Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins

Caleb Stoltzfus; Alexandr Mikhailov; Aleksander Rebane

doi:10.1117/12.2250048

21 February 2017 Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins

Caleb Stoltzfus, Alexandr Mikhailov, Aleksander Rebane

Author Affiliations +

Proceedings Volume 10069, Multiphoton Microscopy in the Biomedical Sciences XVII; 1006924 (2017) https://doi.org/10.1117/12.2250048
Event: SPIE BiOS, 2017, San Francisco, California, United States

Abstract

Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore’s nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.

Citation Download Citation

Caleb Stoltzfus, Alexandr Mikhailov, and Aleksander Rebane "Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins", Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 1006924 (21 February 2017); https://doi.org/10.1117/12.2250048

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