Abstract
Nonlinear 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 nonlinear 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 nonlinear 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 nonlinear 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 nonlinear 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.
Highlights
The basic terminology of non-linear optics will be discussed here, though the in-depth study of non-linear optics is described in Boyd [1]
We have demonstrated that time-correlated single-photon counting (TCSPC)-fluorescence lifetime imaging microscopy (FLIM) system distinguishes the coherent anti-Stokes Raman scattering (CARS) signal from the fluorescence of live Huh7 cells stained with Hoechst3342
The uniqueness and the prospects of nonlinear optical microscopy are elucidated in this review
Summary
The basic terminology of non-linear optics will be discussed here, though the in-depth study of non-linear optics is described in Boyd [1]. In the 1990s, non-linear optical imaging modalities met their need for high peak power lasers, when the mode-locked infrared lasers were invented [2]. The interaction between light and matter under high peak power pulsed laser drives various non-linear optical processes such as multiphoton fluorescence and higher-order harmonic generations [1]. The key advantages of non-linear optical microscopy are deep imaging capability and high spatial resolution of tissue samples when compared to confocal microscopy. Non-linear processes rely on high intensities that are generated by tight focusing of the incoming ultra-fast pulsed lasers through high numerical aperture objective lens. The electrons of the sample respond to the incoming field differently depending upon the strength of electric field. The polarization of medium under the action of the external optical wave can be described by using a power series expansion in an electric field [1]:
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