4 April 1994 Simultaneous lifetime imaging of two fluorophores using a confocal laser microscope
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Abstract
We demonstrate the possibility to study, simultaneously, the variations over the image area of the lifetimes of two different fluorophores, in a confocal laser microscope. In this way information conveyed by the lifetimes can be extracted, in particular complementary information from two different fluorophores. The fluorophores are excited by laser light of two different wavelengths, which are modulated at different frequencies using electro-optical modulators. By frequency-selective detection, using two lock-in amplifiers, it is possible to efficiently separate signals that emanate from each of the fluorophores. Further, by using 2- phase lock-in amplifiers the phase of each of the separated signals is determined. Since the phase shift of the emitted light relative to the exciting light depends on the lifetime of the fluorophore, the technique allows mapping of the lifetime. Two images are obtained, one for each fluorophore. The method can be extended to supply information concerning the excitation cross sections at the two laser wavelengths used.
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Nils R.D. Aslund, Nils R.D. Aslund, Kjell Carlsson, Kjell Carlsson, } "Simultaneous lifetime imaging of two fluorophores using a confocal laser microscope", Proc. SPIE 2184, Three-Dimensional Microscopy: Image Acquisition and Processing, (4 April 1994); doi: 10.1117/12.172108; https://doi.org/10.1117/12.172108
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