Fluorescence microscopy specimen preparation

See also Forensic Sciences Gunshot Residues Hair. Microscopy Applications Environmental. Microscopy Techniques Electron Microscopy Specimen Preparation for Electron Microscopy. X-Ray Fluorescence and Emission Energy Dispersive X-Ray Fluorescence. 

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. 

See also Fluorescence Overview. Microscopy Overview. Microscopy Techniques Specimen Preparation for Eiectron Microscopy. Optical Spectroscopy Refractometry and Refiectometry. Sampling Theory. 

Chandler, Douglas E., and Robert W. Roberson. Bio-imaging Current Concepts in Light and Electron Microscopy. Sudbury, Mass. Jones and Bartlett, 2009. Begins with the history of electron microscopy and examines topics such as specimen preparation, transmission and scanning electron microscopes, and fluorescence microscopy. 

For optical microscopy, hairy root specimens were fixed with 3 % formaldehyde in 50 mol m phosphate buffer (pH 6.8) for 1 h at ambient temperature. Cross-sections of the specimen were prepared using a sliding microtome equipped with a specimen-freezing stage. The sections were examined with microscopes under conventional or fluorescent light (excitation wavelengths of 520-550 nm and emission wavelength of >580 nm). 

Fig. 24.2. Confocal microscopy for the evaiuation of sampie preparations, (a) A f 0 pim mouse tissue section was fixed with -20°C acetone and imaged with a 63x oii objective by confocai microscopy (excitation at 543 nm and detection 570-590 nm). The YFP fiuorescence is observed in the ceii bodies of geneticaiiy iabeied neurons, (b) A second 10 ijim tissue section was prepared by matrix soiution fixation with 25 mg/mi sinapinic acid in -20°C acetone (YFP fluorescence is sensitive to alcohols), and the matrix iayer imaged by excitation with the 488 nm laser line and detection between 490 and 508 nm through 5.5 nm optical sectioning (10 sections), (c) The same specimen was imaged for YFP fluorescence by optical sectioning of the prepared specimen through 9.8 nfn (10 sections) and the fluorescent cell bodies observed. The overlay of total matrix and tissue fluorescence is presented in (d).

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