Light microscopy fluorescence

Fluorescence microscopy is closely allied to transmission (absorption) microscopy in its range of application, but possesses particular advantages (Table 5.17). Because many substances are fluorescent, or can be made so, fluorescence microscopy is widely applicable to all kinds of material. In view of the more complex and expensive instrumentation than conventional transmitted-light microscopy, fluorescence microscopy is usually reserved for those applications in which its high sensitivity is of importance i.e. to examine substances present in low concentrations. Fluorescence microscopy is especially a valuable tool in the biological sciences. 

It is interesting to note the analogy of developments in light microscopy during the last few decades. The confocal microscope as a scaiming beam microscope exceeds by far the nomial fluorescence light microscope in resolution and detection level. Very recent advances in evanescent wave and interference microscopy seem to promise to provide even higher resolution (B1.18). 

Light microscopy allows, in comparison to other microscopic methods, quick, contact-free and non-destmctive access to the stmctures of materials, their surfaces and to dimensions and details of objects in the lateral size range down to about 0.2 pm. A variety of microscopes with different imaging and illumination systems has been constmcted and is conunercially available in order to satisfy special requirements. These include stereo, darkfield, polarization, phase contrast and fluorescence microscopes. 

White, J. G., Amos, W. B. and Fordham, M. (1987). An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light-Microscopy. J. Cell. Biol. 105, 41-8. 

Vukjovic et al.199 recently proposed a simple, fast, sensitive, and low-cost procedure based on solid phase spectrophotometric (SPS) and multicomponent analysis by multiple linear regression (MA) to determine traces of heavy metals in pharmaceuticals. Other spectroscopic techniques employed for high-throughput pharmaceutical analysis include laser-induced breakdown spectroscopy (LIBS),200 201 fluorescence spectroscopy,202 204 diffusive reflectance spectroscopy,205 laser-based nephelometry,206 automated polarized light microscopy,207 and laser diffraction and image analysis.208... 

Microscopic techniques, 70 428 Microscopists, role of, 76 467 Microscopy, 76 464-509, See also Atomic force microscopy (AFM) Electron microscopy Light microscopy Microscopes Scanning electron microscopy (SEM) Transmission electron microscopy (TEM) acronyms related to, 76 506-507 atomic force, 76 499-501 atom probe, 76 503 cathodoluminescence, 76 484 confocal, 76 483-484 electron, 76 487-495 in examining trace evidence, 72 99 field emission, 76 503 field ion, 76 503 fluorescence, 76 483 near-held scanning optical,... 

In contrast to brightfield microscopy, which uses specimen features such as light absorption, fluorescence microscopy is based on the phenomenon in which absorption of light by fluorescent molecules called fluorescent dyes or fluorophores (known also as fluorochromes) is followed by the emission of light at longer... 

Until the past decade, the cytoplasm was widely considered to be structurally unorganized with the main division of labor at the organellar level. Certainly, relatively little was known about the nature of the cyto-skeleton (with the notable exception of the mitotic apparatus and striated muscle), and the dynamics of cytoplasmic behavior were conceptualized vaguely in terms of sol-gel transitions without a sound molecular foundation. Substantial improvements in electron, light, and fluorescence microscopy, as well as the isolation of discrete protein components of the cytoskeleton, have led the way to a much better appreciation of the structural organization of the cytoplasm. Indeed, the lacelike network of thin filaments, intermediate filaments, and microtubules in nonmuscle cells is as familiar today as the organelles identified... 

Shooton, D. (ed.) (1993) Electronic Light Microscopy. The Principles and Practice of Video-Enhanced Contrast, Digital Intensified Fluorescence, and Confocal Scanning Light Microscopy. Wiley-Liss, New York. 

Fluorescent light microscopy distinguishes between the extractable liquids, which fluoresce strongly, and the matrix, which does not. The disruption of weak bonding effected by thermal treatment, which increased the extract yield, was paralleled by changes in fluorescence intensity. The fluorescence spectra of the extracts also reflect the compositional differences between the mobile phase and the solubilised coal network. 

The structure (e.g., number, size, distribution) of fat crystals is difficult to analyze by common microscopy techniques (i.e., electron, polarized light), due to their dense and interconnected microstructure. Images of the internal structures of lipid-based foods can only be obtained by special manipulation of the sample. However, formation of thin sections (polarized light microscopy) or fractured planes (electron microscopy) still typically does not provide adequate resolution of the crystalline phase. Confocal laserscanning microscopy (CLSM), which is based on the detection of fluorescence produced by a dye system when a sample is illuminated with a krypton/argon mixed-gas laser, overcomes these problems. Bulk specimens can be used with CLSM to obtain high-resolution images of lipid crystalline structure in intricate detail. 

Transmission electron microscopy is analogous to light microscopy, with visible light replaced by a beam of electrons produced by a heated metal filament, and glass lenses replaced by electromagnetic coils to focus the beam. An image of the sample is projected onto a fluorescent screen or, for a permanent record, onto film or a CCD detector (Chapter 4, Section ni.C). Alternatively, an image of the sample s diffraction pattern can be projected onto the detectors. 

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