Photobleaching in two-photon excitation microscopy

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.     

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.     

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.     

Polarization of fluorescein fluorescence in single cells.

Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.     

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.     

Photobleaching. A novel fluorescence method for diffusion studies in lipid system.

(1) The fluorescent molecular 12(9-anthroyloxy)-stearic acid dimerises on irradiation with light of 366 nm wavelength. (2) The dimer is nonfluorescent and can be reconverted to the parent compound by irradiation at 254 nm. (3) Kinetic analysis suggests that the dimerisation proceeds by a diffusion-limited second order mechanism in many solvents. (4) Anomalously high rates seen in other systems can be attributed to localised high concentration regions (clusters) of the fluorescent molecule. (5) The analysis has been extended to oriented lipid bilayers and the results suggest that below the gel-liquid crystalline transition temperature the 12(9-anthroyloxy)-stearic acid is excluded by the lipid matrix and forms regions of localised high concentration. (6) In fluid lipid the results suggest an isotropic distribution of the probe. Calculated diffusion coefficients correspond to those found by other techniques.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy.

The decay of evanescent field intensity beyond a dielectric interface depends upon beam incident angle, enabling the 3-d distribution of fluorophores to be deduced from total internal reflection fluorescence microscopy (TIRFM) images obtained at multiple incident angles. Instrumentation was constructed for computer-automated multiple angle-TIRFM (MA-TIRFM) using a right angle F2 glass prism (n(r) 1.632) to create the dielectric interface. A laser beam (488 nm) was attenuated by an acoustooptic modulator and directed onto a specified spot on the prism surface. Beam incident angle was set using three microstepper motors controlling two rotatable mirrors and a rotatable optical flat. TIRFM images were acquired by a cooled CCD camera in approximately 0.5 degree steps for >15 incident angles starting from the critical angle. For cell studies, cells were grown directly on the glass prisms (without refractive index-matching fluid) and positioned in the optical path. Images of the samples were acquired at multiple angles, and corrected for angle-dependent evanescent field intensity using "reference" images acquired with a fluorophore solution replacing the sample. A theory was developed to compute fluorophore z-distribution by inverse Laplace transform of angle-resolved intensity functions. The theory included analysis of multiple layers of different refractive index for cell studies, and the anisotropic emission from fluorophores near a dielectric interface. Instrument performance was validated by mapping the thickness of a film of dihexyloxacarbocyanine in DMSO/water (n(r) 1.463) between the F2 glass prism and a plano-convex silica lens (458 mm radius, n(r) 1.463); the MA-TIRFM map accurately reproduced the lens spherical surface. MA-TIRFM was used to compare with nanometer z-resolution the geometry of cell-substrate contact for BCECF-labeled 3T3 fibroblasts versus MDCK epithelial cells. These studies establish MA-TIRFM for measurement of submicroscopic distances between fluorescent probes and cell membranes.     

A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy.

The aim of this study was to develop a procedure to remove the TO-PRO-3 fluorescent dye from tissue sections and restain with TO-PRO-3, still allowing calculation of DNA content and distribution by confocal laser scanning microscopy (CLSM). This would allow repeated measurements on the same tissue sections and prevents loss of tissue material from valuable clinical samples. Thick sections (14 microm) were cut from a paraffin block of adrenal tissue and stained using TO-PRO-3. Image stacks were acquired by CLSM. Thereafter, three destaining approaches were tested based on incubation, at different temperatures and durations, in the medium that is normally used to dissolve TO-PRO-3. The same areas were imaged again to measure residual fluorescence and were subsequently restained and imaged again. The intensity of the images acquired after initial staining and restaining were compared. A number of 3-D (texture) features computed after segmentation of nuclei were compared as well. The best destaining result was obtained by incubation of sections at 37 degrees C in preheated medium twice for 20 min. On average, the 3-D feature values were comparable with those after initial staining. With the described protocol it is possible to remove TO-PRO-3 fluorescence from tissue sections that can successfully be restained with minimal influence on fluorescence intensity and nuclear chromatin distribution.     

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.     

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.     

Extended resolution fluorescence microscopy.

Fluorescence microscopy is an essential tool of modern biology, but, like all forms of optical imaging, it is subject to physical limits on its resolving power. In recent years, several exciting techniques have been introduced to exceed these limits, including standing wave microscopy, 4Pi confocal microscopy, I5M and structured illumination microscopy. Several such techniques have been definitively demonstrated for the first time during the past year.     

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Summary MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450–500 nm excitation/>510 nm emission) or rhodamine (514 nm excitation/>530 emission) type dyes. A second 5- to 10-fold brighter emission for 450–500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled domes and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 m riboflavin resulted in accumulation in domes of 565±80 m. The transport rate was calculated to be 189±30 pmol/min-cm². Onemm probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10mm nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatable with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.     

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of ±1.5% of full scale in 65 536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 μm when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 · 10^?4 torr^?1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 · 10^?4 torr^?1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficent of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.     

To 5D and beyond: quantitative fluorescence microscopy in the postgenomic era

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.