Instrumentation for multiwavelengths excitation imaging

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 condenser, image the exit face plate of the fibre into the specimen plane of an inverted microscope. This 'critical illumination' yields better homogeneity in the specimen plane than the classical Kohler illumination. Thus, with the Zeiss Fluar objective 40 x, NA = 1.3, fluence rates close to 10(23) photons m(2) s may be achieved at 340 nm. A DOS programme has been written in 'C' which controls both the monochromator and slow scan imaging system. It can acquire up to 13 full frames per s, and in its binning and skipping mode up to 100 subframes per s may be acquired. The frame-transfer structure of the chip allows one to acquire images at wavelength 'B' while simultaneously writing image data previously acquired at wavelength 'A' into the computer.