Fluorescence microscopy sample preparation

Overview. Microscopy Techniques Light Microscopy Sample Preparation for Light Microscopy X-Ray Microscopy. Sample Handling Comminution of Samples. Sampling Theory Practice. Sulfur. X-Ray Fluorescence and Emission Energy Dispersive X-Ray Fluorescence. 

Electron Beam Techniques. One of the most powerful tools in VLSI technology is the scanning electron microscope (sem) (see Microscopy). A sem is typically used in three modes secondary electron detection, back-scattered electron detection, and x-ray fluorescence (xrf). AH three techniques can be used for nondestmctive analysis of a VLSI wafer, where the sample does not have to be destroyed for sample preparation or by analysis, if the sem is equipped to accept large wafer-sized samples and the electron beam is used at low (ca 1 keV) energy to preserve the functional integrity of the circuitry. Samples that do not diffuse the charge produced by the electron beam, such as insulators, require special sample preparation. 

In vitro staining is used to colour cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone can provide. Combined with specific protocols for fixation, sample preparation and investigative methods such as fluorescence microscopy, these standard staining techniques are used as consistent, repeatable diagnostic tools. 

Perhaps one of the most useful aspects of fluorescence microscopy is the ability to determine rapidly the relative spatial localizations of two components within the cell. For poorly characterized macromolecules, this often provides important clues al an early stage of the investigation about functional complexes that can be studied in greater detail with other methods (e.g., biochemistry or genetics). Here, one trades the relatively low resolution of the fluorescence light microscope for the ability to screen rapidly a large number of samples because of the rapid sample preparation and data collection times. ... 

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. 

Filamentous F-actin gels show viscoelastic properties with an average elastic shear modulus in the range of 20-420 Pa [78]. The modulus depends strongly on the length of the filaments and the history of sample preparation, such as the mechanical disruption of actin nanofibrils prior to or during the deformation. Fluorescence microscopy experiments confirmed that applying small shear stresses to F-actin can... 

Both nitric acid digestion and enzyme digestion were tested with liver and lung tissue as well as with cultured cells. Tissue processing with a mixture ofi protease enzymes is preferred because it is applicable to a wide range of particle compositions. Samples were visualized via fluorescence microscopy and transmission electron microscopy to validate the SdFFF results. We describe in detail the tissue preparation procedures and discuss method sensitivity compared to reported levels of nanoparticles in vivo. 

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