di Rienzo C, Gratton E, Beltram F, Cardarelli F.
Super-Resolution in a standard microscope: from fast fluorescence imaging to molecular diffusion laws in live cells.
Super-Resolution Imaging in Biomedicine. By A Diaspro and MAMJ van Zandvoort (Editors). CRC Press, pp. 19-43, 2016. ISBN: 9781482244342Living systems establish local steady-state conditions by maintaining a complex landscape of precisely regulated interactions, which govern the spatial distribution of their molecular constituents. In this regard, fluorescence fluctuation microscopy encompasses a diversified arsenal of analysis tools that provide a quantitative link between cell structural organization and the underlying molecular dynamics. A spatial or temporal analysis of the fluctuating fluorescence signal arising from suitably labeled reporters allows noninvasive measurements of molecular and biochemical parameters such as concentration, diffusion coefficients, binding constants, degree of oligomerization, and so on directly within nonperturbed, live samples. Thus, within the constraints imposed by the requirement of fluorescence labeling, fluctuation microscopy has the potential to add a dynamic molecular dimension to standard fluorescence imaging in vivo. In particular, we shall discuss a fluorescence fluctuation-based method that makes it possible to probe the actual molecular “diffusion law” directly from imaging, in the form of a mean square ... [truncated at 150 words]
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Measurements of fluorescence decay time by the frequency domain method.
Perspectives on Fluorescence: A Tribute to Gregorio Weber (Springer Series on Fluorescence). By DM Jameson (Editors). Springer Berlin Heidelberg, 2016. Among the many contributions of Gregorio Weber to science and technology, the development of frequency domain technology in his lab in 1969 has caused a deep controversy, dividing scientist that will refuse using anything but the frequency domain approach to measure the fluorescence decay from the other camp that simply refuse anything but the time-correlated single photon counting (TCSPC) approach. Although at the time of the major contribution of Gregorio Weber and Richard Spencer in 1969, the TCSPC method was not yet invented, the basic controversy “frequency domain vs time domain” in the field continues today. We have made progress both in the scientific understanding and in describing the technical differences between the two approaches; still it is interesting how scientists continue to be divided. As for many of the contributions of Gregorio Weber that stirred controversy, I would like to mention a common theme of my conversations with Dr. ... [truncated at 150 words]
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Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time.
Perspectives on Fluorescence: A Tribute to Gregorio Weber (Springer Series on Fluorescence). By DM Jameson (Editors). Springer Berlin Heidelberg, 2016. The fundamental unit of biology is unarguably the cell. Thus, as we move forward in our understanding of the processes occurring in the cell, it is crucial to reflect on how much of the cell biophysics remains unexplained or unknown. A ubiquitous observation in cell biology is that the translational motion of molecules within the intracellular environment is strongly suppressed as compared to that in dilute solutions. By contrast, molecular rotation is not affected by the same environment, indicating that the close proximity of the molecule must be aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding, but with expectations that depend on the nanoscale organization assigned to crowding agents. A satisfactory experimental discrimination between possible scenarios has remained elusive due to the lack of techniques to explore molecular diffusion at the appropriate spatiotemporal scale in the 3D-intracellular environment. Here we discuss ... [truncated at 150 words]
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Laurdan identifies different lipid membranes in eukaryotic cells.
Cell Membrane Nanodomains: From Biochemistry to Nanoscopy. By A Cambi and DS Lidke (Editors). CRC Press, pp. 283-304, 2014. ISBN: 9781482209891INTRODUCTION There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluorescent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their lipid composition, it is more useful to use a single probe that can report on the specific properties of the membrane icrodomains, independently of the probe segregation in one specific domain and location in the cell. One fluorescent probe that has been successfully used for this purpose is the lipophilic probe Laurdan (2-dimethylamino-6-lauroylnaphthalene), originally synthesized by Weber and Farris.1 Different membrane environments produce marked changes both in the spectrum and in the fluorescence lifetime of Laurdan. The sensitivity of the emission spectrum ... [truncated at 150 words]
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Fluctuation spectroscopy methods for the analysis of membrane processes.
Cell Membrane Nanodomains: From Biochemistry to Nanoscopy. By A Cambi and DS Lidke (Editors). CRC Press, pp. 215-238, 2014. ISBN: 9781482209891INTRODUCTION In the field of membrane lipid and protein dynamics, fluorescence recovery after photobleaching (FRAP) and single-particle tracking methods have provided ample evidence that lipids and proteins could have their motion restricted by interaction with other lipids and with the cell cytoskeleton.1–8 Fluorescence correlation spectroscopy (FCS) is a relatively new method in this field that has received particular attention recently because of the possible combination with super-resolution microscopy methods.9–14 In this context, the use of very small volumes of excitation or variable volumes of excitation15 was considered necessary to unravel to transport of molecules at the nanoscale. However, most of the FCS studies done so far are based on the original idea of measuring temporal correlation at a single point in the membrane. Measuring a single location in the membrane is restrictive since the temporal fluctuations at one point cannot reveal local microstructures or the anisotropic molecular transport in membranes. ... [truncated at 150 words]
Golfetto O, Hinde E, Gratton E.
The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells.
Methods in Membrane Lipids (Methods in Molecular Biology, Vol. 1232). By DM Owen (Editors). Springer New York, pp. 273-290, 2014. ISBN: 9781493917518In this method paper we describe the spectral phasor analysis applied to Laurdan emission for the assessment of the fluidity of different membranes in live cells. We first introduce the general context and then we show how to obtain the spectral phasor from data acquired using a commercial microscope.
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The phasor approach to fluorescence lifetime imaging: exploiting phasor linear properties.
Fluorescence Lifetime Spectroscopy and Imaging: Principles and Applications in Biomedical Diagnostics. By L Marcu, PMW French, and DS Elson (Editors). CRC Press, pp. 235-248, 2014. ISBN: 9781439861677The phasor approach to fluorescence lifetime imaging (FLIM) is emerging as a practical method for data visualization and analysis. The main feature of the phasor approach is that it is a “fit-free” analysis tool that provides quantitative results about mixtures of fluorophores, Förster resonant energy transfer (FRET), and autofluorescence.
Sánchez SA, Tricerri MA, Gunther G, Gratton E.
Laurdan generalized polarization: from cuvette to microscope.
Modern Research and Educational Topics in Microscopy. Vol. 3: Applications in Physical/Chemical Sciences (Microscopy Book Series, Vol. 3). By A Méndez-Vilas and J Díaz (Editors). Formatex Research Center, Badajoz, Spain, pp. 1007-1014, 2007. ISBN: 9788461194209Laurdan is a fluorescent molecule that detects changes in membrane phase properties through its sensitivity to the polarity of the environment in the bilayer. Polarity changes are shown by shifts in the Laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). This technique was originally developed to be used in a cuvette fluorometer. With the development of twophoton microscopy, Laurdan GP has evolved into a technique capable of spatially resolve micro-domains of different solvent penetration. We discuss here the basic concepts, instrumentation and experimental considerations when transferring the GP technique from the cuvette to the microscope. We also discuss examples of Laurdan GP in membrane model systems using both cuvette and microscope approaches to compare the two methodologies.
Celli A, Lee CY, Gratton E.
Fluorescence microscopy to study pressure between lipids in giant unilamellar vesicles.
Methods in Membrane Lipids (Methods in Molecular Biology, Vol. 400). By AM Dopico (Editors). Humana Press, pp. 333-340, 2007. ISBN: 9781588296627The authors developed a technique to apply high hydrostatic pressure to giant unilamellar vesicles and to directly observe the consequent structural changes with two-photon fluorescence microscopy imaging using high numerical aperture oil-immersion objectives. The data demonstrate that high pressure has a dramatic effect on the shape of the vesicles, and both fluidity and homogeneity of the membrane.
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The RICS method: measurement of fast dynamics in cells with the laser scanning microscope.
Image Analysis in Medical Microscopy and Pathology. By HS Wu and AJ Einstein (Editors). Research Signpost, Kerala, India, pp. 207-224, 2007. ISBN: 8130801167Single point fluctuation correlation spectroscopy (FCS) is an established technique to study diffusion and chemical equilibria in solution. It has limitations when applied to the cell interior. A major difficulty is that the movements of the entire cell or of cellular components are difficult to separate and filter out from the fast dynamics of the molecules moving in the cell. It is the study of these fast dynamics that helps us in understanding molecular interactions. Scanning FCS, in which the laser beam is moved in a circular orbit, provides the fluctuation amplitude and dynamics at many points simultaneously. It can be used to infer cell movement, but has limitations in the time scales accessible. Image correlation spectroscopy, an alternative technique in which the entire field of view is analyzed at once, has the potential to provide detailed maps of the dynamics in a cell, but it suffers from limitations imposed ... [truncated at 150 words]
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Real-time fluorescence lifetime imaging and FRET using fast gated image intensifiers.
Molecular Imaging: FRET Microscopy and Spectroscopy (Methods in Physiology Series). By A Periasamy and RN Day (Editors). Academic Press, pp. 193-226, 2005. ISBN: 9780195177206Summary
This chapter discusses practical features and guidelines for Fluorescence lifetime imaging (FLI) and Förster resonance energy transfer (FRET) that maybe helpful in reading the literature and in interpreting FLI and FRET measurements of complex biological systems. Several reviews are already available (Clegg and Schneider, 1996; [Clegg, et al, 1996] and [Clegg, et al, 2003]; Periasamy et al., 1996; [So, et al, 1996] and [So, et al, 1998]; Schneider and Clegg, 1997; Gadella et al., 2001; Cubeddu et al., 2002; Hink et al., 2002; Peter and Ameer-Beg, 2004). However, we feel that the general reader and aspiring user of FLI with applications to FRET would benefit from a concise, coherent presentation of fundamental aspects of these measurements and interpretations of the data. We will not include particular results from a biological system, nor specifics of new instrumentation. Instead, we focus on basic descriptions of the photophysical measurements and the general characteristics ... [truncated at 150 words]
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Nuts and bolts of excitation energy migration and energy transfer.
Chlorophyll a Fluorescence: A Signature of Photosynthesis (Advances in Photosynthesis and Respiration, Vol. 19). By GC Papageorgiou and Govindjee (Editors). Springer, pp. 83-105, 2005. ISBN: 9781402032172The applications of Fluorescence Resonance Energy Transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. Our understanding of photosynthesis is tightly coupled to our understanding of the transfer of captured energy from the absorption of photons, and following the energy flow through the complex maize of chemical reactions utilizing this energy. Many of these steps involve resonance energy transfer. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation. This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis. Many publications appear weekly using FRET and most of the applications use FRET as a spectroscopic research tool. Photosynthesis holds ... [truncated at 150 words]
Hazlett TL, Ruan Q, Tetin SY.
Application of fluorescence correlation spectroscopy to hapten-antibody binding.
Protein-Ligand Interactions: Methods and Applications (Methods in Molecular Biology, Vol. 305). By GU Nienhaus (Editors). pp. 415-438, 2005. ISBN: 9781588293725Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations ... [truncated at 150 words]
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Reflectance and transmittance spectroscopy.
Laser and Current Optical Techniques in Biology (Comprehensive Series in Photochemical & Photobiological Sciences). By G Palumbo and R Pratesi (Editors). Royal Society of Chemistry, pp. 211-258, 2005. ISBN: 9780854043217This chapter describes several approaches to the optical study of biological tissue using reflectance and transmittance spectroscopy. This topic has spurred significant research efforts as a result of the relevant physiological and metabolic information provided by the optical data, in conjunction with the safe, non-invasive, and costeffective optical approach to the study of tissue. We give a brief historical introduction in Section 11.1, followed by a description of the optical absorption and scattering properties of tissue in Section 11.2. Section 11.3 is devoted to 1 continuous-wave (CW) techniques, which can be applied to the study of relatively superficial tissue layers (as is the case for diffuse reflectance imaging and localized measurements using short separations between the illuminating and collecting optodes), as well as deep tissues on the basis of a modified Beer-Lambert law, transport theory, or diffusion theory. The latter model is commonly employed in time-domain and frequency-domain techniques, which ... [truncated at 150 words]
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Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis.
Epidermal Cells: Methods and Protocols (Methods in Molecular Biology, Vol. 289). By K Turksen (Editors). Humana Press, pp. 413-421, 2005. ISBN: 9781592598304Two-photon fluorescence microscopy is used to detect ultraviolet-induced reactive oxygen species (ROS) in the epidermis and the dermis of ex vivo human skin and skin equivalents. Skin is incubated with the nonfluorescent ROS probe dihydrorhodamine, which reacts with ROS such as singlet oxygen and hydrogen peroxide to form fluorescent rhodamine-123. Unlike confocal microscopic methods, two-photon excitation provides depth penetration through the epidermis and dermis with little photodamage to the sample. This method also provides submicron spatial resolution such that subcellular areas that generate ROS can be detected. In addition, comparative studies can be made to determine the effect of applied agents (drugs, therapeutics) upon ROS levels at any layer or cellular region within the skin.
Dong CY, French TE, So PTC, Buehler C, Berland KM, Gratton E.
Fluorescence-lifetime imaging techniques for microscopy.
Digital Microscopy. A second edition of "Video Microscopy" (Methods in Cell Biology, Vol. 72). By G Sluder and DE Wolf (Editors). Academic Press, pp. 431-464, 2003. ISBN: 9780125641692Three different methods of fluorescence-lifetime imaging for microscopy are presented along with some examples of their use. All three methods use the frequency-domain heterodyning technique to collect lifetime data. Because of the nature of the data collection technique, these instruments can measure the correct lifetime even when the sample undergoes strong photobleaching. Each instrument has unique capabilities that complement the others.
The first microscopic-lifetime imaging technique uses a fast charge-coupled device (CCD) camera and a gated image intensifier. The camera system collects an entire lifetime image in parallel in only a few seconds. This microscope is well suited to kinetic studies of intracellular lifetime changes. The other two techniques use scanned laser source to collect sequential lifetime information pixel by pixel. One microscope uses two-photon excitation to achieve three-dimensional, confocal-like imaging without using detection pinholes. Two-photon excitation also limits the effects of out-of-plane photodamage of the sample. The second laser-scanning microscope ... [truncated at 150 words]
Beechem JM, Gratton E, Ameloot M, Knutson JR, Brand L.
The global analysis of fluorescence intensity and anisotropy decay data: second-generation theory and programs.
Topics in Fluorescence Spectroscopy: "Principles" (Vol. 2). By JR Lakowicz (Editors). Springer US, pp. 241-305, 2003. ISBN: 9780306438752The aim of this chapter is to describe, in detail, the design, application, and "philosophy" behind the current generation of global analysis programs. The sections of this chapter can be summarized as follows:
Section 1: Introduction to time-resolved fluorescence data, some experimental techniques, and some typical examples of how previous works have benefitted from global analysis.
Section 2: Historical overview of how the emphasis of global analysis has evolved from one of multi-dimensional curve fitting to that of multi-dimensional physical model evaluation.
Section 3: General elements required to perform a global analysis of distributed and discrete models. The basic equations of the compartmental approach are examined and the systems theory view of photophysical events is described.
Section 4: In-depth example of the "inner-workings" of the general purpose global analysis program developed at the Laboratory for Fluorescence Dynamics (LFD). Overall flow diagrams as well as specific FORTRAN 77 source codes of key programming sections are ... [truncated at 150 words]
Müller JD, Chen Y, Gratton E.
Fluorescence correlation spectroscopy.
Biophotonics, Part B (Methods in Enzymology, Vol. 361). By G Marriott and I Parker (Editors). Academic Press, pp. 69-92, 2003. ISBN: 9780121822644Introduction: Spontaneous, microscopic fluctuations are an integral part of every fluorescence measurement and add a noise component to the observed fluorescence signal.
Fluorescence correlation spectroscopy (FCS) extracts information from this noise and characterizes the kinetic processes that are responsible for the signal fluctuations. For instance, the dynamic equilibrium between a fluorescent and a nonfluorescent state of a fluorophore introduces fluctuations. Another example is Brownian motion, which leads to the stochastic appearance and disappearance of fluorescent molecules in a small observation volume. FCS characterizes any kinetic process that leads to changes in the fluorescence, because the spontaneous fluctuations at thermodynamic equilibrium are governed by the same laws that describe the kinetic relaxation of a system to equilibrium. Thus, FCS offers a very convenient method for determining kinetic properties at equilibrium without requiring a physical perturbation of the sample. This is especially important for systems in which the use of perturbation techniques in ... [truncated at 150 words]
Bagatolli LA, Sánchez SA, Hazlett TL, Gratton E.
Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers.
Biophotonics, Part A (Methods in Enzymology, Vol. 360). By G Marriott and I Parker (Editors). Academic Press, pp. 481-500, 2003. ISBN: 9780121822637Clegg RM, Holub O, Gohlke C.
Fluorescence lifetime-resolved imaging: measuring lifetimes in an image.
Biophotonics, Part A (Methods in Enzymology, Vol. 360). By G Marriott and I Parker (Editors). Academic Press, pp. 509-42, 2003. ISBN: 9780121822637We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation. Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation–and therefore affect the measured fluorescence lifetime–that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging. FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging ... [truncated at 150 words]
Bell JD, Sánchez SA, Hazlett TL.
Liposomes in the study of phospholipase A2 activity.
Liposomes, Part B (Methods in Enzymology, Vol. 372). By N Duzgunes (Editors). Academic Press, pp. 19-48, 2003. ISBN: 9780121822750Introduction: Phospholipase A2 (PLA2) catalyzes hydrolysis of the sn-2 acyl chain of phospholipids. Physiologically, it appears to be involved in diverse functions such as digestion, membrane homeostasis, production of precursors for synthesis of several lipid mediators, defense against bacteria, clearing of dead or damaged cells, and as ligands for receptors. Three basic types
have been identified: secretory, cytosolic, and intracellular PLA2 (sPLA2, cPLA2, and iPLA2, respectively)...
Tramier M, Holub O, Croney JC, Ishii T, Seifried SE, Jameson DM.
Binding of ethidium to yeast tRNA(Phe): a new perspective on an old Bromide.
Fluorescence Spectroscopy, Imaging and Probes. By R Kraayenhof, AJWG Visser, and HC Gerritsen (Editors). Springer-Verlag Berlin, Heidelberg, New York, pp. 111-121, 2002. ISBN: 9783540427681We have reinvestigated the binding of ethidium bromide (EB) to yeast tRNA(Phe) using frequency domain fluorometry and Global Analysis. Previous fluorescence investigations of EB-tRNA interactions, carried out for more than 30 years, have indicated a "strong" binding site with a lifetime near 26 ns and one or more "weak, non-specific" binding sites with reduced lifetimes. In our study, under specific conditions in which only one EB is bound, a fluorescence lifetime of 27 ns was obtained. However, as the EB/tRNA ratio increased, shorter lifetime components appeared. Global Analysis of the lifetime data was consistent with a model in which the second EB molecule bound has a lifetime of only 5.4 ns. Global Analysis also indicated that this second binding event leads to a reduction in the lifetime of the first EB bound, namely from 27 ns to 17.7 ns. The lifetime decrease associated with the "strong" binding site could be ... [truncated at 150 words]
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Assessment of membrane fluidity in individual yeast cells by Laurdan Generalized Polarization and multi-photon scanning fluorescence microscopy.
Fluorescence Spectroscopy, Imaging and Probes (Vol. 2). By R Kraayenhof, AJWG Visser, and HC Gerritsen (Editors). Springer-Verlag Berlin, Heidelberg, New York, pp. 241-252, 2002. ISBN: 9783540427681Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of sub-populations. Yeast membranes are a primary site of environmental response and adaptation. Using fluorescence spectroscopy with DPH polarization and laurdan Generalized Polarization (GP), we previously found rapid “average” membrane fluidity modulation in yeast populations during growth and in response to nutrients or environmental stresses. To determine whether such responses reflect all cells we conducted the first multi-photon scanning fluorescence microscopy study of yeasts, measuring laurdan GP. We assessed membrane fluidity responses of individual yeasts related to growth phase, heat stress and ethanol stress. Average fluidity decreased as cultures aged, however the decreased fluidity was due in some cases to an increasing proportion of uniformly ... [truncated at 150 words]
Dong CY, Buehler C, So PTC, French T, Gratton E.
Biological applications of time-resolved, pump-probe fluorescence microscopy and spectroscopy in the frequency domain.
Methods In Cellular Imaging (Methods in Physiology Series, Vol. IV). By A Periasamy (Editors). Springer New York, pp. 324-340, 2001. ISBN: 9780195139365In biological applications of optical microscopy, technical developments often lead to novel imaging modalities with significant applications. For example, the development of confocal microscopy led to microscopic imaging with enhanced contrast (see Chapter 5), and the more recent development of two-photon fluorescence microscopy revolutionized fluorescence microscopy by providing an imaging modality capable of high image contrast, reduced photodamage, and exciting possibilities in controlling localized photochemical reactions in three dimensions (see Chapters 9-13).
Parasassi T, Bagatolli LA, Gratton E, Levi M, Ursini F, Yu W, Zajicek HK.
Two-photon excitation microscopy for image spectroscopy and biochemistry of tissues, cells, organelles and lipid vesicles under physiological conditions.
Confocal and Two-Photon Microscopy: Foundations, Applications and Advances. By A Diaspro (Editors). John Wiley & Sons, New York, pp. 469-482, 2001. ISBN: 9780471409205Chen Y, Müller JD, Eid JS, Gratton E.
Two-photon fluorescence fluctuation spectroscopy.
New Trends in Fluorescence Spectroscopy:Applications to Chemical and Life Sciences (Springer Series on Fluorescence). By B Valeur and JC Brochon (Editors). pp. 277-296, 2001. ISBN: 9783540677796Two-photon excitation spectroscopy has inherent 3-D resolution with excitation volumes as small as 0.1 fl. Compared to conventional fluorometers a reduction by a factor of 10^10 of the excitation volume can be achieved. The fluorescence fluctuations within the small excitation volume provide, via fluorescence correlation spectroscopy (FCS), a unique way to study biological phenomena. In this contribution, we outline the instrumentation of two-photon fluorescence correlation spectroscopy and highlight technical details of our experimental setup. We discuss the autocorrelation analysis for single and multiple species with emphasis on the normalized fluctuation amplitude G(0). Furthermore, we revisit another data analysis technique called moment analysis. This method was originally introduced 10 years ago and provides a very fast and convenient characterization of the fluctuation amplitude. We compare experimental results from moment analysis with that of another data analysis method, the photon counting histogram (PCH), and discussed differences between these two methods.
Müller JD, Chen Y, Gratton E.
Photon counting histogram statistics.
Fluorescence Correlation Spectroscopy. Theory and Applications (Springer Series in Chemical Physics, Vol. 65). By R Riegler and EL Elson (Editors). Springer-Verlag, Berlin, pp. 410-437, 2001. ISBN: 9783540674337In the macroscopic world fluctuations are typically exceedingly small and beyond the resolution power of most experiments. Only in special circumstances, such as near the critical point of a liquid, do fluctuations become visible to the naked eye. However, the importance of fluctuations increases once the macroscopic world is left behind and one starts to consider mesoscopic or microscopic systems, where fluctuation phenomena are readily observed. Fluctuation spectroscopy exploits this source of information [20.1] and embraces a diverse field of applications.
Breusegem SY, Loontiens FG, Regenfuss P, Clegg RM.
Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques.
Drug-Nucleic Acid Interactions (Methods in Enzymology, Vol. 340). Academic Press, pp. 212-33, 2001. ISBN: 9780121822415
