Fluorescence fluctuation spectroscopy: a coming of age story.
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Algorithms↗
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Results for “Microscopy, Fluorescence, Multiphoton”
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Photon moment analysis in cells in the presence of photo-bleaching.
The photon counting histogram (PCH) analysis of the fluorescence fluctuations provides the molecular brightness (epsilon) and the average number of fluorophores (N) in an open observation volume. PCH, which is based on the analysis of the whole of the photon counting histogram, has been recently improved by taking into account the detector dead time effect, which is relevant at high fluorescence rates. We investigate here the possibility of quantitatively applying the PCH analysis in the simplified form of photon moment analysis, in which only the first two moments of the photon counting histogram are computed. We have applied this analysis to low fluorescence signals from living cells in the presence of cell micro-movements and molecular photo-bleaching and describe a simple algorithm for its routine application. The algorithm has been tested on Saccharomyces Cerevisiae (yeast) cells labeled with Dimethyl-pepep and Rhodamine 6G, and Chinese Hamster Ovary (CHO) cells stably expressing the regulatory subunit (RII) of protein kinase A fused to the cyan-emitting variant of GFP (CFP). Our statistical analysis allows us to estimate the local concentrations and the brightness of the fluorophores in different cellular compartments (nucleus, membrane, and cytoplasm) despite the occurrence of microscopic cell movements and significant photo-bleaching.
Algorithms↗
Two-point separation in far-field super-resolution fluorescence microscopy based on two-color fluorescence dip spectroscopy, Part I: Experimental evaluation.
The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.
Fluorescent Dyes↗
Limitations arising from two-photon absorption of solvent in pulsed-laser thermal lens detection: determination of the two-photon absorption coefficient of ethanol at 266 nm.
Two-photon absorption of the solvent under pulsed-laser excitation at 266 nm produces a high background thermal lens signal interfering with the analyte signal. Discrimination of both solvent and analyte signals along with calibration of the photothermal response has allowed the determination of the two-photon absorption coefficient of ethanol. The obtained value, 3.0x10(-10) cm W-1, is close to the literature values obtained from transmittance measurements using picosecond or femtosecond laser pulses.
Absorption↗
Modified dual lifetime referencing method for simultaneous optical determination and sensing of two analytes.
Simultaneous fluorometric sensing of two analytes becomes possible using a modified dual lifetime referencing (m-DLR) method. In this scheme, two luminescent indicators are needed that have overlapping absorption and emission spectra but largely different decay times. They are excited by a single light source, and both emissions are measured simultaneously. In the frequency domain m-DLR method, the phase of the short-lived fluorescence of a first indicator is referenced against that of the long-lived luminescence of the second indicator. The analytical information is obtained by measurement of the phase shifts at two modulation frequencies. The method is demonstrated to work for the case of dually sensing oxygen and carbon dioxide. It benefits from simple instrumentation and optical setup. The approach is perceived to be of wide applicability. Examples include (1) analysis of two luminescent analytes, (2) analytical determinations that make use of two probes, and (3) sensing of two species such as carbon dioxide and oxygen (as demonstrated here), or oxygen and chlorophyll, provided the luminophores meet the condition of having largely different decay times and overlapping absorption and emission spectra.
Carbon Dioxide↗
Spectrally resolved frequency domain analysis of multi-fluorophore systems undergoing energy transfer.
Complex systems of fluorophores undergoing energy transfer can exhibit a variety of anomalous lifetime behavior when probed with frequency domain methods. When presented in traditional apparent lifetime format the data from such systems can exhibit "nodal" behavior in which the computed lifetime approaches +/-infinity. The location of the nodes is system and frequency dependent. In addition, simpler systems, not undergoing energy transfer, show ill behavior in the region of zero lifetime (tau(m)) and long lifetime (tau(pi)) due to noise in typical measurements. Here, we systematically investigate systems of multiple fluorophores with and without energy transfer to provide insight into frequency domain investigations of complex systems of fluorophores. The results of simulations are compared to data collected from a multi-fluorophore system designed to exhibit fluorescence resonance energy transfer (FRET) using imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM). The results are applicable to both cuvette and imaging arrangements.
Algorithms↗
Diverse modes of axon elaboration in the developing neocortex.
The development of axonal arbors is a critical step in the establishment of precise neural circuits, but relatively little is known about the mechanisms of axonal elaboration in the neocortex. We used in vivo two-photon time-lapse microscopy to image axons in the neocortex of green fluorescent protein-transgenic mice over the first 3 wk of postnatal development. This period spans the elaboration of thalamocortical (TC) and Cajal-Retzius (CR) axons and cortical synaptogenesis. Layer 1 collaterals of TC and CR axons were imaged repeatedly over time scales ranging from minutes up to days, and their growth and pruning were analyzed. The structure and dynamics of TC and CR axons differed profoundly. Branches of TC axons terminated in small, bulbous growth cones, while CR axon branch tips had large growth cones with numerous long filopodia. TC axons grew rapidly in straight paths, with frequent interstitial branch additions, while CR axons grew more slowly along tortuous paths. For both types of axon, new branches appeared at interstitial sites along the axon shaft and did not involve growth cone splitting. Pruning occurred via retraction of small axon branches (tens of microns, at both CR and TC axons) or degeneration of large portions of the arbor (hundreds of microns, for TC axons only). The balance between growth and retraction favored overall growth, but only by a slight margin. Given the identical layer 1 territory upon which CR and TC axons grow, the differences in their structure and dynamics likely reflect distinct intrinsic growth programs for axons of long projection neurons versus local interneurons.
Animals↗
Fluorescence correlation spectroscopy: a new tool for quantification of molecular interactions.
Fluorescence correlation spectroscopy (FCS) provides a powerful method to measure molecular dynamics and interactions in a wide variety of experimental systems and environments. In this article we focus on the use of FCS methods to quantify molecular interactions, including the use of diffusion analysis and molecular counting. Both autocorrelation and cross-correlation FCS measurements are discussed.
Microscopy, Fluorescence, Multiphoton↗
Visualization of the three-dimensional organization of hypoxia-inducible factor-1 alpha and interacting cofactors in subnuclear structures.
Cells need oxygen (O2) to meet their metabolic demands. Highly efficient systems of O2-sensing have evolved to initiate responses enabling cells to adapt their metabolism to reduced O2 availability. Of central importance is the activation of hypoxia-inducible factor-1 (HIF-1), a transcription factor complex that controls the expression of genes the products of which regulate glucose uptake and metabolism, vasotonus and angiogenesis, oxygen capacity of the blood as well as cell growth and death. Activation of HIF-1 requires the accumulation and nuclear translocation of the HIF-1alpha subunit, its dimerization with HIF-1beta and the binding of co-activator proteins such as p300. In this study we investigated the three-dimensional (3D) distribution of HIF-1alpha within the nucleus and assigned its localization to known nuclear compartments. Using two-photon microscopy we determined the colocalization of HIF-1alpha and -beta subunits within nuclear domains as well as overlaps between HIF-1alpha and p300. Our data provide information on the nuclear distribution of HIF-1alpha with respect to subnuclear domains that could serve as specific locations for hypoxia-induced gene expression.
Cell Line, Tumor↗
Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators.
Genetically encoded Ca2+ indicators (GECIs) based on fluorescent proteins (XFPs) and Ca2+-binding proteins [like calmodulin (CaM)] have great potential for the study of subcellular Ca2+ signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca2+ concentration ([Ca2+]) is complicated by the nonlinear interactions between Ca2+ binding and GECI fluorescence. We have characterized GECIs in pyramidal neurons in cultured hippocampal brain slices, focusing on indicators based on circularly permuted XFPs [GCaMP (Nakai et al., 2001), Camgaroo2 (Griesbeck et al., 2001), and Inverse Pericam (Nagai et al., 2001)]. Measurements of fluorescence changes evoked by trains of action potentials revealed that GECIs have little sensitivity at low action potential frequencies compared with synthetic [Ca2+] indicators with similar affinities for Ca2+. The sensitivity of GECIs improved for high-frequency trains of action potentials, indicating that GECIs are supralinear indicators of neural activity. Simultaneous measurement of GECI fluorescence and [Ca2+] revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively.
Action Potentials↗
Stereotyped odor-evoked activity in the mushroom body of Drosophila revealed by green fluorescent protein-based Ca2+ imaging.
To study the representation of olfactory information in higher brain centers, we expressed a green fluorescent protein-based Ca2+ sensor, G-CaMP, in the Drosophila mushroom body (MB). Using two-photon microscopy, we imaged odor-evoked G-CaMP fluorescence transients in MB neurons [Kenyon cells (KCs)] with single-cell resolution. Odors produced large fluorescence transients in a subset of KC somata and in restricted regions of the calyx, the neuropil of the MB. In different KCs, odor-evoked fluorescence transients showed diverse changes with odor concentration: in some KCs, fluorescence transients were evoked by an odor at concentrations spanning several orders of magnitude, whereas in others only at a narrow concentration range. Different odors produced fluorescence transients in different subsets of KCs. The spatial distributions of KCs showing fluorescence transients evoked by a given odor were similar across individuals. For some odors, individual KCs with fluorescence transients evoked by a particular odor could be found in similar locations in different flies with spatial precisions on the order of the size of KC somata. These results indicate that odor-evoked activity can have remarkable spatial specificity in the MB.
Animals↗
Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM.
We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.
Adenoviruses, Human↗
Na/K pump-induced [Na](i) gradients in rat ventricular myocytes measured with two-photon microscopy.
Via the Na/Ca and Na/H exchange, intracellular Na concentration ([Na](i)) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na](i) might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na](i) in rat ventricular myocytes. In vivo calibration yielded an apparent K(d) of 27 +/- 2 mM Na. Similar resting [Na](i) was found using two-photon or single-photon ratiometric measurements with SBFI (10.8 +/- 0.7 vs. 11.1 +/- 0.7 mM). To assess longitudinal [Na](i) gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na](i) at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na](i) rise was smaller at upstream sites. This resulted in sustained [Na](i) gradients (up to approximately 17 mM/120 microm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10-12 microm(2)/s, significantly lower than in aqueous solutions.
Animals↗
Single-molecule three-color FRET.
Fluorescence resonance energy transfer (FRET) measured at the single-molecule level can reveal conformational changes of biomolecules and intermolecular interactions in physiologically relevant conditions. Thus far single-molecule FRET has been measured only between two fluorophores. However, for many complex systems, the ability to observe changes in more than one distance is desired and FRET measured between three spectrally distinct fluorophores can provide a more complete picture. We have extended the single-molecule FRET technique to three colors, using the DNA four-way (Holliday) junction as a model system that undergoes two-state conformational fluctuations. By labeling three arms of the junction with Cy3 (donor), Cy5 (acceptor 1), and Cy5.5 (acceptor 2), distance changes between the donor and acceptor 1, and between the donor and acceptor 2, can be measured simultaneously. Thus we are able to show that the acceptor 1 arm moves away from the donor arm at the same time as the acceptor 2 arm approaches the donor arm, and vice versa, marking the first example of observing correlated movements of two different segments of a single molecule. Our data further suggest that Holliday junction does not spend measurable time with any of the helices unstacked, and that the parallel conformations are not populated to a detectable degree.
Algorithms↗
Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy.
The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.
Animals↗
Dual-color photon-counting histogram.
We report on the development of dual-color photon-counting histogram (PCH) analysis. Dual-color PCH is an extension of regular PCH and considers the photon counts received in two detection channels instead of one. Because each detection channel records a different color, dual-color PCH distinguishes fluorescent species not only by differences in their brightness, but also according to their color. The additional discrimination by color increases the sensitivity of PCH in resolving a mixture of species considerably. Most dual-color fluorescence fluctuation experiments are performed on fluorophores with overlapping emission spectra. This overlap results in spectral cross talk between the detector channels, which reduces resolvability. Here, we demonstrate that dual-color PCH is able to resolve binary dye mixtures in the presence of cross talk from a single measurement without any additional information about the sample. We discuss the effect of sampling time on the fit parameters of dual-color PCH. Differences between dual-color fluorescence correlation spectroscopy and dual-color PCH will also be addressed. We quantitatively resolve a mixture of the two fluorescent proteins CFP and YFP, which is challenging because of the strong spectral overlap of their emission spectra. Dichroic mirrors are needed to direct the light into the two detection channels. We quantify the influence of these filters on dual-color PCH analysis and determine the optimal transition wavelength of the dichroic mirror for the CFP-YFP pair.
Algorithms↗
A two-photon FRAP analysis of the cytoskeleton dynamics in the microvilli of intestinal cells.
The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.
Actins↗
Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy.
Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.
Biophysics↗