Instrumentation for multiwavelengths excitation imaging

Journal of Neuroscience Methods
P MesslerR Uhl

Abstract

We have developed a fluorescence ratio-imaging system which is based on a 12 bit, 2 MHz slow scan CCD camera and a patented (patent number P 42 28 366.3-52) polychromatic illumination system. The latter produces monochromatic light (12 nm bandwidth) of high intensity (> 3 mW between 300 and 500 nm) and allows one to switch to any wavelength between 260 and 680 nm in less than 3.5 ms in a computer-controlled fashion. The possibility to execute complex wavelength protocols facilitates multiple dye measurements with optimal exposure time for a given wavelength and the return to a dark phase in between exposures. Moreover, it allows sweeping over extended spectral regions in order to determine optimal experimental conditions for a given task. Wavelength selection is performed by a diffraction grating which is mounted onto a galvanometric scanner. The grating is illuminated by white light from a 75 W xenon lamp, using exclusively reflective optics, and the diffracted monochromatic light is coupled into the microscope by means of a single fibre quartz light guide. The epifluorescence optics, a special, achromatic, aplanatic UV condensor, image the exit face plate of the fibre into the specimen plane of an inverted microscope. This 'c...Continue Reading

References

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