Fluorescence microscopy fluorophore selection

Fluorophores can be visualized in fluorescence microscopy using special filter blocks that are composed of the excitation filter, dichroic mirror and emission filter. The excitation filter must select wavelengths of light from a light source that fall in the maximum absorption region of the fluorophore. The emission filter must pass the fluorescent wavelengths but not the excitation wavelengths. The dichroic mirror... 

The use of an evanescent wave to excite fluorophores selectively near a solid-fluid interface is the basis of the technique total internal reflection fluorescence (TIRF). It can be used to study theadsorption kinetics of fluorophores onto a solid surface, and for the determination of orientational order and dynamics in adsorption layers and Langmuir-Blodgett films. TIRF microscopy (TIRFM) may be combined with FRAP ind FCS measurements to yield information about surface diffusion rates and the formation of surface aggregates. 

Just as for any other fluorescence microscopy technique, the choice of the fluorescent probe is of significant importance for PCS. Drops in the autocorrelations curves can occur as a result of photophysical and photochemical processes. In particular, the contribution of saturation effects and triplet blinking has been investigated [97, 98] and the rates of intersystem crossing and triplet decay as well as the excitation cross section of fluorophores could be determined [12]. In addition, antibunching is determined by the photophysics of the fluorophore. Therefore, the choice of appropriate dyes is essential to obtain meaningful results. Apart from that, the fluorescent probe should also serve as a selective label to... 

Detection Fluorescence microscopy investigation of prepared surfaces should be performed before and after immobili2ation of the DNA probe and after the hybridization reaction with complementary and noncomplementary DNA target solutions. For visualization of the results, FITC and Texas Red filters should be mounted in the microscope. The DNA probe is labeled with FITC fluorophore, so it should be visible in the FITC filter. In case of DNA targets, the Cy3 is a selected fluorophore, so it should be visible in the Texas Red filter. In Table 5.1, the possible outcomes of the prepared samples and controls are listed. 

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