Label-Free Non-linear Multimodal Optical Microscopy-Basics, Development, and Applications Nirmal Mazumder, Naveen Balla, Guan-Yu Zhuo, Yury Kistenev, Rajesh Kumar, Fu-Jen Kao, Sophie Brasselet, Viktor Nikolaev, Natalya Krivova To cite this version: Nirmal Mazumder, Naveen Balla, Guan-Yu Zhuo, Yury Kistenev, Rajesh Kumar, et al.. Label- Free Non-linear Multimodal Optical Microscopy-Basics, Development, and Applications. Frontiers in Physics, Frontiers, 2019, 7, 10.3389/fphy.2019.00170. hal-02410671 HAL Id: hal-02410671 https://hal.archives-ouvertes.fr/hal-02410671 Submitted on 13 Dec 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. REVIEW published: 31 October 2019 doi: 10.3389/fphy.2019.00170 Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications Nirmal Mazumder 1*, Naveen K. Balla 2, Guan-Yu Zhuo 3,4, Yury V. Kistenev 5,6, Rajesh Kumar 7, Fu-Jen Kao 8, Sophie Brasselet 9, Viktor V. Nikolaev 5,10 and Natalya A. Krivova 11 1 Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India, 2 Center for Research on Adaptive Nanostructures and Nanodevices, Trinity College of Dublin, Dublin, Ireland, 3 Institute of New Drug Development, China Medical University, Taichung, Taiwan, 4 Integrative Stem Cell Center, China Medical University Hospital, Taichung, Taiwan, 5 Laboratory of Biophotonics, Tomsk State University, Tomsk, Russia, 6 Department of Physics and Mathematics, Siberian Medical State University, Tomsk, Russia, 7 Department of Physics, Norwegian University of Science and Technology (NTNU), Trondheim, Norway, 8 Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, 9 Institut Fresnel, CNRS, Aix-Marseille Université, Ecole Centrale Marseille, Domaine Universitaire St Jérôme, Marseille, France, 10 Laboratory of Molecular Imaging and Photoacoustics, Institute of Strength Physics and Materials Science SB RAS, Tomsk, Russia, 11 Laboratory of Experimental Physiology, Tomsk State University, Tomsk, Russia Non-linear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss Edited by: Stefan G. Stanciu, label-free non-linear optical microscopes including the two-photon fluorescence (TPF), Politehnica University of fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic Bucharest, Romania generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with Reviewed by: various samples. The non-linear signals are generated from collagen in tissue (SHG), Maria Calvo, University of Barcelona, Spain amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), Laura Sironi, elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and University of Milano Bicocca, Italy lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the *Correspondence: Nirmal Mazumder molecular structure of the sample and its relative orientation to the polarization of [email protected] the incident light. Thus, polarization-resolved non-linear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of Specialty section: the advancements on polarization-resolved SHG microscopy including Stokes vector This article was submitted to Optics and Photonics, based polarimetry, circular dichroism, and susceptibility are also presented in this review a section of the journal article. The working principles and corresponding implements of above-mentioned Frontiers in Physics microscopy techniques are elucidated. The potential of time-resolved TPF lifetime Received: 21 March 2019 Accepted: 15 October 2019 imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of Published: 31 October 2019 NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single Citation: laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated Mazumder N, Balla NK, Zhuo G-Y, single-photon counting (TCSPC) in combination with continuum generation from Kistenev YV, Kumar R, Kao F-J, Brasselet S, Nikolaev VV and photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO Krivova NA (2019) Label-Free microscopy is a powerful tool to assess the molecular specificity with high resolution. Non-linear Multimodal Optical Microscopy—Basics, Development, Keywords: non-linear optical microscopy, two-photon fluorescence microscopy, second harmonic generation, and Applications. Front. Phys. 7:170. coherent anti-stokes Raman scattering, fluorescence lifetime imaging, nicotinamide adenine dinucleotide, doi: 10.3389/fphy.2019.00170 collagen Frontiers in Physics | www.frontiersin.org 1 October 2019 | Volume 7 | Article 170 Mazumder et al. Non-linear Optical Microscopy INTRODUCTION excitation requires extremely high photon flux, typically 1020- 1030 photons/(cm2s). A femtosecond pulsed laser is used in The basic terminology of non-linear optics will be discussed here, combination with high-numerical aperture (NA) objective lens though the in-depth study of non-linear optics is described in to generate high photon flux at the focal spot. In TPF, the Boyd [1]. In the 1990s, non-linear optical imaging modalities met excitation wavelengths are chosen for the fluorophores so that their need for high peak power lasers, when the mode-locked they absorb two photons simultaneously. The total energy of the infrared lasers were invented [2]. The interaction between light exciting photons should be equal to or exceed the energy gap and matter under high peak power pulsed laser drives various between the ground state and excited state of the fluorophore for non-linear optical processes such as multiphoton fluorescence TPF to occur. The advantage of fluorescent markers or probes, and higher-order harmonic generations [1]. The key advantages which could be used to selectively tag molecules within the living of non-linear optical microscopy are deep imaging capability system for visualization purposes, has contributed immensely and high spatial resolution of tissue samples when compared toward an understanding of cellular and molecular aspects in to confocal microscopy. Non-linear processes rely on high biological systems. intensities that are generated by tight focusing of the incoming The experimental arrangement of TPF microscope integrated ultra-fast pulsed lasers through high numerical aperture objective with FLIM system is shown in Figure 1A. An inverted confocal lens. Due to the requirement for high intensity, the effective microscope (e.g., Olympus IX71) can be modified for TPF excitation volume in non-linear signal is much smaller than imaging setup. An ultrafast femtosecond laser oscillator is used that of a linear signal [2] and provides 3D imaging capability as the excitation light source. In general, Coherent Mira Optima with subcellular details and high molecular contrast [1]. It have 900-F, Spectra-Physics, Toptica Photonics are the main suppliers opened new routes toward optical diagnostics of complex cellular of femtosecond lasers around the globe [9–13]. The excitation assemblies and tissue imaging [3, 4]. The electrons of the sample wavelength is set according to the absorption wavelength of the respond to the incoming field differently depending upon the fluorophores (two-photon excitation wavelengths are 740 nm for strength of electric field. In case of a weak electric field, the nicotinamide adenine dinucleotide, NADH and 810 nm for Nile electrons follow the oscillations of the field; in case of the strong Red, 860 nm for green fluorescence protein, GFP) and has pulse electric field, they move away from the equilibrium and no longer width of ∼100 fs, with an average power ∼550 mW and repetition follow the incoming field. The polarization of medium under the rate ∼76 MHz. The laser power is controlled by a combination action of the external optical wave can be described by using a of polarizer and half-waveplate. Live cell samples are imaged in power series expansion in an electric field [1]: 4.2 cm2 Lab-Tek Chambers Slide System (NUNC, Rochester, NY) mounted on plan XY microscope stage (IX2-KSP, Olympus) and ˜ = (1) ˜ + (2) ˜ 2 + (3) ˜ 3 + P (t) ǫ0χ E (t) ǫ0χ E(t) ǫ0χ E(t) . , (1) scanned with a laser scanning unit (Olympus, FV300) (as shown in Figure 1). After entering the microscope, the laser beam is where P˜(t) is the induced polarization due to the field E˜ (t) and reflected by a dichroic mirror (690 nm filter) before propagating ǫ is the electric permittivity of free space, χ (1) is the linear 0 through the objective lens (UPlanFLN 40X/N.A. 1.3 oil, Olympus susceptibility and it characterizes the index of refraction and Corp., Japan). The TPF signal is collected through the excitation linear absorption. The second-order optical susceptibility, χ (2), objective lens, in the spectral window depending