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Confocal Laser Scanning Microscopy (CLSM)
Instrumentation
Leica TCS SP2; 8 laser lines for excitations, acousto-optical bveam splitter (AOBS) allows adaptation to different fluoresence dyes without change of filter sets, works in reflection and fluorescence mode. An emission spectrometer is available at the instrument.
Operation Principle
Confocal laser microscopy (CLSM) uses a laser to produce point-probing raster scanning, yielding images with very high contrast in the third dimension. A small aperture at the secondary focus of the objective lens narrows the depth of focus and obstructs most of the light reflected from out-of-focus objects. CLSM is a valuable tool for obtaining high resolution images and 3D reconstructions from surfaces. The key feature of confocal microscopy is its ability to produce blur-free images of thick specimens at various depths. The confocal principle is illustrated schematically below. To image the specimen point by point, a collimated, polarized laser beam is deflected stepwise in the x- and y-direction by a scanning unit before it is reflected by a beam splitter so as to pass through the objective lens of the microscope, and focused onto the specimen. The emitted, longer-wavelength fluorescent light collected by the objective lens passes through the beam splitter and is focused into a small pinhole (i.e., the confocal aperture) to eliminate all the out-of-focus light, i.e., all light coming from regions of the specimen above or below the plane of focus.