FOURIER-TRANSFORM MULTIPIXEL SPECTROSCOPY FOR QUANTITATIVE CYTOLOGY

A Fourier transform multipixel spectroscopy system was set up and appl ied to fluorescence microscopy of single living cells. Continuous fluo rescence spectra for all pixels of the cell image were recorded simult aneously by the system. Multiple frames of data were first acquired an d stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achie ved, typically, for 10(4) pixels in a single cell. The net result was I(x,y,lambda), the fluorescence intensity (I) for each pixel of the im age (x,y), as function of wavelength (lambda). The present study demon strates that multipixel spectroscopy can reveal dynamic processes of t he food-digestive cycle in the unicellular Paramecium vulgaris fed wit h algae. Spectral variability of fluorescence intensity at different c ytoplasmic sites pinpointed the location of cellular deposits of chlor ophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll, Localization of compartmental spectral changes was achie ved using a 'similarity mapping' algorithm, followed by enhanced image construction, Similarity mapping based on the fluorescence spectrum o f native chlorophyll revealed a highlighted image of the cell cytophar ynx structure where algae were ingested. Phagolysosomes, migrating vac uoles and the cytoproct, each containing different ratios of pheophyti n, were similarly imaged.