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24 March 2005 Fluorescence confocal microscopy with spectrally tuned, cryogenically cooled diode lasers
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In confocal microscopy, the laser excitation must be near the fluorochrome absorption peak to efficiently excite the fluorochrome. However, most fluorochromes emitting in the red to infrared have absorption and emission spectra with overlapping wavelength ranges, i.e. small Stokes shifts. As a result, the laser excitation extends into the fluorescence wavelengths hindering separation of the reflected laser signal from the information-containing fluorescence signal. Therefore, compromises are necessary: (1) the entire reflected laser spectrum is filtered, eliminating some of the fluorescence signal; or (2) the entire fluorescence signal is recorded, including unwanted reflected laser light, increasing the background noise. These compromises must be addressed, even with fluorochromes - like Cy5 - having a relatively large Stokes shift. Cryogenically cooled diode lasers eliminate the need for these compromises by tuning the output wavelength away from the fluorochrome emission. By separating the excitation and emission spectra, the confocal fluorescence signal-to-noise ratio can be increased by filtering more of the reflected laser emission, without losing valuable fluorescence information. However, this results in slightly less efficient excitation of the fluorochrome. We will present spectrophotometric analyses of fluorochrome absorption, fluorescence, and diode laser emission as a function of diode operating temperature. We will show that as the diode lasers are cooled their output power increases and more than compensates for the lower fluorochrome excitation, resulting in significantly more intense fluorescence. Thus, by tuning the diode laser, more fluorescence information and less reflected laser light reach the detector, creating images with greater intensity and less background noise.
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Nelson Sivers, Brian Van de Workeen, and Susanne M. Lee "Fluorescence confocal microscopy with spectrally tuned, cryogenically cooled diode lasers", Proc. SPIE 5701, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XII, (24 March 2005);

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