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PubMed1994

Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: a method for reducing fluorescence photobleaching.

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.

3T3 Cells↗
PubMed1998

Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photobleaching (FRAP).

Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to a desired overall biofilm thickness, then employed within classical half-cell diffusion chambers to estimate, from transient solute concentrations, the effective diffusion coefficient for several macromolecules of increasing molecular weight and molecular complexity. Results of traditional half-cell studies were found to be erroneous due to the existence of microscopic water channels or crevasses that perforate the polysaccharidic gel matrix of the biofilm, sometimes completely to the supporting substratum. Thus, half-cell devices measure a composite transfer coefficient that may overestimate the true, local flux of solutes in the biofilm polysaccharide gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale to avoid the errors created by the biofilm architectural irregularities. This technique is based upon the Fluorescence Return After Photobleaching (FRAP), which allows image analysis observation of the transport of fluorescently labeled macromolecules as they migrate into a micro-scale photobleached zone. The technique can be computerized and allows one to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These mappings also indirectly indicate the distribution of water channels in the biofilm, which was corroborated independently by direct microscopic observation of the settling of fluorescently-labeled latex spheres within the biofilm. Fluorescence return after photobleaching results indicate a significant reduction in the solute transport coefficients in biofilm polymer gel vs. the same value in water, with the reduction being dependent on solute molecule size and shape.

Biofilms↗
PubMed1999

In vivo fluence rate and fractionation effects on tumor response and photobleaching: photodynamic therapy with two photosensitizers in an orthotopic rat tumor model.

The effect of fluence rate and light fractionation on phototoxicity was investigated in vivo in an orthotopic rat bladder tumor model. Two photosensitizers, benzoporphyrin derivative monoacid ring A and 5-aminolevulinic acid-induced protoporphyrin IX, were studied. For a given cumulative light dose of 30 J/cm2, enhanced tumor destruction was observed from both photosensitizers when using either lower fluence rates or fractionated light delivery. Photobleaching experiments in vivo demonstrated that the photobleaching rate, however, was not fluence rate dependent. The fluence rate and light fractionation effects on tumor phototoxicity lead to rapid local depletion in oxygen concentration that inhibited subsequent photochemical reactions necessary for efficient photodestruction of tumor cells. Nicotinamide did not enhance photodynamic therapy efficacy, suggesting that the added increase of oxygen within the tumor was not sufficient to enhance photodestruction of hypoxic cell fractions. The independence of the photobleaching rate with fluence rate suggests distinct mechanisms, at least in part, of photodestruction of the tumor and the photosensitizer and that the rate of photosensitizer photo-bleaching may not always be an appropriate monitor for singlet oxygen availability and photodynamic therapy dosimetry.

Aminolevulinic Acid↗
PubMed2000

Wavelength dependence of the time course of fluorescence enhancement and photobleaching during irradiation of ethidium bromide-stained nuclei.

The variation of fluorescence during irradiation of ethidium bromide-stained nuclei with the 458 nm argon laser line was measured at different wavelengths throughout the emission spectrum. When glycerol was used as a mountant, photoenhancement of fluorescence was observed at all wavelengths, but was greater at the shorter wavelengths. Fluorescence increased by almost one order of magnitude at 500 nm after 40 s of irradiation, compared with only about 10% at wavelengths longer than 600 nm after 2-3 s. In nuclei mounted in phosphate buffer, an initial photoenhancement of fluorescence was detected only at the shorter wavelengths, while continuous photobleaching was observed in the rest of the emission spectrum. When the spectra are normalized to maximum, so as to eliminate the effect of the concurrent photobleaching, it appears that the difference between the time course of fluorescence variation in buffer and glycerol depends largely on the lower photobleaching rate in glycerol. The photoenhancement of fluorescence at shorter wavelengths was found to consist of a band peaking at 485-491 nm in glycerol and at 495-496 nm in buffer. Attenuation of the inner-filter effect contributes minimally to the enhancement of fluorescence at shorter wavelengths. Since the dimer is known to be non fluorescent, the light-induced disaggregation of dimers to monomers cannot be an explanation for the large increase of fluorescence at the shorter wavelengths. The same laser beam that was used to excite the fluorescence of stained nuclei was also used for monitoring the concomitant variation of transmitted light, from which the variation of absorptance during irradiation was computed. While the expected decrease of absorptance was observed in glycerol, reflecting the photodestruction of the fluorophore, in buffer solution an unexpected initial increase was found, which may reflect the accumulation of an absorbing photoproduct.

Animals↗
PubMed1982

[Photobleaching quantum yields of visual pigments in the "fauve de Bourgogne" and "albino" rabbit (author's transl)].

Photobleaching quantum yields of visual pigments extracted from retinas of the "fauve de bourgogne" and "albino" rabbit were measured in monochromatic light. For the "fauve de Bourgogne" rabbit, the photobleaching quantum yield was to be 0.65 at different excitation wavelengths (546, 491.6 and 436 nm); at shorter wavelengths, this quantum yield was lower ie. 0.36 at 405 nm. This wavelength effect has been observed on visual pigments extracted from bees retina. In contrast, in the "albino" rabbit, the photobleaching quantum yield was constant and equal to 0.31 whatever the excitation wavelength between 546 and 405 mn.

Animals↗
PubMed2006

MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching.

True confocal microscopy requires point-shaped illumination and detection. To generate an image, a diffraction limited spot is moved over the sample. Single spot scanning has suffered in the past from low image rates; a solution is the employment of very fast scanning devices (resonant scanners) for x-movement. In the process of introducing resonant scanning devices, it was found that both signal yield is improved and bleaching is decreased-in contrary to the assumed performance. This article will show by a simple and well understood model a straightforward explanation for the potential increase of signal yield and decrease in photobleaching. The time that is ruling the dose-rate effects is the effective time; a fluorochrome is illuminated. This time depends on the diameter of the spot that is moved over the sample and the speed at which the spot moves. In essence, the scan process causes a pulsed illumination of the fluorochromes. Various schemes of pulsed illumination are simulated with a fluorescence model. The model includes a dark state, where fluorochromes will exit the fluorescence process and slowly decay back into the ground state. Upon splitting a single dose into two pulses separated by a dark time-reflecting an increased scan speed-the amount of fluorescence emission is increased and bleaching is reduced. These results show a potential increase of fluorescence and a lower photobleaching upon higher scan speed. As illumination during the bleach-phase in a FRAP-experiment is similar to a light pulse, the findings also suggest to critically consider the very beginning of fluorescence recovery in terms of triplet relaxation process that potentially could falsify the measurements.

Fluorescence↗
PubMed2004

Dynamics of proteins in Golgi membranes: comparisons between mammalian and plant cells highlighted by photobleaching techniques.

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.

Animals↗
PubMed2005

Histone dynamics in living cells revealed by photobleaching.

The latest development of imaging technology and fluorescent proteins has enabled us to visualize the dynamics of chromatin proteins in living cells. Particularly, photobleaching techniques like fluorescence recovery after photobleaching (FRAP) revealed the mobility of many nuclear proteins including histones. Although most nucleosomal histones are maintained over cell generations to maintain epigenetic marks on their tails, some exhibit dynamic exchange. In general, histone H3-H4 tetramers stably bind to DNA once assembled during DNA replication; in contrast, the H2A-H2B dimers exchange slowly in euchromatin and are evicted during transcription. Recent data further indicate that different histone variants have different localization and kinetics. The replacement H3 variant, H3.3, is incorporated into transcriptionally active chromatin independently of DNA replication, and the centromeric variant, CENP-A, appears to assemble into nucleosomes in centromeres during G2 phase by replacing canonical H3. Different behaviors of H2A variants are also demonstrated. Importantly, the mobility of histones, and other nuclear proteins, is altered in response to changes in cellular physiology and various stimuli. Whereas we know little about how these dynamics are regulated, distinct complexes that mediate assembly and exchange of specific variants have been isolated, thus future analyses will reveal the molecular mechanisms underlying the phenomena in living cells.

Animals↗
PubMed2005

Three-photon induced photobleaching in a three-dimensional memory material.

The photobleaching properties of a 3D memory material based on pyrylium salts are studied. It is shown that under ultrashort pulsed irradiation photobleaching occurs through two diverse mechanisms. One is major and causes fast bleaching, whereas the other is minor and causes slow bleaching. Furthermore, it is proved that both mechanisms are three- or more-photon processes. This fact gives the capability of significantly increasing data storage density.

Computers, Molecular↗
PubMed2004

In vivo measurement of brain extracellular space diffusion by cortical surface photobleaching.

Molecular diffusion in the brain extracellular space (ECS) is an important determinant of neural function. We developed a brain surface photobleaching method to measure the diffusion of fluorescently labeled macromolecules in the ECS of the cerebral cortex. The ECS in mouse brain was labeled by exposure of the intact dura to fluorescein-dextrans (M(r) 4, 70, and 500 kDa). Fluorescein-dextran diffusion, detected by fluorescence recovery after laser-induced cortical photobleaching using confocal optics, was slowed approximately threefold in the brain ECS relative to solution. Cytotoxic brain edema (produced by water intoxication) or seizure activity (produced by convulsants) slowed diffusion by >10-fold and created dead-space microdomains in which free diffusion was prevented. The hindrance to diffusion was greater for the larger fluorescein-dextrans. Interestingly, slowed ECS diffusion preceded electroencephalographic seizure activity. In contrast to the slowed diffusion produced by brain edema and seizure activity, diffusion in the ECS was faster in mice lacking aquaporin-4 (AQP4), an astroglial water channel that facilitates fluid movement between cells and the ECS. Our results establish a minimally invasive method to quantify diffusion in the brain ECS in vivo, revealing stimulus-induced changes in molecular diffusion in the ECS with unprecedented spatial and temporal resolution. The in vivo mouse data provide evidence for: (1) dead-space ECS microdomains after brain swelling; (2) slowed molecular diffusion in the ECS as an early predictor of impending seizure activity; and (3) a novel role for AQP4 as a regulator of brain ECS.

Animals↗
PubMed2005

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

Biochemistry↗
PubMed2005

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Algorithms↗
PubMed2004

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells.

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

Animals↗
PubMed2004

Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a continuous wave laser: study of oxygen consumption and photobleaching.

Oxygen consumption at the targeted site has a significant effect on dosimetry in photodynamic therapy (PDT). However, oxygen consumption in PDT using a pulsed laser as a light source has not been clarified. We therefore investigated the dependence of cytotoxicity on the oxygen consumption and the photosensitizer photobleaching of PDT using a pulsed laser by comparing with that using a continuous wave (CW) laser. Mouse renal carcinoma cells (Renca) were incubated with a second-generation photosensitizer, PAD-S31. The cells were then irradiated with either a 670-nm nanosecond pulsed light from the 3rd harmonics of a Nd:YAG laser-pumped optical parametric oscillator with a peak fluence rate of approximately 1 MW/cm(2) at 30 Hz or a 670-nm CW diode laser with a total light dose of 40 J/cm(2). Regardless of laser source, cytotoxic effects exhibited cumulative dose responses to the photosensitizer ranging from 12 to 96 microg/ml. However, cytotoxic effect of PDT using the pulsed light was significantly less than that using the CW light with the photosensitizer concentrations of 24 and 48 microg/ml under identical fluence rates. During PDT, the cells exposed to the pulsed light consumed oxygen more slowly, resulting in a lower amount of oxygen consumption when compared with PDT using CW light. In accordance with oxygen consumption, the pulsed light induced significantly less photobleaching of the photosensitizer than the CW light did. These results indicate that the efficiency of PDT using pulsed light is less when compared with CW light, probably being related to suppressed oxygen consumption during the pulsed light irradiation.

Animals↗
PubMed2006

Application of photobleaching for measuring diffusion of prion proteins in cytosol of yeast cells.

Measurement of fluorescence recovery after photobleaching (FRAP) is a non-invasive technique for studying protein dynamics in real time in living cells. FRAP studies are carried out on proteins tagged with green fluorescent protein (GFP) or one of its spectral variants. Illumination with high intensity laser light irreversibly bleaches the GFP fluorescence but has no effect on protein function. By photobleaching a limited region of the cytoplasm, the rate of fluorescence recovery provides a measure of the rate of protein diffusion. A detailed description of the FRAP technique is given, including its application to measuring the mobility of GFP-tagged Sup35p in [psi(-)] and [PSI(+)] cells.

Algorithms↗
PubMed2005

Single molecule blinking and photobleaching separated by wide-field fluorescence microscopy.

Single molecule fluorescence detection of Atto590 in poly(vinyl alcohol) was achieved by using a wide-field epifluorescence microscope with CCD-camera detection. Image sequences are obtained from which the time traces of the detected molecules are built. We find a distinctive difference between the time evolution of the fluorescence originating from the molecules detected in the first image of the sequence compared to the time evolution of the fluorescence of the molecules detected in each image of the sequence. Atto590 shows very long blinking times and photobleaching and photoblinking that are both quadratically dependent on the irradiation power density. Our approach allows kinetic separation of photobleaching from blinking. The possibility of choosing different ensembles of molecules is demonstrated and taken advantage of for this aim. Initially dark molecules or low emitting ones that might be overlooked are important to describe the complete ensemble behavior.

Fluorescent Dyes↗
PubMed2005

Lipase surface diffusion studied by fluorescence recovery after photobleaching.

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.

Adsorption↗
PubMed2006

Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching.

It has not been possible to measure diffusion deep in solid tissues such as tumors because of the limited light penetration of conventional optical techniques. Here we report a microfiberoptic epifluorescence photobleaching (MFEP) method in which photobleaching is done by laser epi-illumination through a multimode fiberoptic whose micron-sized tip can be introduced deep into tissues. We applied MFEP to measure the diffusion of fluorescent macromolecules in tumors in living mice, at depths well beyond those accessible by surface optical measurements. Macromolecule diffusion was slowed about twofold within 200 microm of the surface of a solid tumor, but was slowed greater than tenfold beyond 500 microm. Our results reveal a remarkable and previously unrecognized slowing of diffusion deep in tumors, which correlated with the differing tissue architectures of tumor periphery versus core, and with altered tumor vasculature produced by aquaporin-1 deletion. MFEP should have wide applications for measuring diffusion in organs, solid tumors and other light-inaccessible tissue masses.

Animals↗