Fluorescence microscope

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. 

Fluorescence Microscope. A useful light microscope utilizes UV light to induce fluorescence in microscopic samples (40). Because fluorescence is often the result of trace components in a given sample rather than intrinsic fluorescence of the principal component, it is useful in the crime laboratory for the comparison of particles and fibers from suspect and crime scene. Particles of the same substance from different sources almost certainly show a different group of trace elements. It is also very useful in biology where fluorescent compounds can be absorbed on (and therefore locate and identify) components of a tissue section. 

Somatostatin. Figure 1 Somatostatin-like im mu noreactivity in neurons of the periventricular hypothalamic nucleus of the rat. Coronal brain cryostat sections have been processed for im mu nohistochemistry and sequentially incubated with a primary monoclonal mouse anti human somatostatin antibody and secondary antimouse antibody conjugated with the fluorescence-dye Cy-3. Images have been taken with a Zeiss Axioplan fluorescence microscope. Scale bar, 100 pM. 

Another morphological assay of apoptosis is done with acridine orange, a nuclear staining that reveals chromatin condensation under light and fluorescent microscope. 

One of the early events of the apoptotic process involves the translocation of phosphatidylserine on the surface of cell membranes annexin V binding and propidium iodide uptake reveals various cellular states. After treatment with organotin(IV) compounds the cells could be categorized into populations vital cells (annexin V /P ), early apoptotic cells (annexin V /P ), late apoptotic cells (annexin V /P ), and necrotic cells (annexin V /P" ). Cells are observed with a fluorescence microscope and it is possible to observe translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer one and to see a green stain for annexin V FLUOS bound to PS, and a red stain for propidium iodide. 

Fig. 36—Photograph of nano-particles, (a) SEM photograph, (b) photograph under fluorescence microscope.

Tishler, P. V. Lamborot-Manzur, M. and Atkins, L. "Polymorphism of the Human Y Chromosome Fluorescence Microscopic Studies on the Sites of Morphological Variation". 

Fig. 8.1 (continued) nucleus, (k, 1) Hi hippocampus, (m, n) So supraoptic nucleus, (o, p) Arc hypothalamic arcuate nucleus, (q, r) PH posterior hypothalamic area, (s, t) SN Substantia nigra. AU sections were observed with an Olympus fluorescent microscope (BX 61). Scale bars = 50 mm. (Unpubhshed data)... 

Trimethylaminodiphenylhexatriene chloride (TMADPH Fig. 7.45) is a fluorescent quaternary ammonium molecule that appears to permeate cell membranes [595]. TMADPH fluoresces only when it is in the bilayer, and not when it is dissolved in water. Therefore, its location in cells can be readily followed with an imaging fluorescence microscope. One second after TMADPH is added to the extracellular solution bathing HeLa cell types, the charged molecule fully equilibrates between the external buffer and the extracellular (outer) leaflet bilayer. Washing the cells for one minute removes >95% of the TMADPH from the outer leaflet. If the cells are equilibrated with TMADPH for 10 min at 37°C, followed by a one-minute wash that removed the TMADPH from the outer leaflet, the fluorescent molecule is... 

This substance penetrates into the cell where it is hydrolysed to fluorescein through the action of the enzyme s esterases. The quantity of the fluorescent quinoid form then depends on the local value of pH in the same way as in the case of phenolphthalein. The intensity of the fluorescein radiation is measured with a fluorescence microscope and then processed to a digital image which is the basis of a map of pH distribution in the cell (Fig. 1.13). 

Novel fluorescent anionic surfactants of the types 11.33 and 11.34, where R represents alkyl groups of various lengths, have been applied to wool in order to study their distribution and effects on the physical and chemical properties of the fibre. Sections of the treated fibres were examined under a fluorescence microscope. The intercellular and cell remnant regions appeared to be the preferred locations of the adsorbed surfactants, but the distribution pattern was dependent on the length of the R chain of the surfactant and the conditions of application to wool [52]. 

Fluorescence studies and the binding interaction of Quartz/APES/RB with single- and double-stranded oligonucleotides (ssDNA and dsDNA) in Tris-HCl buffer solution of pH 7.4 were carried out. Quartz/APES/RB exhibited emission at 576 nm, whereas Quartz/APES without BB where nonfluorescent, suggesting that RB successfully assembled on the surface of quartz wafers. By comparison with Aem of 5 x 10 5 M RB solution, which was 588 nm, a hypsochromic shift was found. Considering the fluorescence microscopical image of Quartz/APES/RB, it... 

Xia, Z. and Liu, Y. (2001). Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395-402. 

FLIM systems can be purchased as an add-on for a standard fluorescence microscope. Such a system will consist of a CCD camera coupled to a modulatable image intensifier, an LED light source, and driver electronics. This system will modulate the LED and image intensifier while shifting the phase between them as it takes a series of images (Fig. 2.1). 

In this chapter, it was shown that filterFRET is an easy, intuitive and quantitative alternative to record sensitized emission and FRET efficiency. The major advantages of filterFRET over donor-based FRET detection methods (FLIM) are that it can be carried out with standard wide-held or confocal fluorescence microscopes that are available in most laboratories, and that it yields additional data on the acceptor population. FilterFRET is also fast, requiring just two confocal scans (if need be on a line-by-line basis) which minimizes the risk of artifacts due to, for example, organelle movement in living cells, and acquisition can be optimized for each channel independently. However, quantitative... 

Analyze the protoplasts under a fluorescence microscope suitable for FRET measurements (e.g., Leica SP2/SP5 or Zeiss LSM 510 Meta CLSM). 

Fig. 1 Real-time tracking of cell adhesion [42]. (a) Components of a total internal reflection fluorescent microscope (TIRFM). (b) The cell adhesion process (7) a cell approaches the surface, (2) the cell lands, (3) the cell attaches, and (4) the cell spreads out on the surface. The evanescent field was generated by total internal reflection of a laser beam at the glass-water interface. Cells with fluorescently labeled membranes (dashed lines) were plated on SAMs. Cell membranes within the evanescent field (solid line) were observed by TIRFM. Corresponding TIRFM images are shown below...

Fig. 12 CNTs act as a vector for drug delivery into living cells. After incubation of HeLa cells with AlexaFluor594-labeled SWNTs for 12 h at 37 °C, living cells were observed under confocal fluorescence microscope for a CNT uptake study, (a) Images show dual confocal detection of AlexaFluor594-SWNT (red) internalized into cells with the membrane stained by AlexaFluor488 (green), (b) Series of images of different z-focal scanning planes down through cells. (Adapted from [61])...

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