Basic fluorescence microscopy

It had been found that if bacteria are stained with acridine orange and examined under fluorescent microscopy, viable, as dishnct from dead, cells fluoresce with an orange-led hue. This basic observation has been adapted to an ingenious method of determining bacterial content and may be completed within 1 hour. 

K.J. Stine and C.M. Knobler, Fluorescence Microscopy A Tool for Studying the Physical Chemistry of Interfaces, Ultramicroscopy 47 (1992) 23. (Review short introduction to fluorescence and fluorophores basic instrumentation for fluorescence microscopy and extensions to study dynamics and resonance energy transfer, confocal scanning microscopy results obtained with Langmuir monolayers.)... 

At the most basic level, the data can be visualized (rendered) as an image volume viewed at varying angles and manually compressed or stretched to fit. Alternatively, a more mathematical approach can be taken if the optical aberrations can be measured. In 3D fluorescence microscopy, this measurement is known as the point spread function (psf), and a process of deconvolution can be used to correct any aberrations with a known psf. Once corrected, the data can be rendered for viewing and measurement with confidence. 

Wang, Y.-l. and Taylor, D.L. (1989a). Fluorescence Microscopy of Living Cells in Culture. Part A Fluorescent Analogs, Labeling Cells, and Basic Microscopy. Academic Press, New York. 

Abramowitz, M., Basics and Beyond Series Fluorescence Microscopy, Vol. 4, Olympus America, Inc., Lake Success, New York, 1985. 

Why are chemical stains required for visualizing cells and tissues with the basic light microscope What advantage does fluorescent microscopy provide In comparison to the chemical dyes used to stain specimens for light microscopy What advantages do confocal scanning microscopy and deconvolution microscopy provide in comparison to conventional fluorescence microscopy ... 

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

Fluorescence Microscopy of Living Cells in Culture, Part A Fluorescent Analogs, Labeling Cells, and Basic Microscopy... 

Fluorescence Measurements, Fig. 2 The basic anatomy of a common fluorescence microscopy setup. LS light source, OD optical detector, ExF excitation filter, EmF emission filter, DM dichroic mirror, L lens, EM position of fluorochrome molecules... 

Light microscopy (LM) is regularly used to obtain rapid, inexpensive qualitative and quantitative information in food analysis. The first routine use of LM in food analysis was for the identification of adulteration (e.g., the presence of chicory root in coffee) or contamination (insect, rodent, microbial, and foreign bodies). Bright-field, polarizing, and fluorescent microscopy are the three traditional LM techniques used most frequently in food analysis. The basic instrument is a conventional compound (bright-field) microscope, to which polarizing and fluorescence accessories are easily attached. 

Very interesting work was also reported by Piwnica-Worms and coworkers. They prepared Tat-peptide derivatives like 90 dual-labeled with Tc and fluorescein for scintigraphy and fluorescence microscopy (Scheme 5.45) [218]. Tat-peptide basic regions have been intensely studied because of their ability to translocate across cell membranes, seemingly independent of receptor-mediated endocytosis. The peptides were prepared by SPPS with an N-terminal His ligand, to which Tc(CO)3 or Re(CO)3 is coordinated in solution after cleavage and purification. As an alternative to histidine, diethylenetriamine-pentaacetic acid (DTPA) was also used as an N-terminal metal chelator. The C-terminal cysteine was reacted with fluorescein-5-maleimide already on the resin, thus providing both labels in the same peptide, as shown for 90 in Scheme 5.45. Cellular localization of these... 

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