interface between large vesicles and the aqueous phase. During the early stages of emulsification they observe pearling instabilities. At later stage of emulsification they observe very large tubes with diameters that exceed 50 microns and whose length exceed several order of magnitude its radius.
Michalos A, Paunescu LA, Wolf M, Wolf U, Toronov VY, Franceschini MA, Fantini S, Gratton E.
Assessment of cerebral oxygenation and hemodynamics in obstructive sleep apnea syndrome.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). Optical Society of America, SuH16, 2000. We applied Near-Infrared Spectroscopy for the investigation of cerebral oxygenation during obstructive sleep apnea. We found a relatively large decrease in brain oxygenation during apnea and the peak of deoxygenation occurs after resumption of breathing.
Paunescu LA, Wolf U, Wolf M, Michalos A, Gratton E.
Scattering coefficient - hemoglobin concentration relation determined by frequency-domain spectroscopy during venous occlusion.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). Optical Society of America, TuD2, 2000. We found correlation between reduced scattering coefficient and total hemoglobin concentration measured on muscles by the frequency-domain spectroscopy during venous occlusion protocol. This can be a useful parameter, which can be employed in clinical studies.
Wolf M, Toronov VY, Wolf U, Paunescu LA, Michalos A, Gratton E.
Maps of cerebral hemoglobin concentration changes obtained by near-infrared spectroscopy, Characterization of phase shifts among locations.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). Optical Society of America, pp. 416-418, TuF6, 2000. We obtained maps of cerebral hemoglobin concentration changes by near-infrared spectroscopy during functional stimulation and physiological activity at rest. The correlation and phase shift between locations were characterized by phase portraits and the phase synchronization index.
Wolf U, Wolf M, Toronov VY, Michalos A, Paunescu LA, Gratton E.
Detecting cerebral functional slow and fast signals by frequency-domain near-infrared spectroscopy using two different sensors.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). Optical Society of America, TuF10, 2000. Using near-infrared-spectroscopy a slow signal due to hemodynamic changes following brain activity is well characterized, while a fast signal is not enough investigated. With our frequency-domain-spectrometer and two different sensors we detected both signals.
Toronov VY, Wolf M, Michalos A, Gratton E, Webb A, Hueber DM, Fantini S.
Analysis of cerebral hemodynamic fluctuations measured simultaneously by magnetic resonance imaging and near-infrared spectroscopy.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). Optical Society of America, WA5, 2000. We measure cerebral hemodynamic fluctuations simultaneously by near-infrared spectroscopy and magnetic resonance imaging during functional stimulations. Our repetitive stimulation protocol includes a variety of epochs differing in duration of stimulation and relaxation periods, which allows generating hemodynamic signals highly synchronous with stimulation sequence. Our goals are to compare results of temporal analysis of hemodynamic fluctuations measured by near-infrared spectroscopy and high-speed magnetic resonance imaging, and to compare optical maps of brain activity based on the results of temporal analysis of NIRS signals with magnetic resonance images.
Franceschini MA, Toronov VY, Filiaci ME, Wolf M, Michalos A, Gratton E, Fantini S.
Real-time video of brain activation in human subjects using a non-invasive near-infrared technique.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Miami Beach, Florida, April 2, 2000.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 38). By T Li (Editors). Optical Society of America, ME6, 2000. Near-infrared spectroscopy (NIRS) is a potentially effective technique for non-invasive functional studies of the human brain. In this paper we demonstrate the feasibility of real time mapping of cerebral hemodynamics during brain activation.
Dong CY, Huang H, Sutin JDB, Kwon HS, Cragg GE, Gilbert R, Lee RT, Gratton E, Kamm RD, Lauffenburger DA, So PTC.
Magnetic tweezers microscope for cellular manipulation.
SPIE Photonics West, San Jose, California, 2000.
Optical Diagnostics of Living Cells III (Proceedings of SPIE, Vol. 3921). By DL Farkas and RC Leif (Editors). pp. 176-185, 2000. We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order ... [truncated at 150 words]
Toronov VY, Fantini S, Franceschini MA, Filiaci ME, Wolf M, Gratton E.
Temporal analysis of fluctuations in cerebral hemodynamics revealed by near-infrared spectroscopy.
Saratov Fall Meeting '99: Optical Technologies in Biophysics and Medicine (Proceedings of SPIE, Vol. 4001). By VV Tuchin and DA Zimnyakov (Editors). pp. 224-227, 2000. We have non-invasively studied the motor cortex hemodynamics in human subjects under rest and motor stimulation conditions using a multichannel near-IR tissue spectrometer. We obtained optical maps of oxy- and deoxy-hemoglobin concentration changes in terms of amplitudes of folding average, power spectrum and coherence at the stimulation repetition frequency, and the phase synchronization index. Under periodic motor stimulation conditions, we observed coherence and phase synchronization of the local hemodynamic changes with stimulation.
Franceschini MA, Gratton E, Hueber DM, Fantini S.
Near-infrared absorption and scattering spectra of tissues in vivo.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 526-531, 1999. Tissue inhomogeneity is often neglected in the analysis of the spectral data collected in tissues. We have measured the absorption and reduced scattering spectra of the human forearm in the wavelength range 633-84 1 nm, and in the range of source-detector separations 0.7-3 .3 cm. We found that the layered tissue structure due to the skin and adipose layers may have a significant effect not only on the data collected at different distances, but also on the data at 633 nm, where the optical absorbance is significantly higher than at the longer wavelengths considered. We conclude that the dependence of the optical penetration depth on the absorption and reduced.
Paunescu LA, Casavola C, Franceschini MA, Fantini S, Winter L, Kim J, Wood D, Gratton E.
Calf muscle blood flow and oxygen consumption measured with near-infrared spectroscopy during venous occlusion.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 317-323, 1999. We present non-invasive measurements of the calf muscle blood flow (BF) and oxygen consumption (OC) by near-infrared spectroscopy. We used a frequency domain tissue oximeter (modulation frequency: 1 10 MHz, wavelengths: 758 and 830 nm) to measure in real time (acquisition time: 0.64 s) the hemoglobin concentration and saturation. After 1-mm of baseline acquisition, we achieved venous occlusion by inflating a pneumatic cuff on the subject's thigh to a pressure of 60 mmHg. The cuffwas released after 1 mm. The baseline/inflation/release procedure was repeated 3 times to verify reproducibility. We calculated the BF and OC from the initial rate of increase of the total hemoglobin and deoxy-hemoglobin concentration immediately after the onset of venous occlusion. We examined 8 healthy subjects and 18 patients affected by peripheral vascular disease (PVD) in 1 or 2 legs to investigate whether muscle BF and OC at rest can be useful indicators of vascular insufficiency. ... [truncated at 150 words]
Toronov VY, Filiaci ME, Franceschini MA, Fantini S, Gratton E.
Photon-density wave fluctuation correlation spectroscopy: study of coherence in the brain and muscles.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 244-251, 1999. Using photon-density-wave fluctuation-correlation-spectroscopy, we studied the fluctuations of the optical signal measured in the brain and skeletal muscles. We investigated the autocorrelation power spectra at specific tissue locations, and the coherence between different tissue regions in the frequency band 0-0.3 Hz. We found specific dominant frequency components that can be assigned to vasoconstriction activity. In a measurement protocol involving voluntary motor stimulation (right hand finger movements), we found that the optical fluctuations observed in the left forearm muscle and in the left cerebral motor cortex show different coherence features at rest and during stimulation. These changes are particularly significant at the frequencies associated with vasoconstriction activity. A power-spectrum analysis of the optical fluctuations revealed a resonance-like dependence of the optical signal at the motor cortex on the period of the motor stimulation sequence.
Cerussi AE, Gratton E, Fantini S.
Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology.
Biomedical Imaging: Reporters, Dyes, and Instrumentation (Proceedings of SPIE, Vol. 3600). By DJ Bornhop, CH Contag, and EM Sevick-Muraca (Editors). pp. 171-181, 1999. Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in ... [truncated at 150 words]
Stankovic MR, Hueber DM, Maulik D, Stubblefield PG, Rosenfeld W, Gratton E, Franceschini MA, Fantini S.
Real-time optical imaging and spectroscopy of brain ischemia and hemorrhage.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 676-684, 1999. Perinatal brain injury, a major cause of neurological morbidity in infants, might be prevented or ameliorated if the preceding cerebrovascular disturbances known to cause brain ischemia and hemorrhage could be detected and monitored. We used an ISS tissue oximeter (model 96208, 1 10 MHz modulated light at two NIR wavelengths) to detect and monitor the development of experimental brain ischemia and hemorrhage by frequency domain optical spectroscopy (8-source/i-detector probe) and CW imaging (16-source/2-detector probe) in nineteen instrumented and ventilated newborn piglets. Our study demonstrates: 1) the sensitivity ofthe ISS system to detect <0.04 mL ofblood at a depth of 1-2 cm; 2) the specificity to distinguish subcortical, subarachnoid, and intraventricular hemorrhage; 3) the possibility of using the optically detected arterial pulse signal to differentiate injured from functional brain regions. In the case of subcortical hemorrhage, we observed that the measured cerebral a increased linearly with the sectional area of the ... [truncated at 150 words]
Casavola C, Paunescu LA, Franceschini MA, Fantini S, Winter L, Kim J, Wood D, Gratton E.
Near-infrared spectroscopy and the tilting table protocol: a novel method to study the blood flow and the oxygen consumption in tissues.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 685-692, 1999. We present a novel technique based on tilting the bed where the subject is lying, to non-invasively measure the tissue blood flow (BF) and oxygen consumption (OC) with near-infrared (NIR) spectroscopy. We used a NIR, frequency domain spectrometer to measure the concentrations of oxy-hemoglobin ([Hb02]), deoxy-hemoglobin ([Nb]) and total hemoglobin (THC) in the calf muscle of human subjects. The subject was lying horizontally, and after a baseline acquisition, the bed was tilted by 10 degrees (feet down, head up). This position was kept for 1 mm, then the subject was brought back to the horizontal position. This tilting procedure caused variations in the calf [Hb02], [Hb], and THC similar to those observed during a pneumatic-cuff-induced venous occlusion. The increasing rate of THC and [Hb] caused by tilting allowed the calculation of blood flow and oxygen consumption. We found a quantitative agreement between the values of BF (OC) measured with the ... [truncated at 150 words]
Buehler C, Dong CY, So PTC, French TE, Gratton E.
Frequency-domain pump-probe microscopic imaging using intensity-modulated laser diodes.
Systems and Technologies for Clinical Diagnostics and Drug Discovery II (Proceedings of SPIE, Vol. 3603). By GE Cohn and JC Owicki (Editors). pp. 262-271, 1999. We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.
Hueber DM, Fantini S, Cerussi AE, Barbieri BB.
New optical probe designs for absolute (self-calibrating) NIR tissue hemoglobin measurements.
Optical Tomography and Spectroscopy of Tissue III (Proceedings of SPIE, Vol. 3597). By B Chance, RR Alfano, and BJ Tromberg (Editors). pp. 618-631, 1999. Most of the instruments reported for near infrared absorption measurements in highly scattering media, suitable for noninvasive hemoglobin concentration measurements in living tissues, require some type of instrumental calibration, or knowledge of the initial concentrations of the two species. A new type of probe, which does not require calibration, has been developed. These new probes can provide a rapid and "absolute" measurement of absorption and scattering in tissue when combined with the "multi-distance" method developed, in part, at the Laboratory for Fluorescence Dynamics at the University of Illinois and ISS Incorporated. An initial evaluation of the performance and efficacy of these new probes has been performed using a frequency-domain ISS tissue oximeter. The new probes offer the convenience of not requiring calibration, and also provide more reliable measurements and greater long-term stability compared with the standard multidistance design. The new probes rely on a symmetrical arrangement of light sources and ... [truncated at 150 words]
Toronov VY, Filiaci ME, Franceschini MA, Fantini S, Gratton E.
Exploring tissue dynamics by photon-density-wave fluctuation correlation spectroscopy.
Saratov Fall Meeting '98: Light Scattering Technologies for Mechanics, Biomedicine, and Material Science (Proceedings of SPIE, Vol. 3726). By VV Tuchin, VP Ryabukho, and DA Zimnyakov (Editors). pp. 555-559, 1999. We propose photon-density-wave fluctuation correlation spectroscopy as a method to study the dynamics of tissue optical properties. Phantom measurements have shown that our frequency-domain instrument is appropriate to explore intensity, modulation and phase fluctuations in the frequency band from 0 to 125 Hz. Preliminary in vivo studies have revealed a rich optical dynamics in human tissues.
Lentz BR, Burgess SW, Gratton E.
Concentration dependence of DPHpPC fluorescence lifetime: photophysics and utility for monitoring membrane fusion.
International Symposium on Molecular Mechanisms of Membrane Fusion. April 27-29, 1987. Buffalo, NY.
Molecular Mechanisms of Membrane Fusion. By S Ohki, D Doyle, TD Flanagan, SW Hui, and E Mayhew (Editors). Plenum Publishing Corp., New York, pp. 557-566, 1988. ISBN: 9780306427732Kim KH, So PTC, Kochevar IE, Masters BR, Gratton E.
Two-photon fluorescence and confocal reflected light imaging of thick tissue structures.
Optical Investigations of Cells In Vitro and In Vivo (Proceedings of SPIE, Vol. 3260). By DL Farkas, RC Leif, and BJ Tromberg (Editors). pp. 46-57, 1998. The technology of two-photon excitation has opened a window of opportunity for developing non-invasive medical diagnostic tools capable of monitoring thick tissue biochemical states. Using cellular endogenous chromophores, (beta) -nicotinamide- adenine dinucleotide phosphate [NAD(P)H], the cellular metabolic rates in living human skin were determined. Although important functional information can be obtained from the fluorescence spectroscopy of endogenous chromophores, these chromophores are rather poor contrast enhancing agent for mapping cellular morphology. First, most endogenous chromophores are confined to the cellular cytoplasm which prevents the visualization of other cellular organelles. Second, there is significant variability in the distribution and the quantum yield of endogenous chromophores which depends on tissue biochemistry but prevents consistent comparison of cellular morphology. On the other hand, the deep tissue cellular morphology has been imaged with excellent resolution using reflected light confocal microscopy. In reflected light microscopy, the image contrast originates from the index of refraction differences of ... [truncated at 150 words]
So PTC, Masters BR, Gratton E, Kochevar IE.
Two-photon optical biopsy of thick tissues.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 22). By J Fujimoto and M Patterson (Editors). Optical Society of America, JMA3, 1998. ISBN: 9781557525468Two-photon optical biopsy has been performed in human and mouse skin. Both cellular and extracellular tissue components have been visualized based on endogenous fluorescence.
Fantini S, Walker SA, Franceschini MA, Cerussi AE, Edler J, Moesta KT, Schlag PM, Kaschke M, Gratton E.
Optical characterization of breast tumors by frequency-domain optical mammography.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 22). By J Fujimoto and M Patterson (Editors). Optical Society of America, JTuA5, 1998. ISBN: 9781557525468We present a method aimed at the non-invasive optical characterization of breast tumors. This method is based on using diffusion theory to model the propagation of intensity-modulated light in breast tissue. The tumor is assumed to be a spherical inhomogeneity. We report our frst results obtained in the laboratory on a tissuelike phantom, and in the clinic on a patient affected by breast cancer. The in vivo result indicates that the malignant tumor considered has a higher absorption and a higher reduced scattering coefficient than healthy tissue.
Masters BR, So PTC, Mantulin WW, Gratton E.
Tissue microscopy and spectroscopy: a two-photon approach.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 22). By J Fujimoto and M Patterson (Editors). Optical Society of America, JMA4, 1998. ISBN: 9781557525468Using two-photon excitation, we have demonstrated that While these developments in confocal microscopy fluorescence spectroscopy of endogenous chromophores have succeeded in imaging deep tissue structures in highly such as NAD(P)H can be performed in human skin and scattering biological tissue up to a depth of about 200 to rabbit cornea.
Paunescu LA, Franceschini MA, Fantini S, Cerussi AE, Gratton E.
Effective optical properties of two-layered turbid media using the frequency-domain multi-distance method.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 21). By J Fujimoto and M Patterson (Editors). Optical Society of America, ATuD20, 1998. ISBN: 9781557525468We measured the effective absorption and reduced scattering coefficients on the surface of two-layered turbid media using a frequency-domain multi-distance method. We measured the amplitude and the phase of the modulated intensity at source-detector distances ranging from 1.5 to 3.0 cm. We considered several combinations of one layer (L) (thickness: 0.1-1.6 cm) placed on top of one block (B) (thickness - 5 cm). We found that for layers less than about 0.40 cm thick the frequency-domain multi-distance method yields the optical properties of the underlying block. We also found that for layers more than 1.3 cm thick the frequency-domain multi-distance method yields a reduced scattering coefficient which reproduces that of the superficial layer. For a layer thickness in the range of 1.3-1.6 cm, the effective absorption coefficient approaches the layer absorption coefficient but does not match it to within experimental errors.
Toronov VY, Filiaci ME, Fantini S, Gratton E.
Study of large scale fluctuations in turbid media by photon-density-wave fluctuation correlation spectroscopy.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 21). By J Fujimoto and M Patterson (Editors). Optical Society of America, ASuB7, 1998. ISBN: 9781557525468We propose photon-density-wave fluctuation correlation spectroscopy as a method to study the macroscopic dynamics of tissue optical properties. Phantom measurements have shown that our frequency-domain instrument is appropriate to explore intensity, modulation and phase fluctuations in the frequency band from 0 to 125 Hz. Preliminary in vivo studies have revealed a reach optical dynamics in human tissues.
Franceschini MA, Paunescu LA, Fantini S, Pratesi S, Maier JS, Donzelli GP, Gratton E.
Frequency-domain optical measurements in vitro on two- and three-layerd tissue-like phantoms, and in vivo on infant heads.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 21). By J Fujimoto and M Patterson (Editors). Optical Society of America, ASuB3, 1998. ISBN: 9781557525468We have performed frequency-domain optical measurements on the surface of two- and three-layered turbid media (in vitro) and on infant heads (in vivo). For the in vitro experiment we measured a number of combinations of one layer (thickness in the range 0.1-1.6 cm) on top of one semi-infinite block, with or without a clear layer (thickness of 0.1 cm) sandwiched in between. For the in vivo experiment, we did measurements on 20 preterm infants at the Meyer Children's Hospital in Florence, Italy. In the two-layered samples, we measured the effective absorption and reduced scattering coefficients with a multidistance method based on data taken at source-detector distances in the range 1.5-4.5 cm. We found that the effective optical coefficients reproduce those of the underlying block if the superficial layer is less than about 0.4 cm thick, whereas they are representative of the superficial layer if it is more than about 1.3 ... [truncated at 150 words]
Cerussi AE, Fantini S, Gratton E.
Quantitative fluorescence spectroscopy in strongly scattering media containing multiple fluorophores.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Orlando, Florida
March 8, 1998.
Biomedical Optical Spectroscopy and Diagnostics / Therapeutic Laser Applications (OSA Trends in Optics and Photonics, Vol. 22). By E Sevick-Muraca and J Izatt (Editors). Optical Society of America, BMC3, 1998. We have used diffusion theory to model the fluorescence photon density inside a strongly scattering medium containing multiple fluorophores. Frequency domain parameters were measured as functions of source-detector separation and modulation frequency inside in a turbid medium containing two fluorophores. Our theoretical prediction, based on a model originally proposed by Patterson and Pogue, was found to be in excellent agreement with the experimental data.
Filiaci ME, Toronov VY, Fantini S, Gratton E.
Optical probe and frequency-domain instrumentation to study spatial and temporal correlations of fluctuations in tissues.
Advances in Optical Imaging and Photon Migration (AOIPM), Orlando, Florida, March 8, 1998.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 22). By J Fujimoto and M Patterson (Editors). Optical Society of America, BTuD5, 1998. ISBN: 9781557525468
Fantini S, Moesta KT, Franceschini MA, Jess H, Erdl H, Gratton E, Schlag PM, Kaschke M.
Instrumentation and clinical applications in frequency-domain optical mammography.
19th International Conference - IEEE Engineering in Medicine and Biology society, Oct. 30 - Nov. 2, 1997. Chicago, IL, USA.
Engineering in Medicine and Biology society, 1997. Proceedings of the 19th Annual International Conference of the IEEE (Vol. 6). pp. 2741-2744, 1997. We describe an instrument for frequency-domain light mammography (LIMA), and we present clinical results obtained on patients affected by breast cancer. This instrument uses two laser diodes emitting at 690 and 825 nm. Their intensities are modulated at a frequency of 110 MHz. The amplitude and the phase of the intensity wave are the measured quantities. The breast is slightly compressed between two glass plates and the optical signal is detected in transmission, i.e. on the opposite side of the illuminated spot of the breast. The acquisition time for a full breast image is about 3 minutes. Edge effects (that are mainly due to the breast thickness variability within the scanned area) are corrected by an algorithm of data analysis which enhances the contrast of the optical mammograms. In the clinical applications, we successfully detected 27 out of 37 malignant tumors (73%). This result shows the practical feasibility and the ... [truncated at 150 words]
Franceschini MA, Wallace DJ, Barbieri BB, Fantini S, Mantulin WW, Pratesi S, Donzelli GP, Gratton E.
Optical study of the skeletal muscle during exercise with a second-generation frequency-domain tissue oximeter.
Optical Tomography and Spectroscopy of Tissue: Theory, Instrumentation, Model, and Human Studies II (Proceedings of SPIE, Vol. 2979). By B Chance and RR Alfano (Editors). pp. 807-814, 1997. We present a re-engineered frequency-domain tissue oximeter operating in the near-infrared spectral region. This instrument is based on the multi-distance measurement protocol, which we have implemented in our original design by multiplexing multiple light sources. The new instrument uses intensity modulated (110 MHz) laser diodes emitting at 750 and 840 nm. The laser diodes are coupled to glass optical fibers (600 micrometer core diameter). The average light intensity delivered to the tissue is about 3 mW. The multiplexing electronics are based on solid state switches that allow for acquisition times per point as short as tens of milliseconds. Our tests on phantoms and in vivo with the new oximeter have shown significant improvement in terms of stability, reliability, and reproducibility with respect to the original prototype. Furthermore, by using optical fibers we achieve a high versatility in the design of the measuring probe, permitting custom design for various tissue contours ... [truncated at 150 words]
Maier JS, Cerussi AE, Fantini S, Gratton E.
Experimental recovery of absorption, scattering, and fluorescence parameters in highly scattering media from a single frequency measurement.
Optical Tomography and Spectroscopy of Tissue: Theory, Instrumentation, Model, and Human Studies II (Proceedings of SPIE, Vol. 2979). By B Chance and RR Alfano (Editors). pp. 6-13, 1997. In recent years researchers have made significant progress in understanding the physics of fluorescence in highly scattering materials such as tissues in the near-infrared. We have quantitatively verified a model which describes fluorescence in ideal (homogeneous and infinite) tissue-like media. Given the quantitative accuracy of this model, one can use measurements of the fluorescence of a tissue with a homogeneous distribution of fluorophore to obtain the quantum yield, lifetime of the probe, and the absorption and scattering coefficients of the tissue at the fluorescent wavelength. We demonstrate that this can be done with a simple measurement of the photon density as a function of source- detector separation at the excitation and emission wavelengths. To verify our approach we present the lifetime, quantum yield of the fluorescent probe (rhodamine B), and the absorption and scattering coefficients of the medium at the emission peak wavelength that are obtained by a fit of ... [truncated at 150 words]
Fantini S, Franceschini MA, Gratton E.
Effect of spatially distributed light sources on the frequency-domain solution to the diffusion equation.
Optical Tomography and Spectroscopy of Tissue: Theory, Instrumentation, Model, and Human Studies II (Proceedings of SPIE, Vol. 2979). By B Chance and RR Alfano (Editors). pp. 509-514, 1997. We have investigated the accuracy of the frequency-domain, diffusion equation Green's function in modeling optical signals in turbid media. Our optical measurements were conducted in strongly scattering media having absorption coefficients in the range 0.035 - 0.14 cm-1 and reduced scattering coefficients in the range 5 - 22 cm-1. The optical signal was both delivered to and collected from the sample by means of optical fibers. We have verified that the r-independent factor in the Green's function does not agree with the experimental data. This discrepancy shows that the photons launched in the medium by an optical fiber cannot be modeled by a point source located at a fixed position. These results have no influence on multi-distance measurement protocols (which only employ the r-dependent part of the solution), while they must be considered when calibration measurements on a known sample are performed.
Walker SA, Fantini S, Gratton E.
Effect of index of refraction mismatch on the recovery of optical properties of cylindrical inhomogeneities in an infinite turbid medium.
Optical Tomography and Spectroscopy of Tissue: Theory, Instrumentation, Model, and Human Studies II (Proceedings of SPIE, Vol. 2979). By B Chance and RR Alfano (Editors). pp. 219-225, 1997. Optical inhomogeneities embedded in a turbid medium are characterized not only by their absorption and reduced scattering coefficients, but also by their index of refraction relative to the background medium. Although in diffusion theory it is impossible to separate the index of refraction from the absorption and reduced scattering coefficients in an infinite homogeneous medium, application of boundary conditions for an inhomogeneity adds enough information to separately determine these optical properties. A mismatched index of refraction affects diffuse photon propagation in two ways: photons travel at a different speed inside the inhomogeneity, and photons entering and leaving the inhomogeneity are influenced by Fresnel reflections at the surface of the object. We have integrated these two effects into the analytical solution to the diffusion equation for a cylinder in an infinite medium. Theoretical results are compared with experimental data, and the effect of index of refraction mismatch is evaluated for different ... [truncated at 150 words]
Cerussi AE, Fantini S, Maier JS, Mantulin WW, Gratton E.
Chromophore detection by fluorescence spectroscopy in tissue-like phantoms.
Optical Tomography and Spectroscopy of Tissue: Theory, Instrumentation, Model, and Human Studies II (Proceedings of SPIE, Vol. 2979). By B Chance and RR Alfano (Editors). pp. 139-150, 1997. In the near-infrared spectral region (700 - 900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the dc fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable dc fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in ... [truncated at 150 words]
So PTC, Ragan TM, Gratton E, Carrero J, Voss EW Jr.
Two-photon single particle tracking in 3D.
Functional Imaging and Optical Manipulation of Living Cells (Proceedings of SPIE, Vol. 2983). By DL Farkas and BJ Tromberg (Editors). pp. 45-56, 1997. Transport processes are important in biology and medicine. Examples include virus docking and infection, endocytosis of extracellular protein and phagocytosis of antigenic material. Trafficking driven by molecular motors inside a complex 3D environment is a shared common theme. The complex sequence of these events are difficult to resolve with conventional techniques where the action of many cells are asynchronously averaged. Single particle tracking (SPT) was developed by Ghosh and Webb to address this problem and has proven to be a powerful technique in understanding membrane- protein interaction. Since the traditional SPT method uses wide field illumination and area detectors, it is limited to the study of 2D systems. In this presentation, we report the development of a 3D single particle tracking technique using two-photon excitation. Using a real-time feedback system, we can dynamically position the sub-femtoliter two-photon excitation volume to follow the fluorescent particle under transport by maximizing the detected ... [truncated at 150 words]
König K, Liang H, Kimel S, Svaasand LO, Tromberg BJ, Krasieva TB, Berns MW, Halbhuber KJ, So PTC, Mantulin WW, Gratton E.
Cell damage in UVA and CW/femtosecond NIR microscopes.
Functional Imaging and Optical Manipulation of Living Cells (Proceedings of SPIE, Vol. 2983). By DL Farkas and BJ Tromberg (Editors). pp. 37-44, 1997. Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams ... [truncated at 150 words]
Gratton E, Fantini S, Franceschini MA, Walker SA, Maier JS.
Spectroscopy and tomography of tissues in the frequency-domain.
NATO Advanced Research Workshop on Analytical Use of Fluorescent Probes in Oncology. Miami, Florida. October 14-18, 1995.
Analytical Use of Fluorescent Probes in Oncology (NATO ASI Series A: Life Sciences, Vol. 286). By E Kohen and JG Hirschberg (Editors). Kluwer Academic Press, pp. 41-52, 1997. ISBN: 9780306453694Substantial progress in the field of light spectroscopy and imaging of tissues was achieved when the group of Chance, Patterson and Wilson showed that the optical parameters of a turbid medium can be obtained from time resolved measurements of short light pulses propagating in the medium (Patterson et al, 1991a). Essentially, a fit of the intensity as a function of time, measured at some distance from the source, can provide separately the values of the absorption and of the reduced scattering coefficients. This demonstration was important because the focus was shifted from attempts to separate the scattering from absorption, using empirical corrections to the Beer-Lambert law, to a rigorous application of a physical model. During the same period, our lab proposed employing the Fourier transform equivalent concept using an intensity modulated light source (Gratton et al, 1990). Since frequency domain methods have better resolution and sensitivity and are much faster ... [truncated at 150 words]
König K, So PTC, Mantulin WW, Gratton E.
Cell damage in two-photon microscopes.
Optical Biopsies and Microscopic Techniques (Proceedings of SPIE, Vol. 2926). By IJ Bigio, WS Grundfest, H Schneckenburger, K Svanberg, and PM Viallet (Editors). pp. 172-176, 1996. We report on power thresholds for cell damage in femtosecond two-photon NIR microscopes and discuss interactions between vital cells and high intense femtosecond kW pulses in the TW/cm2 (1012 W/cm2) range. The influence of femtosecond NIR pulses on cell metabolism and cell vitality was studied by autofluorescence imaging, morphology studies, employment of vitality kits and sensitive cell cloning assays. We show that cells remain unaffected by high intense NIR femtosecond pulses below certain power thresholds. This allows nondestructive nonlinear 3D fluorescence imaging of vital cells. Above these thresholds, giant cell growth, failed cell division, complete cell destruction, and intracellular plasma formation occur.
Franceschini MA, Fantini S, Cerussi AE, Barbieri BB, Chance B, Gratton E.
Contrast enhancement by edge effect corrections in frequency-domain optical mammography.
Advances in Optical Imaging and Photon Migration (OSA Trends in Optics and Photonics, Vol. 2). By RR Alfano and JG Fujimoto (Editors). Optical Society of America, pp. 160-163, 1996. ISBN: 9781557524256Gratton E, Mantulin WW, Weber G, Royer CA, Jameson DM, Reininger R, Hansen RWC.
Fluorescence dynamics of biological systems using synchrotron radiation.
Synchrotron Radiation Instrumentation, Argonne National Laboratory, Oct 18-20, 1995.
Rev Sci Instrum. 1996; 67(9): 3363.A beamline for time-resolved fluorescence spectroscopy of biological systems is under construction at the Synchrotron Radiation Center. The fluorometer, operating in the frequency domain, will take advantage of the time structure of the synchrotron radiation light pulses to determine fluorescence lifetimes. Using frequency-domain techniques, the instrument can achieve an ultimate time resolution on the order of picoseconds. Preliminary experiments have shown that reducing the intensity of one of the fifteen electron bunches in the storage ring allows measurement of harmonic frequencies equivalent to the single-bunch mode. This mode of operation of the synchrotron significantly extends the range of lifetimes that can be measured. The wavelength range (encompassing the visible and ultraviolet), the range of measurable lifetimes, and the stability and reproducibility of the storage ring pulses should make this beamline a versatile tool for the investigation of the complex fluorescence decay of biological systems.
Maier JS, Walker SA, Gratton E.
Frequency-domain optical spectroscopy and imaging of tissues.
Biomedical Optical Instrumentation and Laser-assisted Biotechnology. Erice, Italy. Nov 10-22, 1995.
Biomedical Optical Instrumentation and Laser-assisted Biotechnology (NATO Science Series E, Vol. 325). By AMV Scheggi, S Martellucci, AN Chester, and R Pratesi (Editors). Kluwer Academic Press, pp. 121-142, 1996. ISBN: 0792341724INTRODUCTION: The use of light for the investigation of the interior of the human body has a relatively long history. In clinical practice, transillumination has been performed since 1929 to detect tumors in the breast and other organs although with limited success [I]. There are a number of methods based on light, including continuous-monitor instruments, to detect variations in the concentration of some substances such as hemoglobin and its oxidation state (8, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 181. A typical example this type of instrument is the common pulse oximeter. The vast literature on the subject can roughly be divided into two major topics: the use of light to detect anatomical structures, which we will refer to as tomography or imaging, and the use of light to perform spectroscopy of the tissue with the purpose of detecting and quantifying different molecular species which we refer ... [truncated at 150 words]
Gratton E, Dong CY, So PTC.
Fluorescence lifetime microscopy: a stimulated emission approach.
Optical Diagnostics of Living Cells and Biofluids (Proceedings of SPIE, Vol. 2678). By DL Farkas, RC Leif, AV Priezzhev, T Asakura, and BJ Tromberg (Editors). pp. 98-109, 1996. The stimulated emission process has been rarely exploited in spectroscopy and microscopy. Instead, a spectroscopy method based on the pump-probe principle has been frequently used to observe picosecond and femtosecond processes. This common approach has not been applied to microscopy due to the relatively slow acquisition time and the lack of 3D information. We have exploited an idea originally proposed by F. Lytle group in which two pulsed lasers are simultaneously focused on the sample. One laser is used to excite a population of molecules and the second laser to induce stimulated emission. The stimulated radiation is carried away in the same direction of the stimulating laser beam. By collecting the fluorescence emission in other directions, we observe a modulation of the fluorescence signal as a function of the delay between the two laser pulses. The repetition rate of the two lasers is slightly different producing a frequency beating at ... [truncated at 150 words]
Maier JS, Cerussi AE, Fantini S, Franceschini MA, Gratton E.
Quantitative fluorescence in tissue-like media.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Orlando, Florida, March 18, 1996.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 3). By E Sevick-Muraca and D Benaron (Editors). Optical Society of America, FG6, 1996. ISBN: 9781557525475We present a study focused on quantitative fluorescence in tissues at near-infrared wavelengths. We demonstrate the quantitative description of fluorescence in tissue-like media provided by a model derived from difhsion theory. Based on this model we conclude that the spatial distribution of the fluorescent light does not depend on the lifetime of the probe. We also show that lifetime and quantum yield or probe concentration can be determined in a hghly scattering medium using frequency-domain techniques, without the use of a reference probe.
Franceschini MA, Fantini S, Cerussi AE, Barbieri BB, Chance B, Gratton E.
The effect of water in the quantitation of hemoglobin concentration in a tissue-like phantom by near-infrared spectroscopy.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Orlando, Florida, March 18, 1996.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 3). By E Sevick-Muraca and D Benaron (Editors). Optical Society of America, AP16, 1996. ISBN: 9781557525475We have investigated the contribution of water in the quantitative determination of hemoglobin concentration and saturation by near-infrared spectroscopy in turbid media. Our study consists of in vitro measurements on an aqueous suspension containing Liposyn, bovine blood, and yeast, buffered at pH 7.2. The optical coefficients of the medium (µa~0.03-0.08 cm-1, µs'~6 cm-1) match those of biological tissue in the near-infrared, and the hemoglobin concentration (23 µM) is also similar to that found in tissues. We were able to reversibly saturate and desaturate hemoglobin in the full range 0-100% by flowing either oxygen or nitrogen through the medium. We found that water absorption must be taken into account to obtain accurate results of hemoglobin concentration and saturation. However, for the two wavelengths employed in this study (715 and 825 nm), the water correction has a small effect on deoxy-hemoglobin concentration, and on hemoglobin saturation in the high saturation limit. Oxy-hemoglobin ... [truncated at 150 words]
Cerussi AE, Maier JS, Fantini S, Franceschini MA, Gratton E.
The Frequency-domain multi-distance method in the presence of curved boundaries.
Biomedical Optical Spectroscopy and Diagnostics (BOSD), Orlando, Florida, March 18, 1996.
Biomedical Optical Spectroscopy and Diagnostics (OSA Trends in Optics and Photonics, Vol. 3). By E Sevick-Muraca and D Benaron (Editors). Optical Society of America, AP9, 1996. We present experiments designed to test the applicability of the frequency-domain semi-infinite multi-distance method to cylindrical boundaries. Material with absorption and reduced scattering coefficients similar to those of tissue exposed to near-infrared light was cast into five cylindrical phantoms ranging in diameter from 4.2 to 10.2 cm, and also into a semi-infinite control. Measurements performed along the long axis of symmetry on the larger diameter cylinders reliably recovered both the absorption and the reduced scattering coefficients within 10% of the control values. Measurements performed along the circumference yielded values for these optical coefficients that differed significantly from the control values. These findings are explained by observing the measured frequency-domain parameters as functions of source-detector separation.
König K, So PTC, Mantulin WW, Gratton E, Krasieva TB, Berns MW, Tromberg BJ.
Two-photon excited cellular autofluorescence induced by CW and femtosecond NIR microradiation.
Optical and Imaging Techniques for Biomonitoring (Proceedings of SPIE, Vol. 2628). By HJ Foth, R Marchesini, H Podbielska, M Robert-Nicoud, and H Schneckenburger (Editors). pp. 12-19, 1996. We report the first two-photon excited autofluorescence measurements in single cells induced by continuous wave NIR laser microbeams. In particular we demonstrate NIR-excited NAD(P)H fluorescence of motile spermatozoa in a single-beam gradient force optical trap ('optical tweezers'). In addition, two-photon NIR excited autofluorescence imaging and 'tau- mapping' of CHO cells was performed with laser scanning microradiation of a tunable femtosecond-Ti:sapphire laser. Cellular response on autofluorescence to two-photon absorption of NIR radiation was compared with exposure to UVA light (one-photon absorption).
Mantulin WW, Fantini S, Franceschini MA, Walker SA, Maier JS, Gratton E.
Tissue optical parameter map generated with frequency-domain spectroscopy.
Biomedical Optoelectronic Instrumentation (Proceedings of SPIE, Vol. 2396). By JA Harrington, DM Harris, and A Katzir (Editors). pp. 323-330, 1995. Near infrared optical imaging is emerging as a potentially important imaging modality, because it offers real time data access, portability, cost-effectiveness, and the relatively safe use of non-ionizing radiation. Reconstruction of images by optical tomography is complicated by the diffusive character of light propagation in optically heterogeneous tissue. The spatial volume element probed by the light path between the light source and optical detector is rather wide and depends on a variety of experimental and instrumental factors. We have published an optical image of the hand in air based on photon density wave distribution characteristics, using both steady-state (intensity) and frequency-domain (phase and modulation) experimental conditions. Since then, we have developed new instrumentation, better measurement protocols, some reconstruction algorithms and a more complete theoretical understanding of photon diffusion in both homogeneous and heterogeneous media. We have now performed frequency-domain measurements (at a modulation frequency of 160 MHz with 760 nm ... [truncated at 150 words]
Ferrari M, de Blasi RA, Fantini S, Franceschini MA, Barbieri BB, Quaresima V, Gratton E.
Cerebral and muscle oxygen saturation measurement by a frequency-domain near-infrared spectroscopic technique.
Optical Tomography, Photon Migration, and Spectroscopy of Tissue and Model Media: Theory, Human Studies, and Instrumentation (Proceedings of SPIE, Vol. 2389). By B Chance and RR Alfano (Editors). pp. 868-874, 1995. Absorption and reduced scattering coefficients at 715 and 825 nm as well as hemoglobin saturation and content of the forehead and the forearm were measured by a 110 MHz frequency-domain multisource instrument. The absolute data obtained by the frequency- domain spectrometer were compared with oxygenation changes measured by a continuous wave instrument during quadriceps ischemia and postural changes. These preliminary results indicate that portable frequency-domain instruments could be very helpful to investigate brain and muscle pathophysiology.
Walker SA, Cerussi AE, Gratton E.
Back-projection image reconstruction using photon density waves in tissues.
Optical Tomography, Photon Migration, and Spectroscopy of Tissue and Model Media: Theory, Human Studies, and Instrumentation (Proceedings of SPIE, Vol. 2389). By B Chance and RR Alfano (Editors). pp. 350-357, 1995. The reconstruction of scattering and absorption inhomogeneities in tissues generally involves the solution of the inverse scattering problem. This is a computationally intesive task that cannot be easily performed during image acquisition. Instead, we obtain approximate spatial maps of absorption and scattering coefficients using a back-projection algorithm, similar in principle to that used in computerized tomography. Given the nonlinear nature of light propagation in tissue, we expect that this approach can only give a first approximation solution of the reconstruction problem. Our preliminary results indicate that relatively accurate maps are rapidly obtained. We have reconstructed, to a first approximation, the optical parameters and positions of scattering and partially absorbing objects. Our back-projection approach employs frequency-domain methods using a light emitting diode as the light source (100 MHz modulation frequency, peak wavelength 715 nm). Data is collected from multiple linear scans of the investigated area at different projection angles, as in ... [truncated at 150 words]
Fantini S, Franceschini MA, Walker SA, Maier JS, Gratton E.
Photon path distributions in turbid media: applications for imaging.
Optical Tomography, Photon Migration, and Spectroscopy of Tissue and Model Media: Theory, Human Studies, and Instrumentation (Proceedings of SPIE, Vol. 2389). By B Chance and RR Alfano (Editors). pp. 340-349, 1995. Near-IR optical tomography is thwarted by the highly scattering nature of light propagation in tissue. We propose a weighted back-projection method to produce a spatial map of an optical parameter which characterized the investigated medium. We have studied the problem of the choice of the back-projection weight function for the absorption coefficient ((mu) a) and for the reduced scattering coefficient ((mu) s') of tissuelike phantoms. Working in frequency-domain optical imaging, we have initially approached the problem of quantifying the effect caused by a small absorbing defect embedded in the medium on the measured DC intensity, AC amplitude, and phase. The collection of DC, AC, and phase changes during a 1 mm step raster scan of the absorbing defect provides information on the photon path distributions and, in general, on the probed spatial region when DC, AC, and phase are, respectively, the measured parameters. We report experimentally determined weight functions for ... [truncated at 150 words]
Franceschini MA, Fantini S, Walker SA, Maier JS, Mantulin WW, Gratton E.
Multichannel optical instrument for near-infrared imaging of tissue.
Optical Tomography, Photon Migration, and Spectroscopy of Tissue and Model Media: Theory, Human Studies, and Instrumentation (Proceedings of SPIE, Vol. 2389). By B Chance and RR Alfano (Editors). pp. 264-273, 1995. Our research is aimed at the development of a frequency-domain instrument for conducting non-invasive, real-time, near-infrared, optical tomography of tissue in vivo. Our goal is to reconstruct a spatial map of the optical properties of a strongly scattering medium in a semi-infinite-geometry sampling configuration. Specifically, we focus our attention on the absorption coefficient ((mu) a) and the reduced scattering coefficient ((mu) s') of the medium. We have developed a frequency- domain measurement protocol (which we call precalibrated), which permits one to recover the values of (mu) a and (mu) s' of a uniform tissue-like phantom from a measurement at a single source-detector separation and a single modulation frequency. It requires a preliminary reference measurement on a calibration sample of known optical properties before the measurement on the investigated sample. This approach is in principle rigorous only in macroscopically homogeneous media. We have verified that the equations valid for uniform media ... [truncated at 150 words]
Maier JS, Barbieri BB, Chervu A, Chervu I, Fantini S, Franceschini MA, Levi M, Mantulin WW, Rosenberg A, Walker SA, Gratton E.
In vivo study of human tissues with a portable near-infrared tissue spectrometer.
Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases II (Proceedings of SPIE, Vol. 2387). By RR Alfano (Editors). pp. 240-248, 1995. In this paper, we present a series of measurements made with a portable frequency-domain near-infrared tissue spectrometer (OMNIA). This is the first application of the OMNIA in a clinical setting. All of the measurements presented here were taken in vivo, most were on human subjects. We report the results of three experiments: (1) A simple ischemia/plethysmography experiment, which indicates ability of the instrument to noninvasively, continuously monitor the hemoglobin saturation of a limb. (2) A survey of hemoglobin saturation in patients with peripheral vascular disease. (3) An animal experiment to demonstrate the correlation of our instrument readings with results from established techniques for measuring hemoglobin saturation. We measured the absorption and reduced scattering coefficients of the tissue at two wavelengths (715 nm and 850 nm). From the absorption coefficients, we calculated the concentrations of oxygenated and deoxygenated hemoglobin ([HbO2] and [Hb]), which immediately yield the hemoglobin saturation (Y) and the ... [truncated at 150 words]
Fantini S, Franceschini MA, Maier JS, Walker SA, Gratton E.
Frequency-domain multisource optical spectrometer and oximeter.
Photon Transport in Highly Scattering Tissue (Proceedings of SPIE, Vol. 2326). By S Avrillier, B Chance, GJ Mueller, AV Priezzhev, and VV Tuchin (Editors). pp. 108-116, 1995. We have designed and constructed a near-infrared spectrometer for the non-invasive optical study of biological tissue. This instrument works in the frequency-domain and employs multiple source-detector distances to recover the absorption coefficient ((mu) (alpha )) and the reduced scattering coefficient ((mu) s') of tissue. The light sources are eight light emitting diodes (LEDs) whose intensities are modulated at a frequency of 120 MHz. Four LEDs emit light at a peak wavelength of 715 nm ((lambda) 1), while the other four LEDs emit at a peak wavelength of 850 nm ((lambda) 2). From the frequency-domain raw data of phase, dc intensity, and ac amplitude obtained from each one of the eight light sources, which are located at different distances from the detector fiber, we calculate (mu) (alpha ) and (mu) s' at the two wavelengths (lambda) 1 and (lambda) 2. The concentrations of oxy- and deoxy-hemoglobin, and hence hemoglobin saturation, are ... [truncated at 150 words]
Gryczynski Z, Fronticelli C, Gratton E, Lubkowski J, Bucci E.
Effect of disordered hemes and dimerization in isolated α-subunits of hemoglobin detected by time-resolved fluorescence spectroscopy in the picosecond range.
Time-Resolved Laser Spectroscopy in Biochemistry IV (Proceedings of SPIE, Vol. 2137). By JR Lakowicz (Editors). pp. 129-134, 1994. Our recent linear dichroism study of transition moment directions for protoporphyrin derivatives [1,2] demonstrate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e. on the k2 orientation parameter of the Forster equation. Using the atomic coordinates of human hemoglobin and taking into account the direction of the transition moment of the near UV (300-380 nm) heme absorption band we have estimated the rate of energy transfer from tryptophan to heme in the isolated a chains, which are a single tryptophan protein. It appears that the rate of energy transfer is very sensitive to the orientation of the transition moment of the heme and similarly to myoglobin [3] natural heme ... [truncated at 150 words]
So PTC, French TE, Gratton E.
Frequency domain time-resolved microscope using a fast-scan CCD camera.
Time-Resolved Laser Spectroscopy in Biochemistry IV (Proceedings of SPIE, Vol. 2137). By JR Lakowicz (Editors). pp. 83-92, 1994. Time-resolved fluorescence imaging can enhance the contrast of microscope images and it can also provide important information about the micro-environment in cellular systems. We have developed a fluorescence microscope which can measure fluorescence lifetimes over an entire image. Fluorescence lifetimes are measured by using heterodyne frequency domain techniques. Heterodyning is accomplished by using an intensity modulated laser light source and a fast scan CCD camera coupled with a gain modulated microchannel plate as the detector. The high duty cycle of this method allows us to generate a phase resolved image with about five seconds integration time. Operating in the fast scan mode, the systematic uncertainties in lifetime determination caused by photobleaching are less severe than those of slow-scan cameras. The microchannel plate can be modulated at frequencies up to 300 MHz, which allows us to measure lifetimes as short as 500 ps with resolution of 50 ps. The modulation of ... [truncated at 150 words]
Fantini S, Franceschini MA, Fishkin JB, Gratton E.
Absolute measurement of absorption and scattering coefficients spectra of a multiply scattering medium.
Biomedical Fiber Optic Instrumentation (Proceedings of SPIE, Vol. 2131). pp. 356-364, 1994. On the basis of the diffusion theory model, frequency-domain spectroscopy allows for a quantitative determination of the absorption ((mu) a) and scattering ((mu) s') coefficient spectra of a homogeneous multiple scattering medium. We performed measurements using an intensity modulated light emitting diode (LED) as the light source. The LED's spectral distribution permits the study of a spectral region extending for about 80 nm. Data sets (phase shift and average intensity) at two different source-detector distances are acquired: the absorption and scattering coefficient spectra of the medium are then calculated from analytical expressions for (mu) a and (mu) s'. Methylene blue (peak absorption wavelength 656 nm) is used as a test absorbing material. The methylene blue is dissolved in an aqueous Liposyn solution which serves as the multiple scattering medium. The relative amounts of absorber and scatterer are chosen such that the values of (mu) a and (mu) s' match typical ... [truncated at 150 words]
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Spectroscopic innovations at the Laboratory for Fluorescence Dynamics.
ell and Biotissue Optics: Applications in Laser Diagnostics and Therapy (Proceedings of SPIE, Vol. 2100). By VV Tuchin (Editors). pp. 6-18, 1994. The emergence of time-resolved fluorescence techniques has been very important to the study of biochemical, biophysical and biomedical research areas. Many of the advances in these fields have been technology driven. The Laboratory for Fluorescence Dynamics has been actively involved in development of frequency domain technology for a variety of applications. Our recent work on microwave super heterodyning detectors, fluorescence lifetime resolved spectroscopy, frequency domain fluorescence imaging microscopy, fluorescence lifetime resolved stopped-flow kinetics and global methods of data analysis is summarized in this report. We provide examples of how these new technologies are applied.
Fantini S, Franceschini MA, Fishkin JB, Mantulin WW, Gratton E.
Absorption spectra of a chromophore in highly scattering media.
ell and Biotissue Optics: Applications in Laser Diagnostics and Therapy (Proceedings of SPIE, Vol. 2100). By VV Tuchin (Editors). pp. 114-123, 1994. Frequency domain spectroscopy provides a quantitative measure of the optical properties, namely (mu) a and (mu) s' coefficient spectra, of multiply scattering, macroscopically homogeneous media or tissue. The diffusion model for photon transport provides the theoretical framework for the analytical expressions of the optical properties. Experimentally, we intensity modulated (60 MHz) a light emitting diode, which emits between 620 - 700 nm. From data sets of relative phase shifts and average intensity at different source- detector separations, we calculated on the basis of the analytical expressions a wavelength resolved absorption (mu) a ((lambda) ) and scattering (mu) s' ((lambda) ) coefficient spectrum. The test material was methylene blue, whose absorption spectrum (maximum 656 nm) closely matches the wavelength profile of the diode source. The multiply scattering, macroscopically homogeneous medium for dissolving the methylene blue was provided by a diluted fat emulsion, Liposyn III. The concentrations of both the absorbing and ... [truncated at 150 words]
Franceschini MA, Fantini S, Gratton E.
LEDs in frequency-domain spectroscopy of tissues.
Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases (Proceedings of SPIE, Vol. 2135). By RR Alfano (Editors). pp. 300-306, 1994. Light spectroscopy in the frequency-domain has been used to study the optical properties of biological tissues. We have analyzed the possibility of using LEDs as intensity modulated light sources for frequency-domain spectroscopy. The use of LEDs presents several advantages: one LED's output covers a spectral region of about 80 nm, and commercially available LEDs allow for the coverage of the spectral range from 550 to 900 nm, which is a region of interest in near-IR medical applications; the light output of an LED is stable with respect to that of lasers and lamps; the wide angular distribution make LEDs safe for in vivo studies. Furthermore, LED frequency-domain spectroscopy is a relatively inexpensive technique. We describe some circuits we used to modulate the intensity of LEDs at radio frequency, and point out the possibility of building a multisource spectrometer. Some applications of LED frequency-domain spectroscopy, both in vitro and in vivo, ... [truncated at 150 words]
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Frequency-domain methods in optical tomography: detection of localized absorbers and a backscattering reconstruction scheme.
Photon Migration and Imaging in Random Media and Tissues (Proceedings of SPIE, Vol. 1888). By B Chance and RR Alfano (Editors). pp. 440-451, 1993. We studied the ability to detect small absorbing objects embedded in a highly scattering medium. Absorbing spheres of varying size, from 0.8 mm to 6.8 mm radius, submerged in a solution of highly scattering, low absorbing liquid: skim milk, were studied in a trans-illumination geometry. Groups of more than one sphere and a single circular disk, with radius identical to that of one of the spheres, were also studied. Single linear raster scans in the plane of the sphere, with the spheres centered between the source and detector, were made. Data was taken in the frequency-domain, yielding profiles of the objects in each of the three measurable quantities: DC intensity, phase, and modulation. The diffraction pattern from the sphere differed from that of the disk, demonstrating a volume effect associated with photon diffusion. The diffraction pattern of multiple spheres differed from that of single spheres. We have also performed a ... [truncated at 150 words]
Mantulin WW, Fishkin JB, So PTC, Gratton E, Maier JS.
Quantitative diffusive wave spectroscopy in tissues.
Photon Migration and Imaging in Random Media and Tissues (Proceedings of SPIE, Vol. 1888). By B Chance and RR Alfano (Editors). pp. 420-427, 1993. High frequency, intensity-modulated light waves are attenuated and phase-shifted by the absorption and scattering properties of highly scattering media, such as tissue. The simultaneous measurement of the average light intensity, modulation amplitude, and phase- shift at a fixed distance from a sinusoidally modulated light source, permits a quantitative determination of the absolute values of the absorption and scattering coefficients from a frequency-domain scan. Our studies have established the range of modulation frequencies that give the highest sensitivity to changes of the optical parameters in model systems. We have measured the optical absorption spectra of dyes suspended in highly scattering media. These spectra match those found in non-scattering media. This frequency-domain approach provides a simple method to perform quantitative spectroscopy in highly scattering media.
Mantulin WW, French TE, Gratton E.
Optical imaging in the frequency domain.
Medical Lasers and Systems II (Proceedings of SPIE, Vol. 1892). By DM Harris, CM Penney, and A Katzir (Editors). pp. 158-166, 1993. Our laboratory has developed a modular laser tomography system, with pulsed or amplitude modulated (MHz to GHz), near infrared lasers that deliver a probing beam to the tissue of interest through a fiber optic. After the incoming light is scattered and attenuated by the tissue, a detector or imaging fiber optic bundle delivers it to a point detector (photomultiplier tube) which is heterodyned with the modulation frequency to yield the phase delay and demodulation resulting from the light-tissue interaction. The CCD electronics are phase-locked with those of the digitizer to minimize pixel jitter and, in addition, an external clock synchronizes the detector units with the modulated laser source. The digitized time slices are integrated into four bins corresponding to four quadrants of the cross correlation period. The final processing step, a fast Fourier transform, generates the phase shift, demodulation, and average intensity data suitable for image reconstruction.
Reinhart GD, Gratton E, Mantulin WW.
Dissociation of large oligomeric proteins by high hydrostatic pressure: dynamic light scattering studies.
NATO Advanced Study Institute. Aquafredda di Maratea, Italy. September 20-October 3, 1992.
High Pressure Chemistry, Biochemistry and Materials Sciences (NATO Science Series C, Vol. 401). By R Winter and J Jonas (Editors). Kluwer Academic Press, pp. 619-626, 1993. ISBN: 9780792322900In the study of oligomeric protein association, the combined approach of high hydrostatic pressure perturbation with optical spectroscopic detection has provided great insight into structure and dynamics of these complex systems. Various methods offer advantages in specific cases. For example, a decrease in the light scattering intensity tracks the pressure induced dissociation of oligomeric proteins. However, optical artifacts complicate the interpretation of light scattering experiments under pressure. Dynamic light scattering offers the possibility of directly detecting changes in the translational diffusion coefficient, rather than the total scattered intensity associated with oligomer dissociation. The translational diffusion coefficient of the protein is related to its molecular weight and hydrodynamic volume, both of which change with oligomer dissociation. Dynamic light scattering offers greater sensitivity for the study of very large oligomers, that are not readily accessible using other spectroscopic methods. In addition, dynamic light scattering is conveniently used in conjunction with high hydrostatic ... [truncated at 150 words]
Clegg RM, Feddersen BA, Gratton E, Jovin TM.
Time-resolved imaging fluorescence microscopy.
SPIE Conference, Los Angeles, California, 1990.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 448-460, 1992. The extension of microscope luminescence measurements into the temporal domain provides the possibility of determining time-resolved properties of microscope samples and their surrounding environments, and thereby extends the conventional steady state measurements. 'Time resolved imaging microscopy' is a relatively new technique whereby fast kinetic and luminescence decay parameters (decay times and the corresponding time or phase resolved amplitudes) are directly and simultaneously measured throughout an image, pixel by pixel, in an optical microscope. Molecular rotation, solvent and matrix relaxation, quenching mechanisms, reactions, and energy transfer are examples of molecular spectroscopic processes that can be studied best by directly measuring the time dependent properties. Dynamic measurements are generally much more informative than their steady state counterparts. The goal of our work is to develop time-resolved methods that can be applied conveniently and routinely to biological material in the microscope over a wide time domain. In addition to the augmented purely spectroscopic ... [truncated at 150 words]
van der Meer BW, Raymer MA, Wagoner SL, Hackney RL, Beechem JM, Gratton E.
Models for fluorescence energy transfer between moving donors and acceptors.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 220-229, 1992. A number of theoretical models are introduced in which donors and acceptors perform lateral or rotational motion during the time in which fluorescence energy transfer takes place. Each case is reduced to an eigenvalue problem, and for each model calculations are made of observables, such as fluorescence intensities and anisotropies, by employing matrix methods. It is shown that the observables depend on the size of the motional step only if fluorescence energy transfer occurs. This finding indicates that fluorescence energy transfer studies may reveal whether the dynamics of a system (e.g., a protein) is better described in terms of transitions between a number of discrete states or in terms of diffusion equations. Some theoretical tools are offered for analyzing fluorescence energy transfer data without restrictive assumptions for motional averaging regimes or the orientation factor.
French TE, Gratton E, Maier JS.
Frequency-domain imaging of thick tissues using a CCD.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 254-261, 1992. Imaging of thick tissue has been an area of active research during the past several years. Among the methods proposed to deal with the high scattering of biological tissues, the time resolution of a short light probe traversing a tissue seems to be the most promising. Time resolution can be achieved in the time domain using correlated single photon counting techniques or in the frequency domain using phase resolved methods. We have developed a CCD camera system which provides ultra high time resolution on the entire field of view. The phase of the photon diffusion wave traveling in the highly turbid medium can be measured with an accuracy of about one degree at each pixel. The camera has been successfully modulated at frequencies on the order of 100 MHz. At this frequency, one degree of phase shift corresponds to about 30 ps maximum time resolution. Powerful image processing software displays ... [truncated at 150 words]
vandeVen MJ, Barbieri BB, Gratton E, Maier JS.
Analysis of noise sources in frequency-domain fluorometry.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 332-341, 1992. In frequency domain fluorometry, as well as all other spectroscopic techniques, the noise ultimately limits the sensitivity of the instrument and the precision of the measurement. The analysis of the sources of noise in different instruments has revealed that the noise is due to a number of different instrumental factors rather than photon statistics. The ultimate goal is to eliminate those factors to achieve a situation in which the limit is the detector intrinsic noise. We have developed a system, based on digital signal processing, in which the influence of several spurious noise sources has been reduced. A study of the range of cross-correlation frequencies used to obtain the best signal-to-noise ratio is presented.
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Diffraction of intensity-modulated light in strongly scattering media in the presence of a "semi-infinite" absorbing or reflecting plane bounded by a straight edge.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 362-367, 1992. A simple model is developed, based on the diffusion approximation to the linear transport equation, which gives analytic expressions that describe the coherent propagation of sinusoidally intensity-modulated light through a strongly scattering, low absorbing, homogeneous, infinite medium in terms of the interaction coefficients of the medium. Previously, the analytical multiform solutions to Laplace's equation and the Helmholtz equation were derived respectively by Sommerfeld and Carslaw in their studies of electrostatics, sound, and heat to model intensity modulated light in the presence of an absorbing or reflecting `semi-infinite' plane bounded by a straight edge that is immersed in an infinite, strongly scattering, low absorbing, homogeneous medium. The model predictions are in good agreement with the present results of experiments performed on media consisting of IntralipidTM and skim milk emulsions containing minute quantities of black India ink and with Monte Carlo simulations. These studies provide a theoretical basis for the understanding of ... [truncated at 150 words]
Berland KM, Gratton E, vandeVen MJ.
Laser heterodyning detector for frequency-domain ultrafast spectroscopy.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 370-378, 1992. The attainable frequency range of ordinary heterodyning and super-heterodyning frequency- domain fluorescence instrumentation is limited by the response of the optical detector rather than by the harmonic content of the light source. The replacement of the photomultiplier detector by a 6 (mu) microchannel plate detector has improved the frequency response from 500 MHz to 10 GHz. A new method is developed to detect fast, excited state processes by extending to the frequency-domain the well known pump/probe (absorption) technique used in the time-domain. The upper frequency limit attainable with this method is limited only by the pulse width of the light sources. For picosecond pulse lasers this limit extends to hundreds of gigahertz. A theoretical determination of the basic equations is given, and data are shown for the excited state decay of a rhodamine 6G sample in ethylene glycol.
Bucci E, Gryczynski Z, Gratton E, Tenenholz T.
Heme-tryptophan relationships in hemoglobin explored by frequency-domain time-resolved fluorescence at 10-GHz resolution.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 784-791, 1992. The time-resolved fluorescence spectroscopy of human and bovine oxy- and deoxyhemoglobins was measured in either 0.03 M phosphate buffer or 0.03 M borate buffer between pH 6.5 and 9.2. A frequency resolved fluorometer was used with bandwidth up to 10 GHz. Excitation was at 294 nm, the emission was monitored through a broad band interference filter centered at 335 nm, coupled to a cut-off filter at 316 nm. In all cases, the best simulations were obtained with two discrete exponential decays, one near 30 ps, and the other of several hundred ps. In human hemoglobin, the longer component showed a substantial lengthening upon removal of oxygen. In bovine hemoglobin, the shorter component decreased upon deoxygenation. It was possible to infer that the shorter lifetimes originated from the average intrachain distances. However, the hemes at this longer distance would become the main acceptors of energy transfer only when the energy transfer ... [truncated at 150 words]
Gratton E, Mantulin WW, vandeVen MJ, Fishkin JB, Maris MB, Chance BC.
The possibility of a near-infrared optical imaging system using frequency domain method.
3rd International Conference for Peace through Mind/Brain Science. August 5-10, 1990. Hamamatsu City, Japan.
Mind Brain Imaging Program. 1990; : 183-189.Imaging of the interior of the human body is of paramount importance in medicine. Current methods are based on x-rays, nuclear magnetic resonance, ultrasound and other specialized methods. Most of these methods provide images of anatomical details. Optical imaging in the near-infrared has been suggested as an attractive, alternative method. The idea was put forward by the group of B. Chance in Pennsylvania a few years ago. In addition to anatomical features, near infrared imaging carries information about metabolism based on the oxygenation state of hemoglobin.
Barbieri BB, de Piccoli F, vandeVen MJ, Gratton E.
What determines the uncertainty of phase and modulation measurements in frequency-domain fluorometry.
SPIE Conference, Los Angeles, California, 1990.
Time-Resolved Laser Spectroscopy in Biochemistry II (Proceedings of SPIE, Vol. 1204). By JR Lakowicz (Editors). pp. 158-170, 1990. Introduction: The basic theory of a frequency domain fluorometer has been known since the original work of Gaviola. With the introduction of multifrequency capabilities and the use of ultra high speed detectors, the basic understanding of the details of the instrumentation has become essential. The ultimate time resolution and separation capabilities for complex decay species critically depends on the noise characteristics of the instrument's various components. In frequency domain fluorometry, it is customary to measure the standard deviation of the phase and modulation at each individual frequency point.
Gratton E, Feddersen BA, vandeVen MJ.
Parallel acquisition of fluorescence decay using array detectors.
SPIE Conference, Los Angeles, California, 1990.
Time-Resolved Laser Spectroscopy in Biochemistry II (Proceedings of SPIE, Vol. 1204). By JR Lakowicz (Editors). pp. 21-25, 1990. Frequency domain fluorometry provides an alternative method for the recording of fluorescence decay kinetics (1), The major advantages of this method are the time resolution and the fast measurement of lifetime values. There are several systems, where a change in lifetime occurs across the emission band. These systems include heterogeneous ground state systems, where species emit at different wavelengths; excited state reactions, with products emitting at different parts of the spectrum; and dipolar relaxation processes, where the emission spectra change with time. To characterize the decay of the systems, it is necessary to collect the emission decay at several wavelengths. Generally, this process is obtained by successive measurements using a monochromator or a series of bandpass filters. For steady-state spectra, optical multichannel analyzers (OMA) are available, which can collect the entire emission spectra at once. By gating the image intensifier, used with some of these analyzers, it is possible to ... [truncated at 150 words]
Gratton E, Alcala JR, Prendergast FG.
Protein dynamics: fluorescence lifetime distributions.
International Symposium in Honor of Gregorio Weber's Seventieth Birthday. September 9-12, 1986. Bocca di Magra, Italy.
Fluorescent Biomolecules: Methodologies and Applications. By DM Jameson and GD Reinhart (Editors). Plenum Press, pp. 17-32, 1989. ISBN: 9780306431135It is now well established that proteins in their native conformation can exist in a large number of subconformations slightly different one from the other (Lakowicz & Weber, 1973; Austin et al., 1975; Careri et al., 1975, 1979; Karplus & McCammon, 1983). Subconformations originate from small structural fluctuations around the main conformation. The protein structure is very flexible and it allows rotations around the phi and psi angles of the polypeptide chain and around the C-alpha carbon on the side chain. The stabilization of one particular native structure depends on a large number of interactions which are affected by solvent, ions of the medium and chemico-physical parameters. Some parts of the protein structure are more stable than others due to a more favorable interaction between the amino acid residues and they form structural domains. Frequently,
these domains are associated with secondary structural elements such as alpha helical segments or beta sheets....
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Lipid transfer reactions: fluorescence studies.
International Symposium in Honor of Gregorio Weber's Seventieth Birthday. September 9-12, 1986. Bocca di Magra, Italy.
Fluorescent Biomolecules: Methodologies and Applications. By DM Jameson and GD Reinhart (Editors). Plenum Press, pp. 115-132, 1989. ISBN: 9781468456219In cells the transport of lipid molecules from sources of biosynthesis or ingestion to the various membranes and organelles represents an important component of metabolism (Pagano and Sleight, 1985). The analogous problems of protein synthesis, membrane assembly, turnover, and secretion are also being actively studied by cell biologists. The important sites of lipid trafficking include the plasma compartment of blood and the cells of the vascular and extravascular compartment. The lipid composition of these sites includes cholesterol, cholesteryl esters, triglycerides, fatty acids, and phospholipids. Figure 1 schematically illustrates several pathways of lipid transfer in a membrane; these include lateral diffusion of the lipid in the membrane plane, spontaneous desorption into the surrounding aqueous compartment with subsequent uptake by an adjacent membrane surface, and bilayer translocation, which is commonly referred to as flip-flop. This latter mechanism is often accelerated by enzymes termed “translocases”. Other lipid transfer mechanisms not pictured include transient ... [truncated at 150 words]
Gratton E, Alcala JR, Marriott GM.
Time resolved fluorescence anisotropy decay studies in proteins.
First EBSA Workshop, a Marcus Wallenberg Symposium, Stockholm, Sweden, July 6-10, 1986.
Structure, Dynamics, and Function of Biomolecules (Springer series in biophysics). By A Ehrenberg (Editors). Springer-Verlag, 1987. ISBN: 9783540172796Gratton E, Alcala JR, Marriott GM, Prendergast FG.
Fluorescence lifetime distributions of single tryptophan proteins: a protein dynamics approach.
First EBSA Workshop, a Marcus Wallenberg Symposium, Stockholm, Sweden, July 6-10, 1986.
Structure, Dynamics, and Function of Biomolecules (Springer series in biophysics). By A Ehrenberg (Editors). Springer-Verlag, 1987. ISBN: 9783540172796Glaser M, Gratton E, Parasassi T, Sapora O, Conti F.
Spectroscopic studies of biological systems "in vivo".
2nd Conference on "The State of Art of Research in the Chemistry Department", Rome, Italy, June 1-2, 1987.
In the last years a large interest has been devoted to the investigation of the structural and dynamical modifications of biological membranes during physiological processes such as the cell duplication cycle and the differentiation. Preliminary our studies considered membrane model systems constituted of phospholipid vesicles of various composition...
Gratton E, Alcala JR, Marriott GM.
Rotations of tryptophan residues in proteins.
617th Meeting, Dundee.
Biochem Soc Trans. 1986; 14(5): 835-838.A classic study of the application of dynamic fluorescence depolarization methods to the investigation of protein internal rotations was reported some years ago by Munro et al. (1979). The relative large width of the excitation pulse used in those studies limited the time resolution to about 0.1 nanoseconds. Sommer et al. (1985) later measured the anisotropy decay of the single tyrosine residue in lima bean trypsin inhibitor using a femtosecond laser and streak camera. More recently, Harris et al (1986) have utilized time-correlated single photon counting with the narrow pulses obtained from a mode-locked laser to determine the rotational correlation times of the single tryptophan residue in mutants of T4 lysozyme. On the other hand, the use of differential phase and modulation ratio fluorometry to measure rotational properties of intrinsic fluorophores in proteins has been well established in our laboratory (Alcala et al., 1985, Gratton et al., 1985, Jameson et ... [truncated at 150 words]
Gratton E, Alcala JR, Marriott GM, Prendergast FG.
Fluorescence studies of protein dynamics.
Computer analysis for life science, Okayama, Japan, July 9-12, 1985.
Computer analysis for life science: Progress and challenges in biological and synthetic polymer research: proceedings of the International Symposium on. By C Kawabata and AR Bishop (Editors). Ohmsha, pp. 1-11, 1986. ISBN: 9784274072611Gratton E, Alcala JR, Marriott GM.
Rotational motions of tryptophan and tyrosine residues in proteins.
International Symposium on Structure and Dynamics of NucIeic Acids, Proteins, and Membranes. Aug 31-Sep 5, 1986, Riva del Garda, Italy.
Structure and Dynamics of Nucleic Acids, Proteins, and Membranes. By E Clementi and S Chin (Editors). Plenum Press, New York, pp. 149-151, 1986. ISBN: 030642553XProteins are flexible structures; their flexibility is crucial for biological function....
