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Fourier Transform Multipixel Spectroscopy and Spectral Imaging of Protoporphyrin in Single Melanoma Cells
Author(s) -
Malik Zvi,
Dishi Michal,
Garini Yuval
Publication year - 1996
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1996.tb05663.x
Subject(s) - protoporphyrin , fluorescence , chemistry , protoporphyrin ix , subcellular localization , porphyrin , biophysics , spectroscopy , cytoplasm , photochemistry , optics , biochemistry , biology , physics , quantum mechanics
Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo‐PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge‐coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (10 3 ‐ − 4 × 10 3 point pixels in a single cell). The net result was the intensity I (x, y, λ), for each pixel of the image (x, y), at any wavelength (λ). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5‐aminolevulinic acid (ALA). The fluorescence image of ALA‐treated cells revealed endo‐PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one sub‐cellular site to another. Photoirradiation of the cells induced point‐specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral‐image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal‐lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo‐products could be visualized at sites of formation in suhcellular compartments.