Time-resolved spectral imaging: better photon economy, higher accuracy

Farzad Fereidouni; Keimpe Reitsma; Gerhard A. Blab; Hans C. Gerritsen

doi:10.1117/12.2080392

5 March 2015 Time-resolved spectral imaging: better photon economy, higher accuracy

Farzad Fereidouni, Keimpe Reitsma, Gerhard A. Blab, Hans C. Gerritsen

Proceedings Volume 9329, Multiphoton Microscopy in the Biomedical Sciences XV; 93290H (2015) https://doi.org/10.1117/12.2080392
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

Lifetime and spectral imaging are complementary techniques that offer a non-invasive solution for monitoring metabolic processes, identifying biochemical compounds, and characterizing their interactions in biological tissues, among other tasks. Newly developed instruments that perform time-resolved spectral imaging can provide even more information and reach higher sensitivity than either modality alone. Here we report a multispectral lifetime imaging system based on a field-programmable gate array (FPGA), capable of operating at high photon count rates (12 MHz) per spectral detection channel, and with time resolution of 200 ps. We performed error analyses to investigate the effect of gate width and spectral-channel width on the accuracy of estimated lifetimes and spectral widths. Temporal and spectral phasors were used for analysis of recorded data, and we demonstrated blind un-mixing of the fluorescent components using information from both modalities. Fractional intensities, spectra, and decay curves of components were extracted without need for prior information. We further tested this approach with fluorescently doubly-labeled DNA, and demonstrated its suitability for accurately estimating FRET efficiency in the presence of either non-interacting or interacting donor molecules.

Citation Download Citation

Farzad Fereidouni, Keimpe Reitsma, Gerhard A. Blab, and Hans C. Gerritsen "Time-resolved spectral imaging: better photon economy, higher accuracy", Proc. SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV, 93290H (5 March 2015); https://doi.org/10.1117/12.2080392

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