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A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy r(inf) was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.

Fluorescence Microscopy and Fluorescence Probes, edited by Jan Slavik, 1996. Plenum Press, New York and London. 306 pages. (hardback, $95)This volume is a compilation of abstracts of papers presented at the Fluorescence Microscopy and Fluorescence Probes Conference held in Prague in June 1995. This conference was one of the first tangible results of the end of the Cold War in the field of microscopy. As such, it attracted a great many of those who constitute the vanguard of progress in the field of fluorescence microscopy, on both sides of the old East-West divide. About one-third of the 127 contributors were from the West.

Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate measurements. Researchers hoping to obtain precise kinetic data from fluorescence recovery after photobleaching or photoactivation experiments need an easily controllable system for stimulation of a specific region and subsequent imaging, and the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) with SIM scanner makes this possible. We describe here how to precisely measure off rates using a cytosolic photoactivatable probe that binds endosomal membranes.

Coherent control based on a feedback-controlled self-learning loop was applied to enhance the ratio of two-photon (2P) fluorescence from a fluorescent label enhanced green fluorescence protein and three-photon (3P) fluorescence from the essential amino acid L-Tryptophan. The two biosamples were mixed in a buffer solution contained in a quartz cuvette and exposed to near-infrared laser pulses from a femtosecond (fs) oscillator. However, the enhancement of the 2P/3P fluorescence ratio was always accompanied by a significant loss of the valuable 2P fluorescence. To achieve a trade-off between the 2P/3P fluorescence ratio and the 2P fluorescence intensity, we then engineered the cost function in the self-learning algorithm. The optimal pulse shape obtained by use of the engineered cost function could be useful for 2P fluorescence imaging of living cells with reduced phototoxicity, because DNA and protein can be directly damaged by 3P absorption of fs laser according to an excitation band ~270nm.

We present a method for recovering the intrinsic fluorescence coefficient, defined as the product of the fluorophore absorption coefficient and the fluorescence energy yield, of an optically thick, homogeneous, turbid medium from a surface measurement of fluorescence and from knowledge of medium optical properties. The measured fluorescence signal is related to the intrinsic fluorescence coefficient by an optical property dependent path-length factor. A simple expression was developed for the path-length factor, which characterizes the penetration of excitation light and the escape of fluorescence from the medium. Experiments with fluorescent tissue phantoms demonstrated that intrinsic fluorescence line shape could be recovered and that fluorophore concentration could be estimated within ±15%, over a wide range of optical properties.

Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca(2equation)](i)) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400-500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca(2+)](i) distributions with three-dimensional submicrometer resolution and 10% precision are obtained at100-µM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca(2+)](i) in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.

SUMMARY The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general-purpose host-computer CPU, the video kinetics imaging can be performed in near real-time. The method is applied to chick embryo myotubes labelled with fluorescein-conjugated α-bungarotoxin. The pre-bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde-induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.

The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D 5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.

Fluorescence recovery after photobleaching (FRAP) experiments to measure the mobility of cell surface components require a brief, but intense, pulse of light to photobleach the fluorescence in a restricted area of the cell. We studied possible photodamage to the cell surface during the photobleaching step using light and scanning electron microscopy (SEM) and various FRAP measurements themselves. The cell membrane was left impermeable to trypan blue after photobleaching. SEM studies show that the morphology of the cell surface is not altered by photobleaching. Cells can be repeatedly photobleached and/or photobleached using longer bleach times and greater intensities without systematically altering FRAP kinetics. Singlet oxygen quenchers or free radical traps designed to inhibit putative photoreagents produced during photobleaching do not markedly affect the results. Fluorescein and rhodamine labels give similar results. All of these results, obtained with several different monolayer cultures, suggest that photodamage induced during photobleaching is not a serious artefact in the cellular FRAP results obtained to date.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.

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We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260–3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10–12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.

Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.

International audience; Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

The dipole potential (Psi(d)) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Psi(d) changes by a variation of the intensity ratio of their two well-separated fluorescence bands. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Psi(d) in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced. (c) 2005 Elsevier B.V. All rights reserved.

— We describe a simple method for determination of the dependence of fluorophore photo-bleaching on the intensity of the incident light. A sample of fluorophore is illuminated uniformly with an intensity which is sufficient only to bleach a small fraction of the fluorophore during a fluorescence lifetime period. The long-term decay of fluorescence due to photobleaching is monitored before and after a stepped increase in the incident intensity. The order of the photobleaching reaction is determined by taking the ratio of the logarithms of the limiting values of the slope of the fluorescence bleaching curve and the absolute fluorescence change. The method is applied to fluorescein, bound to dextran or ovalbumin, in aqueous solution, and the order of the reaction in both cases is shown to be unity.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.

Publisher Summary The fluorescence redistribution after photobleaching (FRAP) method permits nucleocytoplasmic transport dynamics to be determined from the transient behavior of a spatial gradient of fluorescence photochemically induced in a nucleus by a short, intense burst of laser light. Recovery of fluorescence within the bleached area, due to the spatial redistribution of surrounding intact fluorescently labeled molecules, is measured by the repetitive scanning of the nucleus with an attenuated laser beam. The rate and extent of recovery permits the calculation of a permeation rate constant and the fraction of tracer molecules free to move. The method depends on the detection of specific fluorescent probes. The use of this noninvasive analytical approach permits the rapid evaluation of nuclear transport for all types of macromolecules, for example, proteins and RNA both in vitro and in vivo , offering higher resolution in time and space when compared with approaches employing microinjection and thin-section autoradiography. Of equal significance is the advantage that all types of nuclei are examined for transport dynamics without the size limitations or potential damage resulting from microinjection methods.

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. We employ digitized fluorescence microscopy (DFM) alone or in combination with fluorescence recovery after photobleaching (FRAP) to investigate individual cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to define antigens or fluorescent analogs of cellular constituents as well as ultra-sensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells.

n-Prop]yl gallate (0.1 to 0.25 molar, in glycerol) reduces by a factor of 10 the rate of fading of fluorescence of cell structures labeled with tetramethylrhodamine or fluorescein-conjugated antibodies. Hence, prolonged photographic exposure of immunofluorescently labeled cells in the fluorescence microscope yields images with increased sensitivity, making feasible multiple data collection, as with serial optical sectioning.

Fluorescence rapidly returns to spots bleached by a laser beam in the continuous fluorescence of cultured cells labeled on the surface with fluorescein isothiocyanate. The rate of recovery of fluorescence after bleaching can be interpreted as a measure of the lateral diffusion of integral membrane proteins labeled with fluorescein.

The conventional method of studying mass transport in membranes by spot photobleaching and then following the recovery of fluorescence has disadvantages. Among them, the need for a high density of fluorescent molecules, the measurement of the beam profile, and a knowledge of the photobleaching processes are of a crucial importance. The application of a planar fringe pattern of light both for the bleaching and the monitoring of the fluorescent molecules solves these three major difficulties. Brownian diffusion coefficients and flow velocities can be measured independently and are averaged over the whole fringe pattern volume. These transport coefficients are explored over the wide range of experimentally accessible distances (from an interfringe spacing 0.5-50 micron). The quantification of the mobile and immobile components is further simplified by scanning the fringe pattern and detecting only a modulated fluorescence recovery signal. The fringe pattern photobleaching method is particularly adapted to the measurements of diffusion coefficients and flow velocity of membrane components, as well as of cytoplasmic proteins. The theoretical results and the test experiments with fluorescent bovine serum albumin are described.

Rates of photodecomposition were studied in some fluorophores during short (milliseconds) and longer (minutes) illumination periods. A xenon burner served as light source, and care was taken to obtain optimum conditions for activation. The fluorophores studied included (i) the formaldehyde-induced fluorescent product from 5-hydroxytryptamine in mast cells, (ii) Berberine sulphate bound to mast cell polyanions, (iii) Feulgen-Pararosaniline-stained DNA, and (iv) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. All fluorophores showed a significant fading during 3 min illumination. The Fluorescein isothiocyanate-conjugate faded the most rapidly; its fluorescence intensity was reduced to 50% of the initial value after 2 sec continuous illumination. No fluorophore faded significantly during the initial few milliseconds of illumination. On the basis of these findings, an inexpensive measuring device was constructed. It contained a peak-reader and memory circuit triggered by the flash synchronization tap of a camera shutter positioned in the activation beam. The peak-reader has a response time of about 2 msec. Repeated measurements on the various fluorophores indicate that this peak-reading device may be used to measure fluorescence intensity without fading.

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.

Single terrylenediimide molecules diluted in a 20-nm-thick polyvinylbutyral polymer film were localized and observed by scanning confocal fluorescence microscopy. A modular and compact confocal microscope and the high optical stability of the molecules allowed a repeated imaging and observation over >5 h at room temperature. Most of the molecules showed several “on-off-on” transitions (blinking) on a time scale from seconds to hours, before permanent bleaching occurred. We determined that >1.5 × 10^7 fluorescence photons are emitted from the most-stable molecules before the final bleaching step occurs. Despite the “on-off-on” transitions, however, the overall change in fluorescence intensity, either integrated over each image of a time series or summed for several individual molecules, resembled an exponential-like decay, familiar from measurements of many-molecule ensembles. We also observed the polarization of the fluorescence from single molecules during excitation with circular polarized light. From these measurements, possible rotations of the molecular dipoles were studied. Over a span of 5 h, the polarization angle in most cases did not change by >15-20°. This may explain the slow and small intensity changes but excludes molecular rotation as a reason for the blinking behavior.

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Multichannel-detection for simultaneous registration of all information on a signal burst (intensity, lifetime, anisotropyr and spectral range) is used for analytics and studies of conformational dynamics of a single fluorescent labeled molecule in solution.

Dye photobleaching is a photochemical reaction that can be investigated locally using fluorescence microscopy techniques. In this study, a user-friendly computational tool to assist photobleaching experiments called Photobleaching Lifetime Imaging Microscopy (PbLIM) is presented. With this tool it is possible to recover the photobleaching kinetics spatially, where a photobleaching lifetime is generated for each pixel of the image. Our model was applied to the photobleaching process of thionine encapsulated into the one-dimensional nano-channels of Zeolite L (ZL), from where we gained insight into the molecular oxygen distribution inside the ZL channels, as well as the detailed photobleaching of the confined thionine. © The Royal Society of Chemistry and Owner Societies 2016.

Highlights • Development of a tool to track viral RNA in living cells. • Tick-borne encephalitis virus as a model system to study Flaviviral RNA dynamics. • FRAP and FLIP to measure the dynamic properties of viral RNA., Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication are believed to trigger the innate immune response through interaction with cytoplasmic cellular sensors. Therefore, understanding the subcellular distribution and dynamics of Flavivirus RNAs is of paramount importance to understand the interaction of the virus with its cellular host, which could be of insect, tick or mammalian, including human, origin. Recent advances on the visualization of Flavivirus RNA in living cells together with the development of methods to measure the dynamic properties of viral RNA are reviewed and discussed in this essay. In particular the application of bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are analysed in the context of tick-borne encephalitis virus replication. Conclusions driven by this approached are discussed in the wider context Flavivirus infection.

It is shown that all-trans-retinal under model conditions of its excessive accumulation in photoreceptor membranes interacts with amino groups of rhodopsin and lipids, forming at least three distinct fluorophores with fluorescence quantum yield 20–40 times higher than that of free all-trans-retinal. These retinal derivatives are likely precursors of photo- and cytotoxic fluorophores of lipofuscin and in particular of A2E. Spectral characteristics of fluorophores have been described. Picosecond time-resolved laser fluorescence spectroscopy was used to study kinetics of fluorescence decay of both free and bound all-trans-retinal; fluorophores were determined and their lifetimes have been measured. Based on calculations it is shown that the decay kinetics of all-trans-retinal derivatives consists of three components with lifetimes equal to 48, 208, and 900 ps; kinetics of free all-trans-retinal is monoexponential with lifetime of 31 ps. The chemical nature of fluorophores with the lifetimes obtained is discussed.

The fluorescence spectra of the title compound have been measured in ethanol and in aqueous solutions of different pH. In ethanol the neutral molecule's fluorescence (at 360 nm) is observed, whereas in aqueous solutions emission occurs from the anion (at 428 nm) and from a "tautomeric" species (at 460 nm), but not from the neutral molecule. An aqueous solution at pH 3, which contains about 30% of ethanol gives a broad fluorescence band ranging from the near uv to the orange region of the spectrum. Contrarily, 7-methoxylepidone has no fluorescence maximum in the visible part of the spectrum.

Partial recovery of fluorescence intensity after irradiation-induced fading of lipopigment autofluorescence is reported, during a subsequent period of 60 sec when each specimen was in darkness. This phenomenon may have significance for various techniques of quantitative fluorescence microscopy.

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein localization in cellular membranes. In this study, we developed numerical simulations of lateral diffusion of a fluorophore in a membrane with arbitrary bleach geometry and exchange of the fluorophore with cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. Based on our model simulations, we designed and performed FRAP experiments with varying bleach region sizes on plasma-membrane localized EGFP:Ras2 in live yeast cells to investigate the mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated from a 1 micron x 1 micron bleach region-of-interest (ROI) size successfully predicted the 0.5 micron x 0.5 micron bleach ROI experiment without additional fitting. Successful prediction of the second experiment without data fitting shows the agreement of the experiment with the theory and excluded alternative models including both diffusion and binding, which were also tested against the experimental data. We also performed Fluorescence Correlation Spectroscopy (FCS) experiments as an independent method to measure the mobility of EGFP:Ras2. We show that simulation of FRAP experiments based on the mobilities and fluorophore fractions derived from FCS model fits enables the validation of the FCS model. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on widely available commercial confocal laser scanning microscopes.

Continuous fluorescence microfotolysis (CFM) is a technique suited especially for measuring lateral diffusion in membrane systems. Unlike the fluorescence recovery after photobleaching (FRAP) method it monitors the continuous competition between bleaching and diffusion of labelled molecules in a small laser spot, and is therefore not a relaxation procedure. The laser intensity is constant and low, but provokes a certain photobleaching. As time proceeds, more and more fresh molecules from areas at a farther distance diffuse into the illuminated spot. Therefore the initial decreasing fluorescence signal finally levels at a steady-state value.