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Studies of porphyrin‐containing specimens using an optical spectrometer connected to a confocal scanning laser microscope
Author(s) -
TREPTE O.,
ROKAHR I.,
ANDERSSONENGELS S.,
CARLSSON K.
Publication year - 1994
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1994.tb03521.x
Subject(s) - confocal , spectrometer , microscope , optics , laser , materials science , porphyrin , fluorescence , detector , laser scanning , microscopy , scanning electron microscope , chemistry , physics , photochemistry
A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user‐defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 350 at λ = 400 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three‐dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photo‐dynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and δ‐amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3–5 h before they were killed. The organs were removed directly after, and snap‐frozen in carbon dioxide ice with isopentane. No further staining or fixation procedures were adopted.