Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

Abstract

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 label-free non-linear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The non-linear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved non-linear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy

“…For clinical applications, it can be translated into nonlinear endomicroscopy (Lin et al, 2018;Zhuo et al, 2020) benefited from cheap and high-power femtosecond fiber laser systems to perform real-time and deep-tissue intravital imaging. Additionally, multimodal NLO imaging (Yue et al, 2011;Mazumder et al, 2019) enables simultaneous acquisition of the structural and chemical information of cells and tissues with sub-micrometer resolution, which is helpful for the researches in oncology, neurology, regenerative medicine, and developmental biology.…”

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

“…These studies suggest that the assessment of the heterogeneity of the cell population, their capability for collagen synthesis and remodeling, and variation in their differential potential can be assessed via MPM techniques (20,64,65). Considering the utility of TPEF and CARS to detect adipogenic differentiation of hMSCs and assess lipid amount, organization, orientation, and concentration (20,25,26,36,64), MPM-based imaging of lipid deposition may be useful in monitoring CAVD progression. MPM imaging has already been used to visualize elastin and collagen microstructures in heart valves using TPEF and SHG, respectively (17,30,45,66).…”

“…This technique involves estimating the fluorescence lifetime decay rates, which are sensitive to microenvironmental changes such as pH or protein binding (11). Of note, FLIM of NAD(P)H can be used to distinguish its bound and free states through a biexponential least-squares fit of the lifetime decay curves (11,19,(24)(25)(26). Free NAD(P)H has a mean lifetime of 0.3-0.4 ns, while protein-bound NAD(P)H has a mean lifetime of 1.9-5.7 ns (11).…”

“…The proportion of free NAD(P)H tends to increase when cells are undergoing glycolysis, while bound NAD(P)H often increases with increases in the rate of oxidative phosphorylation. FLIM is advantageous because it is independent of fluorophore concentration, laser intensity fluctuations, and the effects of tissue adsorption and scattering (18,19,(24)(25)(26)(27).…”

Coherent Anti-Stokes Raman Scattering Microscopy and Its Applications

“…As a new nonlinear optical microscopy technique, coherent Raman scattering microscopy overcomes the limitation of the imaging speed of spontaneous Raman micro-imaging [2][3][4][5][6][7][8]. In coherent Raman scattering microscopy, typically, two beams of light are used to simultaneously excite the sample, one beam is referred to as the pump beam and the other is the Stokes beam.…”