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REVIEW: Label-Free Multiphoton Microscopy: The Origin of Fluorophores and Capabilities for Analyzing Biochemical Processes

E. A. Shirshin1,2,a*, B. P. Yakimov1, M. E. Darvin3, N. P. Omelyanenko4, S. A. Rodionov4, Y. I. Gurfinkel5, J. Lademann3, V. V. Fadeev1, and A. V. Priezzhev1

1Lomonosov Moscow State University, Faculty of Physics, 119991 Moscow, Russia

2Institute of Spectroscopy, Russian Academy of Sciences, 108840 Troitsk, Moscow, Russia

3Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1, 10117 Berlin, Germany

4N. N. Priorov National Medical Research Center of Traumatology and Orthopaedics, 127299 Moscow, Russia

5Medical Scientific-Educational Center of Lomonosov Moscow State University, 119192 Moscow, Russia

* To whom correspondence should be addressed.

Received August 22, 2018; Revised September 25, 2018; Accepted September 25, 2018
Multiphoton microscopy (MPM) is a method of molecular imaging and specifically of intravital imaging that is characterized by high spatial resolution in combination with a greater depth of penetration into the tissue. MPM is a multimodal method based on detection of nonlinear optical signals – multiphoton fluorescence and optical harmonics – and also allows imaging with the use of the parameters of fluorescence decay kinetics. This review describes and discusses photophysical processes within major reporter molecules used in MPM with endogenous contrasts and summarizes several modern experiments that illustrate the capabilities of label-free MPM for molecular imaging of biochemical processes in connective tissue and cells.
KEY WORDS: molecular imaging, multiphoton microscopy, FLIM, autofluorescence, optical harmonic generation, fluorophores

DOI: 10.1134/S0006297919140050