LFD Publications

Nanometer-scale imaging of collagen fibers using gold beads

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Bo Chen and Enrico Gratton.
Nanometer-scale imaging of collagen fibers using gold beads.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 926-Pos.

The 3D spatial position of a particle can be determined by scanning the excitation volume of a 2-photon microscope in a three-dimensional orbit around the particle and by subsequently analyzing the fluorescence intensity profile along the orbit. We track the movement of gold beads moving along collagen fibers by 3D particle tracking method. As the particle moves on the fiber, the particle trajectory maps the substrate with high-resolution (2-20 nm). When the particle moves in close proximity to the collagen, it locally couples and excites to the weak fluorophores on the collagen. This method provides the possibility to characterize the interactions between particle and substrate even further. We can obtain the dynamic structure information of collagen fibers with nanometer resolution in real time. More interestingly, the gold beads move not at random but in specific directions under two photon laser excitation. We were able to move the gold particle very fast along the collagen fibers parallel to the scanning line direction. Based on these results, we can control the velocity and direction of gold beads at our own will. Work supported in part by NIH-P41 P41-RRO3155 and P50-GM076516.

In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Francesco Caldarelli and Enrico Gratton.
In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3904-Pos.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. We recently introduced a method based on pair correlation functions (pCF) which measure the time the same molecule takes to migrate from one location to another within the cell 1. The spatial and temporal correlation among two arbitrary points in the cell can provide a map of molecular transport, and also highlight the presence of barriers to diffusion with very high time resolution (in the microsecond scale) and spatial resolution (limited by diffraction). Here we report the use of this method to directly monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which SPT has given results that cannot be confirmed by traditional FCS measurements because of the lack of spatial resolution. Our substrate is composed by a GFP linked to a functional nuclear localization sequence (NLS) and transfected into living CHO-K1 cells: the recombinant NLS-GFP protein can bind to molecular carriers mediating cytoplasm-to-nucleus active import as well as shuttle across the NPC by passive diffusion (its molecular weight is below the cut-off size limit of the NPC). We show that obstacles to molecular flow can be detected ... [truncated at 200 words]

N&B and cross-N&B analysis detect oligomerization of Huntingtin in live cells

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Giulia Ossato, Michelle A Digman, Tamas Lukacsovich, J Lawrence Marsh, and Enrico Gratton.
N&B and cross-N&B analysis detect oligomerization of Huntingtin in live cells.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3388-Pos.

Aggregation of misfolded proteins is a hallmark of several neurodegenerative diseases such as Huntington's disease (HD). HD is caused by a mutation of Huntingtin caused by an elongation of a polyglutamine (polyQ) sequence in the protein. Here we describe the application of the recently developed Number and molecular Brightness method (N&B) to monitor the aggregation process of Huntingtin exon1 (Httex1). N&B measures the molecular brightness of the protein aggregates in the entire cell non-invasively based on the fluctuation dynamics at each pixel of an image. This analysis provides a map of aggregation with pixel resolution. We observed the behavior of Httex1-97QP-EGFP this is a construct with 97 polyQ repeats corresponding to Juvenile onset of the disease. We preformed experiments in ST14A cells transfected with Httex1-97QP-EGFP. We establish that the process of nucleation leading to inclusion formation has four phases: i) Initially only monomers are present; ii) Following an increase in protein concentration (~1 mM), due to protein accumulation, small oligomers (8-15 proteins) form throughout the cell; iii) At higher protein concentrations, an inclusion is formed in the cytoplasm; iv) The inclusion recruits most of the Httex1 protein in the cell, including those in the nucleus, leaving only monomers at very low concentration. We ... [truncated at 200 words]

Dynamic imaging and fluctuation spectroscopy on single microvilli in opossum kidney cells by the modulation tracking method

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Luca Lanzanò, Peter T Fwu, Hector Giral, Moshe Levi, and Enrico Gratton.
Dynamic imaging and fluctuation spectroscopy on single microvilli in opossum kidney cells by the modulation tracking method.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3906-Pos.

Regulation of renal tubular inorganic phosphate (Pi) transport occurs via the proximal tubular apical brush border membrane (BBM) sodium gradient-dependent Pi (NaPi) cotransport proteins. Distinct families of NaPi cotransporters show differential regulation under dietary and hormonal stimuli, but the way this is accomplished, for instance through localization in distinct BBM micro- or nano-domains and/or preferential interaction with different PDZ proteins, is not yet understood. Crucial information could come from the application of single molecule fluctuation correlation spectroscopies on the BBM of living cultured Opossum Kidney (OK) cells expressing NaPi co-transporters with different GFP constructs. The BBM is composed of many microvilli, several micron long structures with a diameter of about 100nm. The microvilli show a relatively fast motion (in the seconds time scale) that makes the use of fluctuation spectroscopy difficult. None of the current nano-resolution optical methods seems capable of measuring the clustering dynamics of proteins on the surface of rapidly moving microvilli. We developed an optical imaging technique called Modulation Tracking (MT) in which we track the center of mass of the microvillus at an arbitrary point along its length while the laser spot rapidly oscillates perpendicularly to the surface and the changes in the modulation are used to measure ... [truncated at 200 words]

Modulation particle tracking

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Peter T Fwu, Luca Lanzanò, and Enrico Gratton.
Modulation particle tracking.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 929-Pos.

In this study, we present a novel optical imaging method that makes use of high precision particle tracking of fluorescent particles to obtain images of nanometer size structures in live cells. Particle tracking not only provides the trajectory of the center of mass but also the particle orientation and size can now be observed, in vivo and real time with the nanometer resolution. This method helps in further understanding of the dynamics of the small particles in biological systems, which was hard to achieve by the current optical techniques. The method is based in rapidly modulating the position of the laser beam around small structures on the order of 100nm in size. When the laser spot oscillates in the direction toward the particle surface, the fluorescence of the particle is modulated. The modulation, which is the ratio of the alternating part to the average fluorescence intensity, is a function of the distance of the particle from the center of mass to the oscillation. In order to track the particle, we circularly moved the oscillating laser spot around the moving particle, and at the same time, analyzed the modulation in the frequency spectrum of the intensity along the orbit to perform ... [truncated at 200 words]

Scanning laser image correlation (SLIC) measurements in Zebra Fish larvae

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Molly J Rossow, William W Mantulin, and Enrico Gratton.
Scanning laser image correlation (SLIC) measurements in Zebra Fish larvae.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3004-Pos.

Scanning Laser Image Correlation (SLIC) is a technique to measure the flow of small particles and to observe local flow patterns over an area. SLIC can be applied to situations including blood cells flowing through blood vessels or tracer particles flowing through microfluidic channels. The main advantage of SLIC over other flow measurement techniques is that SLIC can be scaled to measure flow in areas ranging from microns to centimeters wide. To accomplish this, an image is first acquired through laser scanning and analyzed with number and brightness analysis (N&B) to identify regions of flow. This is used as a guide to manually (or potentially automatically) select a pattern within the image, such as a line along the center of a channel, that is then scanned repeatedly with the laser beam. Since the entire image is not scanned in each measurement SLIC measurements can be obtained quickly and efficiently. The results of these scanned patterns are analyzed with the recently developed pair correlation technique to extract the rate of flow and to identify characteristic flow patterns such as turbulence, particles that adhere to the channel walls, and variable velocity along the length of the channel. In the work presented here we ... [truncated at 200 words]

Fluctuation analysis with the spinning disk confocal microscope

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Francesco Cutrale and Enrico Gratton.
Fluctuation analysis with the spinning disk confocal microscope.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 942-Pos.

Analysis of the fluctuations in time and space of confocal images has the potential to provide information about molecular diffusion and molecular interactions directly in live cells. Fluctuation image analysis has been commonly done in the laser scanning microscope. In the slow regime, when the fluctuations are slower than the frame rate, the time correlation between the same pixel in different frames of an image stack provides all the information about diffusion and brightness. In the fast regime, for example for molecules diffusing in the cytoplasm, the frame rate is too slow to follow the fluctuations due to diffusion. In the raster scan confocal microscope, these fluctuations are detectable because of the correlation of the intensity with the next pixel in the same line or in the next line. In fluctuation spectroscopy an important parameters is the sampling time that must be shorter that the time of the decay of the fluctuation. In the spinning disk confocal microscope, the sampling time at each pixel is very short. However, in the normal data acquisition protocol of the spinning disk microscope the intensity at one pixel is averaged with the intensity at the same pixel after the disk has performed several rotations. ... [truncated at 200 words]

Multiple components mapping of live tissue by phasor analysis of fluorescence lifetime imaging

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Chiara Stringari, Michelle A Digman, Peter Donovan, and Enrico Gratton.
Multiple components mapping of live tissue by phasor analysis of fluorescence lifetime imaging.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 1116-Plat.

In fluorescence lifetime microscopy (FLIM) of live tissues a major issue is the assignment of autofluorescence to specific molecular components and their interactions within the physiological context. Here we use the phasor approach to fluorescence lifetime imaging to analyze complex decays in a live tissue. The tissues used were seminiferous tubules from the testes of wild type mice or mice expressing GFP from an Oct4 transgene. Lifetime images were acquired in the time domain and analytically transformed in the phasor representation. By examination of the clustering of the phasors we identified different molecular components: auto fluorescence, GFP, collagen and retinol. Each chemical species was identified and categorized by its specific location in the phasor plot. This phasor fingerprint reduces the importance of knowing the exact lifetime distribution of the fluorophores and emphasizes the contribution of the species to the signal. To better identify specific tissue components we also used spectral imaging and second harmonic generation microscopy. Linear combinations in the same pixel of molecular species were recognized and their relative fraction was calculated and mapped. The analysis of the fluorescence decay with higher harmonics of the phasor plot separates different molecular components that have the same location in the phasor plot ... [truncated at 200 words]

Self-referencing differential spectroscopy analysis in breast translational research

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Shanshan Xu, Albert Cerussi, and Enrico Gratton.
Self-referencing differential spectroscopy analysis in breast translational research.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3896-Pos.

A self-referencing differential spectroscopy analysis approach has been developed for broadband near-infrared (NIR 650-1000 nm) absorption spectra to reveal intrinsic optical breast cancer biomarkers. Through the application of this method that accounts for inter-patient variability using the normal tissue as an internal control, we have characterized the metabolic differences between malignant and normal tissues that result from subtle alterations in molecular disposition. From a pilot study of 15 cancer patients performed in 2007, absorption signatures, not arising from the individual abundance in the four major chromophores (lipid, oxy-hemoglobin, deoxy-hemoglobin and water), have been demonstrated to successfully differentiate the normal and malignant tissues. Based on the data acquired from a NIR Diffuse Optical Spectroscopy Imaging instrument, specific spectral signatures containing specific NIR absorption bands are located in regions at about 760, 930, and 980 nm indicative of lipid biomarkers or water in abnormal state. The shape of the fingerprint spectra, namely specific tumor component (STC) spectra, is highly reproducible and exhibits consistent and particular wavelength-dependent characteristics. STC index algorithm was set up to quantitatively computing the residual due to components that are unaccounted for by the basis spectra. A 61 subject retrospective study aiming to distinguish between benign and malignant breast tumors was ... [truncated at 200 words]

Alpha hemolysin induces an increase of erythrocytes calcium: a fluorescence lifetime imaging microscopy study

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Susana A Sánchez, Laura Bakás, Enrico Gratton, and Vanesa Herlax.
Alpha hemolysin induces an increase of erythrocytes calcium: a fluorescence lifetime imaging microscopy study.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 2992-Pos.

α--hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a series of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin causes the lysis of several cells as erythrocytes, granulocytes, monocytes, endothelial cells and renal epithelial of different species and low concentrations induces the production of cytokines and apoptosis. Eriptosis, the apoptosis process in erythrocytes, can be induced by several toxins and the increase in calcium concentration inside the cell is being postulated as the trigger of this process. In this context, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA; calcium concentration was monitored following the changes in lifetime of the calcium indicator Green 1 using fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report we present evidences that, at sublytic concentrations, HlyA induces an increase in Calcium concentration in rabbit erythrocytes in the first 5 minutes. Results are discussed in relation to the difficulties of measuring Calcium concentrations in erythrocytes where hemoglobin is present, the ... [truncated at 200 words]

Caveolin-1 boosts clustering of mu (µ) Opioid receptors in the plasma membrane

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Parijat Sengupta, Susana A Sánchez, and Suzanne F Scarlata.
Caveolin-1 boosts clustering of mu (µ) Opioid receptors in the plasma membrane.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 2564-Pos.

Caveolin-1 (Cav1), is a structural protein component of many mammalian cell plasma membrane and is known to be involved in lipid and protein sorting, receptor desensitization, receptor trafficking, cell migration and many other cellular events. Here we determine if stable expression of Cav1 in cells alters the receptor organization prototype on the membrane. We use two different cell lines for this study: Fisher Rat Thyroid (FRTwt) cells that do not express detectable level of Cav1 and a sister line that is stably transfected with canine Cav1 protein (FRTcav). We express µ opioid receptors (MOR) tagged with either YFP (MOR-YFP) or CFP (MOR-CFP) in cells for different experiments. Förster resonance energy transfer (FRET) measurement between MOR-CFP and Gai-YFP in FRTwt and FRTcav cells shows receptor sequestration in the presence of Cav1. We find that diffusion of MOR-YFP in plasma membrane of FRTcav cells is slower compared to FRTwt cells by scanning fluorescence correlation spectroscopy (scanning-FCS) experiments. Photon counting histogram (PCH) analyses provide higher average brightness for MOR-YFP in FRTcav cells. Taken together these data provide evidence for caveolin-assisted enhanced clustering of G-protein coupled receptors on the plasma membrane.

Biometallic nano-structures: a thousand-fold fluorescence enhancement with nanopetals

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Michelle A Digman, Chi-Cheng Fu, Giulia Ossato, Luke P Lee, Enrico Gratton, and Michelle Khine.
Biometallic nano-structures: a thousand-fold fluorescence enhancement with nanopetals.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3883-Pos.

Although, metal nanoparticles have been shown to increase emission fluorescence of molecules due to the propagation of electron density waves, i.e. surface plasmons, at the interface between the metal and substrate, biometallic nanostructures yield a higher emission enhancement and can be suitable for measuring cell surfaces at the nano scale. We have developed a method to attain bimetallic structures on the surface of memory polymers in order to achieve sharp bi-layered uniaxial and biaxial nanopetals. The sharp edges of the nanopetals exhibit remarkable increase of emission intensity of fluorescent molecules. We observe several thousand fold increase in intensity at the edges or “hotspots” of both uniaxial and biaxial nanopetals. The fluorescence intensities observed at the hotspots are brief bursts of intensity as the molecules diffuse through the structures. These bursts are below the resolution limit of our optics and possibly be due to single molecular emission. The intensity of the bursts increases non-linearly with increase laser intensity suggesting that the events may be attributable to stimulated emission, excited-state absorption, or saturation intensity dependent 2-photon emission cross-section. We also show a decrease in the excited-state lifetime of the fluorescence particles, fluorescein, revealing strong plasmonic interactions. Our findings reveal an ultra-sensitive and ... [truncated at 200 words]

Spatial correlation of speckle fluctuations reveals thickness and features of the ocular surface tear film

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Kaveh Azartash and Enrico Gratton.
Spatial correlation of speckle fluctuations reveals thickness and features of the ocular surface tear film.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 2100-Pos.

Here we present Fluctuation Analysis of Spatial Image Correlation (FASIC), a non-invasive method for evaluating the complex dynamics of the tear film surface by spatial correlation analysis. Tear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. Dry Eye Syndrome (DES) refers to abnormalities of tear film secretion and/or stability diagnosed by conventional methods such as the Schirmer test and tear break-up time (TBUT). Several different physical methods have been developed to measure non-invasively the structure and function of the tear film including high-speed videokeratography and dynamic wavefront aberrometry. Interferometry and optical coherence tomography are amongst new proposed methods to measure tear film thickness that have remained in research phase. With FASIC, a series of images are obtained using a laser illumination and a cMOS camera. The spatial correlation is calculated for every frame. A sinusoidal background due to interference of the tear film appears in this spatial correlation together with other features. We have developed a mathematical model to obtain the thickness of the tear film from this sinusoidal background. The model includes the macroscopic dynamics of small lipid droplets in the tear film. Consistent data with ... [truncated at 200 words]

Receptor-ligand interactions in the plasma membrane of live cells resolved in space and time by N&B analysis

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Christian Hellriegel, Valeria R Caiolfa, Nicolai Sidenius, Enrico Gratton, and Moreno Zamai.
Receptor-ligand interactions in the plasma membrane of live cells resolved in space and time by N&B analysis.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3899-Pos.

In this presentation we show how we push the Number and Brightness analysis (N&B) to the limits of applicability. We demonstrate that by N&B we can observe how a GFP labeled membrane receptor (namely uPAR) dimerizes upon ligand binding in live cells. We show how we obtain real time, spatially and temporally resolved images of the molecular reorganization of uPAR in the cell membrane. These results are backed by extensive simulations, and by well-defined live cell calibration experiments (using monomeric and dimeric GFP-uPAR constructs ). N&B quantifies the amplitudes of fluorescence intensity fluctuations as individual fluorescent species diffuse in and out of a pixel in a series of images. The basic idea is that the amplitude fluctuations of a diffusing molecule labeled with two dyes (e.g. a dimer, or a bound ligand-receptor pair) will be twice as large as the amplitudes of a molecule with only one dye (i.e. a monomer, or the unbound ligands and receptors), simply because the doubly labeled object is twice as bright as the individual one. N&B is related to fluctuation spectroscopy such as fluorescence correlation spectroscopy FCS and photon counting histogram, PCH. These methods can resolve molecule-molecule interactions, but are usually restricted to the acquisition at ... [truncated at 200 words]

Single point FCS on a commercial confocal laser scanning microscope with analog detectors

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Iyrri L Salvemini, Enrico Gratton, and Pierre D J Moens.
Single point FCS on a commercial confocal laser scanning microscope with analog detectors.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 935-Pos.

Fluorescence Correlation Spectroscopy is a technique invented in the early 1970s to measure diffusion coefficient, chemical reaction rates and photo physical processes. It is a common belief that in order to obtain single point FCS data, one needs either a sophisticated FCS instrument with photon counting detectors or avalanche photon detectors or an instrument custom made for this type of experiments. Here we show that we can obtain single point FCS data on a commercial confocal laser scanning microscope without any modifications (Nikon C1). We successfully measured the diffusion coefficient and the concentration of Rhodamine B in solution for concentrations ranging from 5 nM to 280 nM. We also determined the diffusion coefficient of two different labeled lipid analogs (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and BODIPY TMR phosphatidylinositol (4,5) bisphosphate) incorporated in the membrane of giant unilamellar vesicles. The results obtained for these lipid analogs are in good agreement with previously published data. Finally, we highlighted the fact that the actual proportion of labeled lipid analogs incorporated in the membrane of the giant unilamellar vesicle (formed by the electroformation method) is significantly different than the proportion of these lipids in the organic solvent stock solution.

The pair-correlation approach to FCS

Sat, 20 Feb 2010 00:00:00 -0800

Abstract

Enrico Gratton and Michelle A Digman.
The pair-correlation approach to FCS.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; [epub ahead of print], 3959-Plat.

Molecular diffusion and transport processes are fundamental in physical, chemical, biochemical and biological systems. Current approaches to measure molecular transport in cells and tissues based on perturbation methods like fluorescence recovery after photobleaching are invasive, fluctuation correlation methods are local and single particle tracking requires the observation of isolated particles for relatively long periods of time. We propose to detect molecular transport by measuring the time cross-correlation of fluctuations at a pair of locations in the sample. When the points are further than two times the size of the point spread function, the maximum of the correlation is proportional to the average time a molecule takes to move from a specific location to another. We demonstrate the method with simulations, using beads in solution and by measuring the diffusion of molecules in cellular membranes. The spatial pair cross-correlation method detects barriers to diffusion and heterogeneity of diffusion because the time of the correlation maximum is delayed in the presence of diffusion barriers. This non-invasive sensitive technique follows the same molecule over a large area producing a map of molecular flow and does not require isolated molecules thereby many molecules can be labeled at the same time and within the point ... [truncated at 200 words]

In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions

Tue, 08 Dec 2009 00:00:00 -0800

Abstract

Francesco Caldarelli and Enrico Gratton.
In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. We recently introduced a method based on pair correlation functions (pCF) which measure the time the same molecule takes to migrate from one location to another within the cell (1). The spatial and temporal correlation among two arbitrary points in the cell can provide a map of molecular transport, and also highlight the presence of barriers to diffusion with very high time resolution (in the microsecond scale) and spatial resolution (limited by diffraction). Here we report the use of this method to directly monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which SPT has given results that cannot be confirmed by traditional FCS measurements because of the lack of spatial resolution. Our substrate is composed by a GFP linked to a functional nuclear localization sequence (NLS) and transfected into living CHO-K1 cells: the recombinant NLS-GFP protein can bind to molecular carriers mediating cytoplasm-to-nucleus active import as well as shuttle across the NPC by passive diffusion (its molecular weight is below the cut-off size limit of the NPC). We show that obstacles to molecular flow can be detected ... [truncated at 200 words]

Modulation tracking of cellular adhesions in 3D collagen matrices

Tue, 08 Dec 2009 00:00:00 -0800

Abstract

Michelle A Digman, Luca Lanzanò, and Enrico Gratton.
Modulation tracking of cellular adhesions in 3D collagen matrices.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.

Cell mobility, proliferation, and cell adhesions are fundamental for cancer metastasis, wound healing, stem cell differentiation, and development. The dynamics of protein interactions and cell migrations have been extensively studied in the 2D environment. We have developed methods applicable to 2D raster scan images (RICS and scanning FCS) that have given information about the formation of complexes responsible for cell adhesion and migration. It is now established that cell movement in 3D matrices and in tissues have different dynamics. In 3D the search of the adhesions site with the extracellular matrix is a daunting problem. Cells make only few transient contacts and the entire cell moves in 3D going out of focus in conventional confocal or 2-photon excitation microscopy. There is no simple way to use the raster scan fluctuations techniques in the 3D environment that were successful in determining protein complexes and interaction in 2D. We have developed an imaging method that produces high resolution 3D images with time resolution comparable to the raster scan methods used in 2D. The method is based on a tracking-imaging principle. Using this approach we are able to follow the aggregation of proteins in the point of contacts between cell filopodia and the ... [truncated at 200 words]

Fluorescence fluctuation analyses reveal that FAK-paxillin and α5 integrin-talin form complexes within adhesions

Sun, 06 Dec 2009 00:00:00 -0800

Abstract

Colin K Choi, Alexia Bachir, Jessica Zareno, Michelle A Digman, Enrico Gratton, and Alan R Horwitz.
Fluorescence fluctuation analyses reveal that FAK-paxillin and α5 integrin-talin form complexes within adhesions.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.

Transient, localized protein interactions are central to adhesion formation, turnover, and signaling during cell migration. In highly motile cells, initial contact with the extracellular matrix is established via nascent adhesions in the lamellipodium. These adhesions function as signaling hubs that regulate protrusion and adhesion and also serve as traction points for migration. Paxillin and its putative binding partner FAK have emerged as critical regulators of migration. Using dual-color total internal reflection fluorescence (TIRF) and confocal imaging modalities, we measured the simultaneous fluctuations of paxillin and FAK fluorescence intensity and used cross-variance and cross-correlation techniques to detect their interactions and kinetics in living cells at high spatiotemporal resolution. The TIRF-based cross-variance analysis shows that paxillin and FAK reside and exchange as multimeric complexes in nascent adhesions. A phosphomimetic mutant of paxillin (Y31E, Y118E), which increases protrusion and adhesion assembly and turnover, significantly increases the complex size. In contrast, a non-phosphorylatable paxillin mutant (Y31F, 118F) that suppresses adhesion turnover reduces the paxillin aggregation nearly 2-fold. In addition, confocal-based cross-correlation analysis (ccRICS) showed only minimal levels of wild-type paxillin and FAK interactions in the cytosol; however, the phosphomimetic mutant forms robust, freely diffusing complexes. We also investigated the associations between talin and α5 ... [truncated at 200 words]

Molecular basis of the potent membrane remodeling activity of the epsin1 ENTH domain

Sun, 01 Nov 2009 00:00:00 -0800

Journal Article

Youngdae Yoon, Jiansong Tong, Park Joo Lee, Alexandra Albanese, Nitin Bhardwaj, Morten Källberg, Michelle A Digman, Hui Lu, Enrico Gratton, Yeon-Kyun Shin, and and Wonhwa Cho.
Molecular basis of the potent membrane remodeling activity of the epsin1 ENTH domain.
J Biol Chem. 2009; [epub ahead of print].

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin1 ENTH domain has potent vesicle tubulation activity despite lack of intrinsic molecular curvature. EPR revealed that the N-terminal alpha-helix penetrates the PtdIns(4,5)P(2)-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.

N&B analysis of Huntingtin aggregation in live cells

Mon, 12 Oct 2009 00:00:00 -0800

Abstract

Giulia Ossato, Michelle A Digman, Charity Aiken, J Lawrence Marsh, and Enrico Gratton.
N&B analysis of Huntingtin aggregation in live cells.
NIH NCRR/NIBIB P41 Principal Investigator Meeting. Bethesda, Maryland. October 2009.

Aggregation of misfolded proteins is a hallmark of several neurodegenerative diseases such as Huntington's disease. Here we describe the utility of the recently developed Number and molecular Brightness method (N&B) to monitor the aggregation process of Hungtintin exon 1 (Httex1). N&B measures the molecular brightness of the protein aggregates in the entire cell non-invasively based on the fluctuation dynamics at each pixel of an image. Dynamic imaging by the N&B method allowed detection of monomers, oligomers and inclusions in different regions of the cell simultaneously at different time points. We observed the behavior of Httex1-EGFP, with polyglutamine (polyQ) repeats of different lengths (Httex1-97QP-EGFP, Httex1-46QP-EGFP, Httex1-25QP-EGFP in transfected COS-7 cells. We found that Httex1 is present throughout the cell and that the non-pathogenic protein (Httex1-25QP-EGFP) does not aggregate while the pathogenic proteins (Httex1-46Q-EGFP and Httex1-97QP-EGFP) form inclusions. We establish that the process of nucleation leading to inclusion formation has four phases: i) Initially only monomers are present; ii) Following an increase in protein concentration, due to protein accumulation, small oligomers (8-15 proteins) form throughout the cell; iii) At higher protein concentrations, an inclusion is formed in the cytoplasm; iv) The inclusion recruits most of the Httex1 protein in the cell, including ... [truncated at 200 words]

Coherent movement of cell layers during wound healing by image correlation spectroscopy

Wed, 07 Oct 2009 00:00:00 -0800

Journal Article

Kandice Tanner, Donald R Ferris, Luca Lanzanò, Berhan Mandefro, William W Mantulin, David M Gardiner, Elizabeth L Rugg, and Enrico Gratton.
Coherent movement of cell layers during wound healing by image correlation spectroscopy.
Biophys J. 2009; 97(7): 2098-2106. PMCID: PMC2756390

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume (~37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury.

Real-time nanomicroscopy via three-dimensional single-particle pracking

Mon, 05 Oct 2009 00:00:00 -0800

Journal Article

Yoshihiko Katayama, Ondrej Burkacky, Martin Meyer, Christoph Bräuchle, Enrico Gratton, and Don C Lamb.
Real-time nanomicroscopy via three-dimensional single-particle pracking.
ChemPhysChem. 2009; 10(14): 2458-2464.

We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution.

Imaging nanometer structures using modulation tracking (opening lecture)

Sun, 20 Sep 2009 00:00:00 -0800

Abstract

Enrico Gratton, Luca Lanzanò, and Peter T Fwu.
Imaging nanometer structures using modulation tracking (opening lecture).
International Symposium - Analysis of Biomolecular Machines in the Nanometer Range. September 20-23, 2009. Jena, Germany.

We describe a new modality of imaging nanostructures using modulation tracking based on optical feedback. The method uses a variant of circular tracking that we have developed some years ago for tracking particles and single molecules in cells. Since the method does not use the diffraction properties of light, we are not breaking the diffraction limit. We will show nanometer images of microvilli in living cells. The microvilli have a diameter of approximately 100 nm and length of several microns. The method allows the observation of patches of fluorescently labeled protein on the microvilli surface and the diffusion of fluorescent proteins along the microvilli membranes.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy, applications in systems biology

Tue, 01 Sep 2009 00:00:00 -0800

Journal Article

Michelle A Digman and Enrico Gratton.
Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy, applications in systems biology.
WIREs Syst Biol Med. 2009; 1(2): 273-282.

This article focuses on methods based on fluctuation correlation spectroscopy to determine the formation of protein complexes in living cells. We present the principles of the fluctuation method applied to cells. We discuss the novelty and the promises of this approach. The emphasis is in the discussion of the underlying statistical assumptions of the image correlation spectroscopy analysis rather than in reviewing applications of the method. Although one example of the application of the fluctuation method is given, this article also contains simulations that are better suited to illustrate and support the basic assumptions of the method.

Photophysical properties of blue-emitting silicon nanoparticles

Thu, 06 Aug 2009 00:00:00 -0800

Journal Article

Manuel J Llansola Portolés, Felipe Rodriguez Nieto, Delia B Soria, Javier I Amalvy, Pablo J Peruzzo, Daniel O Mártire, Mónica Kotler, Oliver Holub, and Mónica C Gonzalez.
Photophysical properties of blue-emitting silicon nanoparticles.
J Phys Chem C. 2009; 113(31): 13694–13702.

Silicon nanoparticles with strong blue photoluminescence were synthesized by electrochemical etching of silicon wafers and ultrasonically removed under N2 atmosphere in organic solvents to produce colloids. Thermal treatment leads to the formation of colloidal Si particles of 3 ± 1 nm diameter, which upon excitation with 340−380 nm light exhibited room temperature luminescence in the range from 400 to 500 nm. The emission and the one- and two-photon excitation spectra of the particles are not sensitive to surface functionalization with methyl 2-methylprop-2-enoate. However, the derivatized particles show higher emission quantum yields in air-saturated suspensions (44%) than the underivatized particles (27%), as well as higher stability of its dispersions. FTIR and XPS spectra indicate a significant surface oxidation of the particles. The Si:O:C ratio at the surface of the derivatized particles estimated from XPS is Si3O6(C5O2Hy)1, with y = 7−8. Vibronic spacing is observed in both the emission and excitation spectra. The information obtained from one-photon excitation experiments (emission and excitation spectra, photoluminescence quantum yields, luminescence decay lifetimes, and anisotropy correlation lifetimes), as well as from two-photon excitation fluorescence correlation spectroscopy (brightness and diffusion coefficients) and TEM, indicate that the blue-emitting particles are monodisperse and ball-shaped. Particle size clearly determines the ... [truncated at 200 words]

Imaging barriers to diffusion by pair correlation functions

Wed, 22 Jul 2009 00:00:00 -0800

Journal Article

Michelle A Digman and Enrico Gratton.
Imaging barriers to diffusion by pair correlation functions.
Biophys J. 2009; 97(2): 665-673. PMCID: PMC2711318

Molecular diffusion and transport are fundamental processes in physical, chemical, biochemical, and biological systems. However, current approaches to measure molecular transport in cells and tissues based on perturbation methods such as fluorescence recovery after photobleaching are invasive, fluctuation correlation methods are local, and single-particle tracking requires the observation of isolated particles for relatively long periods of time. We propose to detect molecular transport by measuring the time cross-correlation of fluctuations at a pair of locations in the sample. When the points are farther apart than two times the size of the point spread function, the maximum of the correlation is proportional to the average time a molecule takes to move from a specific location to another. We demonstrate the method by simulations, using beads in solution, and by measuring the diffusion of molecules in cellular membranes. The spatial pair cross-correlation method detects barriers to diffusion and heterogeneity of diffusion because the time of the correlation maximum is delayed in the presence of diffusion barriers. This noninvasive, sensitive technique follows the same molecule over a large area, thereby producing a map of molecular flow. It does not require isolated molecules, and thus many molecules can be labeled at the same time ... [truncated at 200 words]

Measuring diffusion by spatial-cross-correlation

Sat, 11 Jul 2009 00:00:00 -0800

Abstract

Enrico Gratton and Michelle A Digman.
Measuring diffusion by spatial-cross-correlation.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S38, O-14.

Fluorescence correlation spectroscopy (FCS) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. It provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. Single particle tracking (SPT) is also a highly sensitive technique to measure particle movement. However, the FCS method suffers in spatial resolution while the SPT technique only allows for the tracking of isolated molecules. Here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. Our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. Therefore we can trace the average path of the particles. For example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. This information cannot be obtained with the FRAP (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method.

Determining stoichiometry of molecular complexes in live cells

Sat, 11 Jul 2009 00:00:00 -0800

Abstract

Michelle A Digman and Enrico Gratton.
Determining stoichiometry of molecular complexes in live cells.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S47, O-47.

In this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. The method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described N&B (Number and Brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. The new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. While the original N&B method was developed for one color, i.e., a single molecular species, the new method, ccN&B, extends the analysis to two colors and introduces the concept of cross-variance. This method is similar in concept to the two-color PCH analysis. However, the covariance based ccN&B method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. The method is highly sensitive and has relatively high temporal resolution. We have applied this method to adhesion complexes in cells. In addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers ... [truncated at 200 words]

Oligomerization of Concanavalin A in live cells detected by fluctuation analysis

Sat, 11 Jul 2009 00:00:00 -0800

Abstract

Valeria Vetri, Giulia Ossato, Valeria Militello, Michelle A Digman, Maurizio Leone, and Enrico Gratton.
Oligomerization of Concanavalin A in live cells detected by fluctuation analysis.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S137, P-393.

We report an experimental study on ConcanavalinA (ConA) aggregation in live cells. In vitro, close to physiological temperature, ConA readily forms fibrils involving secondary structure changes leading to β-aggregate structures. The effect of ConA on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. In particular, we monitored protein aggregation in live cells by means N&B analysis, Cross-N&B and RICS. N&B showed the aggregation kinetic and the progressive formation of ConA oligomers at cell surface. This suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. In parallel, the morphology of the cells changes indicating the progressive cell compaction and death. Aggregation and binding of small aggregates to the cell surface were assessed by RICS: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. Oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. Moreover, aggregates stoichiometry was measured during the kinetic by Cross-Variance N&B.