Epi illumination

Epi-illumination Subcellular imaging structures Freeze fracture Preparation of cellular ultrastructures in frozen-hydrated and living state for electron microscopy macromolecular organization of bilayer membranes... 

The fluorescence spectra of the monomeric cyanine nucleic acid stains family (PO-PRO-1, BO-PRO-1 and YO-PRO-1) introduced by Molecular Probes (http // probes.invitrogen.com) cover the entire visible wavelength range. These dyes may also be used with ultraviolet trans- or epi-illuminator excitation sources. The monomeric cyanine nucleic acid stains exhibit large degrees of fluorescence enhancement upon binding to DNA (or RNA) up to 1,800-fold. Consequently,... 

Figure 7.6 shows the intensity / that would be detected in the glass at a particular supercritical angle, given an excitation intensity that is not a function of z (e.g., epi-illumination rather than TIR). Only the results for perpendicular dipoles are shown (so that averaging over azimuthal is unnecessary). The result for parallel dipoles is qualitatively similar except that the metal film case would be very much reduced in overall intensity. 

In a microscope, standard polarized epi-illumination cannot distinguish order from disorder in the polar direction (defined as the optical axis) because epi-illumination is polarized transverse to the optical axis and observation is along the optical axis at 180°. However, microscope TIR illumination can be partially polarized in the optical axis direction (the z-direction of Section 7.2) and can thereby detect order in the polar angle direction. Timbs and Thompson(102) used this feature to confirm that the popular lipid probe 3,3 -dioctadecylindocarbocyanine (dil) resides in a supported lipid monolayer with its dipoles parallel to the membrane surface, but labeled antibodies bound to the membrane exhibit totally random orientations. 

One TIRF study found that some membrane proteins behave just oppositely to AChR they avoid the cell/substrate contact regions/ 1 When endothelial cells are grown on a bare glass surface or are brought into suspension, a specific membrane protein marked with antibodies appears all over the cell surface, as evidenced by epi-illumination and TIRF. However, when the cells are grown on (or returned to) a surface coated with their own extracellular matrix material, the protein disappears from the basal (cell/substrate-contacting) side of the cells. 

TIRF is an experimentally simple technique for selective excitation of fluorophores on or near a surface. It can be set up on a standard upright or inverted microscope, preferably but not necessarily with a laser source, or in a nonmicroscopic custom setup or commercial spectrofluorimeter. In a microscope, the TIRF setup is compatible and rapidly interchangeable with bright-field, dark-field, phase contrast, and epi-illumination and accommodates a wide variety of common microscope objectives without alteration. 

A. L. Stout and D. Axelrod, Evanescent field excitation of fluorescence by epi-illumination microscopy, Appl. Opt 28, 5237-5242 (1989). 

The laser beam can be directed along one of several optical paths available in either the upright or inverted microscope configurations. For fluorescence applications, it is often convenient to make use of the standard epi-illumination... 

In one application, an epi-illuminated optical trap was used in conjunction with an inverted fluorescence microscope so that individual cells could be identified with FITC-conjugated antibodies and subsequently trapped for further micromanipulation (24). In the first part of the experiment, as a demonstration of a sorting technique, the trapped cells were transferred into a capillary tube, that was located in the Petri dish along with the cells. The optical trap was used to lift... 

Fig. 7. Schematic diagram of forces exerted on a cell when using an inverted microscope with (A) epi-illumination (i.e., laser focused through the objective) or (B) transillumination (i.e., laser focused through the condenser). is the axial force, and Fl is the lateral trapping force. Curved arrows represent the laser beam waist and point in the direction of light propagation.

The gels contain inhomogeneities with a characteristic size of about 1 um Under favorable circumstances, this structure can be visualized in the wet gel by epi-illumination microscopy. When it can be visualized, the structure has a "spongy appearance, consistent with what one might expect of a system that has undergone spinodal decomposition. So far, however, there is no concrete evidence to support this mechanism of phase separation. 

In the case of aqueous thin films between oil droplets (Figure 5), the interferometer beam is brought into the microscope through the epi-illumination attachment whereby the objective lens is used to both observe the film and focus the interferometer beam. The contrast of the observed image is much improved in stray light is minimized by positioning a pinhole at the image plan of the epi-illumination device. The thickness calculations remained the same as for the a/w films as the refractive index of the aqueous thin film was the same in both cases. 

Since its development by Delhaye and Dhamelincourt in 1975 [1] the epi-illumination Raman microprobe has become one of the most important input systems in Raman spectroscopy and is the instrument around which most Raman imaging systems are constructed. Epi-illumination instruments are almost always constructed around research-grade commercially available fluorescence microscope frames, with input optics modified to accept an exciting laser and with output optics modified to direct backscattered Raman signal to a spectrograph. 

Recently, Qi et al. made measurements on approximately 70 serum samples using an epi-illuminated waveguiding sample chamber, 160 mW of 830 nm excitation, up to 150 s of spectral integration, and PLS leave-one-out... 

Two methods have been developed in fluorescence microscopy transmitted light fluorescence and epi-illumination fluorescence. In the first method, the specimen is excited by light passing through the condenser lens, and fluorescence emission is captured by the objective lens (Figure 16.5). 

In the epi-illumination method, a dichroic beam splitter comprises two parts one acting as an excitation filter and the second as an emission filter (Figure 16.6). 

Total Bacteria Count The TBC is determine by directly counting the actual number of microorganisms collected on a filter after it is used to filter a sample of the water in question.5 The sample is stained with acridine orange and viewed with an epi-illuminated fluorescent microscope. This technique is more accurate and quicker than the culture technique, but is not as practical for field work. 

Fig. 4.16. Fluctuation of product formation from dehydro-rhodamin [130 nM] by a single horse radish peroxidase molecule in the presence hydrogen peroxide [10p,M]. Observation by confocal epi-illumination (a) Autocorrelation function of product fluctuation (b) [39]...

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