![]() The techniques to be reviewed encompass the most basic (such as wide-field fluorescence microscopy) to cutting edge techniques like Stimulated Emission Depletion (STED) microscopy. The object of this review is to provide non-expert microscopists a concise description and guide to select techniques that may have the widest appeal and that are, or will soon be, commonly used in most light microscopy core facilities or advanced biological research labs. In this review, knowledge of the basics of fluorescence microscopy (including wide-field microscopy) presented in that paper will be assumed. This review is intended to expand and build-upon the last review of fluorescence microscopy in this series ( Coling and Kachar, 1997) which provided a foundation for understanding fluorescence microscopy and the basics of immuno-labelling. Table 1 outlines how these factors differ between the various commercialized microscopy techniques discussed in this work. The best image is one that can balance these factors to obtain the necessary information while avoiding photobleaching or phototoxic effects. On top of these basic variables other secondary variables also can become important such as the cost of the necessary equipment and the difficulty of the technique.ĭiagram of some of the critical opposing factors in an imaging experiment. Given these constraints, these variables are difficult to balance and require careful attention to detail and systematic empirical testing. This can lead to destruction of the fluorophore and unwanted biological consequences leading to cell death or changes in the physiology of the cells or tissue being illuminated. In many experiments, light levels at the diffraction limited spot (focused by the objective) can be very high. This is bounded by the limits imposed by photo-bleaching and/or photo-toxicity. In a very simple form, the ideal light microscopy experiment can be viewed as optimizing the competing properties of image resolution (in the XY or lateral direction as well as the Z or axial dimension), imaging speed (and/or acquisition time), and the amount of signal collected from the fluorescing sample ( Figure 1). Optimal use of fluorescence microscopy requires a basic understanding of the strengths and weaknesses of the various techniques as well an understanding of the fundamental trade-offs of the variables associated with fluorescent light collection. However, for the biologist inexperienced in light microscopy, matching the best technique to the biological experiment can prove to be difficult. Many of the new advanced techniques are now being commercialized, opening their use to a large fraction of modern biologists. These advances include the wide-spread use of fluorescent proteins (for review see ( Shaner et al., 2005), the myriad number of new fluorophores available (for reviews see ( Eisenstein, 2006 Suzuki et al., 2007), the growth of the utility of the basic confocal microscope, the use of multi-photon microscopy to optically image far deeper into tissues, and the breaking of the diffraction limit for “super-resolution”. Many new techniques have been developed over the last decade which enable more comprehensive exploitation of light for biologic imaging. ![]() This versatility explains why thousands of papers a year are published using variants of fluorescent microscopy techniques. Fluorescence microscopy enables the study of diverse processes including protein location and associations, motility, and other phenomenon such as ion transport and metabolism. ![]() This allows direct visualization of the inner workings of physiological processes at a systems level context in a living cell or tissue. It provides a window into the physiology of living cells at sub-cellular levels of resolution. Fluorescence microscopy is a powerful tool for modern cell and molecular biologists and, in particular, neurobiologists.
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