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      Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging

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          Abstract

          A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

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          Two-photon laser scanning fluorescence microscopy.

          W Denk, J H Strickler, W. Webb (1990)
          Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
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            Über Elementarakte mit zwei Quantensprüngen

            Maria Göppert-Mayer (1931)
            Article 0 comments Cited 449 times – based on 0 reviews     Review now
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              Deep tissue two-photon microscopy.

              Fritjof Helmchen, Winfried Denk (2005)
              With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.
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                Author and article information

                Contributors
                Journal
                Journal ID (nlm-ta): Front Bioeng Biotechnol
                Journal ID (iso-abbrev): Front Bioeng Biotechnol
                Journal ID (publisher-id): Front. Bioeng. Biotechnol.
                Title: Frontiers in Bioengineering and Biotechnology
                Publisher: Frontiers Media S.A.
                ISSN (Electronic): 2296-4185
                Publication date (Electronic): 09 October 2020
                Publication date Collection: 2020
                Volume: 8
                Electronic Location Identifier: 585363
                Affiliations
                [1] 1Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano , Milan, Italy
                [2] 2Istituto di Fotonica e Nanotecnologie (IFN) – CNR , Milan, Italy
                [3] 3Department of Physics, Politecnico di Milano , Milan, Italy
                Author notes

                Edited by: Paolo Bianchini, Italian Institute of Technology (IIT), Italy

                Reviewed by: Jayaramakrishnan Velusamy, University of Guanajuato, Mexico; Shuwen Zeng, Centre National de la Recherche Scientifique (CNRS), France

                *Correspondence: Valentina Parodi, valentina.parodi@ 123456polimi.it

                †These authors have contributed equally to this work

                This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

                Article
                DOI: 10.3389/fbioe.2020.585363
                PMC ID: 7581943
                PubMed ID: 33163482
                SO-VID: 05522e84-0e4c-4dd9-9eb2-1fb786422a95
                Copyright © Copyright © 2020 Parodi, Jacchetti, Osellame, Cerullo, Polli and Raimondi.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                Date received : 20 July 2020
                Date accepted : 16 September 2020
                Page count
                Figures: 6, Tables: 4, Equations: 0, References: 89, Pages: 18, Words: 0
                Categories
                Subject: Bioengineering and Biotechnology
                Subject: Review

                Keywords: nonlinear microscopy,stem cells,tissue engineering,label-free microscopy,live imaging
                Data availability:
                Keywords: nonlinear microscopy, stem cells, tissue engineering, label-free microscopy, live imaging

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