42] Calibration of microspectrophotometers as it applies to the detection of lipofuscin and the blue- and yellow-emitting fluorophores in situ

Abstract

Publisher Summary This chapter discusses the importance of careful calibration in spectrofluorometric work. Lipofuscin, the “wear and tear pigment” of classical pathology, which accumulates with age, is studied. Metabolically active postmitotic cells of numerous tissues have the greatest accumulations of these heterogeneous lipophilic granules. These lysosomal residual bodies are thought to contain indigestible remnants of damaged cellular membranes that were autophagized or phagocytized. Fluorescence microscopists identify lipofuscin granules by their yellow–orange autofluorescence under near-ultraviolet (UV) excitation. A major contradiction to this observation exists however. Reports on the fluorescence properties of the lipid extracts of lipfuscin-laden cells show peak emission in the blue (415–490 nm) region of the visible spectrum. Lipofuscin, the age pigment, fluoresces yellow when excited with ultraviolet in vitro as well as in situ . To determine whether the UV-excited fluorescence is yellow or blue, in situ corrected emission and excitation spectra using microspectrofluorometry is obtained. To obtain accurate excitation and emission spectra, it is necessary to calibrate and correct for the spectra sensitivity of the photomultiplier, the excitation intensities, the spectral characteristics of excitation and emission “monochromators,” and the transmission properties of the light path through the microscope.