Optical spectrometer for a confocal scanning laser microscope with applications in porphyrin-containing specimens

A spectrometer has been developed for the Phoibos confocal scanning laser microscope. With this spectrometer, spectral information from a single point, or a user defined region, within the microscope specimen, can be recorded. The spectrometer is based on an integrated spectrometer module, manufactured by Carl Zeiss, Germany. The module takes its light input signal through a fiber with an entrance diameter of 0.5 mm. Integrated in the spectrometer module are dispersing optics, based on a grating, as well as preamplifier electronics. A regulated cooling unit keeps the detector at -4°C, thereby allowing longer integration times. The spectral resolution, defined as the minimum distance between two peaks (Rayleigh criterion) is approximately 10 nm. The entrance of the optical fiber is employed as a pinhole. With different magnification in the optical path leading the light to the spectrometer, the entrance can either be employed as a pinhole of the same size as the one used during conventional confocal scanning, i.e. the 3D spatial resolution will be retained, or the light throughput can be increased at the expense of optical resolution. With the described equipment, studies of rodent lung and liver specimens containing porphyrins have been made. Organs from animals injected with δ-amino levulinic acid, a precursor to protoporphyrin IX and haem in the haem cycle, have been studied. Spectroscopic detection is necessary in order to separate the porphyrin signal from other fluorescent components in the specimen.