Fluorescence microscopy

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.     

Fluorescence spectroscopy

Some practical aspects of the use of spectrophotofluorometers as applied to the organic analyses of returned lunar samples and other geological materials have been discussed. Because of the single beam nature of spectrophotofluorometers many instrumental artifacts such as grating anomalies, irregular spectral output of light sources and response characteristics of photodetectors are apparent in sample spectra. In order to avoid some of these instrumental artifacts from influencing sample spectra, the performance characteristics of each optical component have been described and the necessity of instrumental calibration has been emphasized.     

Fluorescence lifetime-resolved imaging

This is a short account of fluorescence lifetime-resolved imaging, in order to acquaint readers who are not experts with the basic methods for measuring lifetime-resolved signals throughout an image. We present the early FLI (fluorescence lifetime imaging) history, review shortly the instrumentation and experimental design, discuss briefly the fundamentals of the measured fluorescence response, and introduce the basic measurement methodologies. We also emphasize the complex nature of the fluorescence response in FLI signals, and introduce certain analysis methods that are appropriate and informative for complex fluorescence decays. The advantages of model independent analyses are discussed and examples given.     

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.     

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex.Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)^1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible³. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions⁴. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions^5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET)⁷. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments⁸. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.Download video file.^{(55M, mov)}     

Fluorescence crossmatch testing

This paper is a short evaluation of the crossmatch testing performed with the fluorescence cytotoxic test to detect HLA compatibility of the platelet concentrates administered. From a total of 1,500 reactions between HLA incompatible donors and recipients mutual agreement was achieved in 96% of the reactions. HLA compatibility between donors and recipients was achieved in 97.31%. Advantages of the fluorescence method were stressed out.     

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.     

Comparative Studies on Photosynthetic Fluorescence Spectra and Fluorescence Kinetics of Bryophytes

Bryophytes are the transitional forms from water habitants to terrestrials, however, there have been only a few works on their photosynthesis. It was the first time to study on photosynthetic fluorescence spectra and fluorescence kinetics of primitive and advanced species comparatively. Both the primitive and advanced ones had the same fluorescence spectra at room temperature, which contained two maximum emissions: F_686-690 from the Photosystem II and F_736-740 from the Photosystem I. And then, there were three maximum emissions in the fluorescence spectra at 77K :F_687-689 and F_697-699 from Photosystem II, and F_723-734 from Photosystem I. The first two maximum emissions were the same for both the primitive and advanced species. According to the third maximum emission the bryophytes under study fell into two categories: The first one possessing the maximum emission around 725 nm, including Ditrichum flexicaule , Didymodon icmadophyllum , Didymodon rigidicaulis, Aloina obliquifolia, Plagiomnium confertidens and Marchantia polymorpha, which were primitive mosses and advanced liverwort. The second one possessing the maximum emission around 732nm, including Thuidium delicatulum , Pylaisia brotheri , Myuroclada maximowiczii , Taxiphyllum taxirameum, Gollania neckerella, Eurohypnum leptothallum, which were advanced mosses, and the primitive one Plagiomnium rostratum. The characteristics of fluorescence spectra implied that the Photosystem II was conservative and Photosystem I was changeable during bryophyte evolution. The primitive mosses possess mainly the PSI core complex (CPI) and then the advanced species contain both CPI and LHC-I. In analysis of photosynthetic fluorescence kinetics, Fv/(Fc+Fv) is a measure of the activity of the Photosystem II; Fv/Fm is dependent on efficiency of primary photoconversion in the Photosystem II; Fm/(Fo+Fv) is related to photosynthetic carbon assimilation; and Fd/Fs is a measure of the potential photosynthetic quantum conversion. The fluorescence kinetics of the bryophyte photosynthesis showed that the Photosystem II activity, the efficieiency of primary photoconversion in Photosystem II, the photosynthetic carbon assimilation and the efficiency of the potential photosynthetic quantum conversion in primitive species, such as Ditrichum flexicaule, Didymodon icmadophyllus, D. rigidicaulis, Plagiomnium rostratum and the liverwort Marchantia polymorpha, were lower than those in the advanced species, Myuroclada maximowiczii, Pylaisia brotheri , Gollania neckerella Taxiphyllum taxirameum , Thuidium delicatulum. However, the primitive Plagiomnium confertidens was of the high activities and efficiencies and the advanced Eurohypnum leptothallum was of low ones. It seemed that P. confertidens and E. leptothallum were an intermediatefrom the primitive to the advanced.     

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

双分子荧光互补技术

双分子荧光互补（bimolecular fluorescence complementation, BiFC）是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术.该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新形成能具有活性的荧光蛋白.BiFC方法简单直观,既可以检测蛋白之间的相互作用,也可以定位相互作用蛋白质的位点.多色BiFC系统共用或与荧光共振能量转移（FRET）技术联用,还可以检测细胞内多个蛋白质的相互作用.