Nonlinear Optical Microscopy Techniques Research Articles

Nonlinear dynamics of femtosecond laser interaction with the central nervous system in zebrafish

Understanding the photodamage mechanism underlying the highly nonlinear dynamic of femtosecond laser pulses at the second transparent window of tissue is crucial for label-free microscopy. Here, we report the identification of two cavitation regimes from 1030 nm pulses when interacting with the central nervous system in zebrafish. We show that at low repetition rates, the damage is confined due to plasma-based ablation and sudden local temperature rise. At high repetition rates, the damage becomes collateral due to plasma-mediated photochemistry. Furthermore, we investigate the role of fluorescence labels with linear and nonlinear absorption pathways in optical breakdown. To verify our findings, we examined cell death and cellular responses to tissue damage, including the recruitment of fibroblasts and immune cells after irradiation. These findings contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing precise, and localized injuries using near-infrared femtosecond laser pulses.

Nonlinear Optical Microscopy of Interface Layers of Epitaxial Garnet Films

The structure of magnetic domains is an exciting research object that shows an enormous variety of delightful patterns. Epitaxial garnet is one of the most studied magnetic dielectrics with well-recognized bulk domains, while the magnetic composition at the surface is less investigated. Here we apply the nonlinear optical microscopy technique for the visualization of the interface magnetic domains of 10 μm thick (LuBi)3Fe5O12 film and prove that it is qualitatively similar for both garnet/air and garnet/substrate interfaces. As an efficient extension of the second harmonic generation microscopy, we suggest and demonstrate the possibilities of the third harmonic generation one, which provides higher resolution of the method.

Application of Ultrashort Lasers in Developmental Biology: A Review

The evolution of laser technologies and the invention of ultrashort laser pulses have resulted in a sharp jump in laser applications in life sciences. Developmental biology is no exception. The unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of transparent material without disrupting the surrounding tissues makes ultrashort lasers a versatile tool for precise microsurgery of cells and subcellular components within structurally complex and fragile specimens like embryos as well as for high-resolution imaging of embryonic processes and developmental mechanisms. Here, we present an overview of recent applications of ultrashort lasers in developmental biology, including techniques of noncontact laser-assisted microsurgery of preimplantation mammalian embryos for oocyte/blastomere enucleation and embryonic cell fusion, as well as techniques of optical transfection and injection for targeted delivery of biomolecules into living embryos and laser-mediated microsurgery of externally developing embryos. Possible applications of ultrashort laser pulses for use in Assisted Reproductive Technologies are also highlighted. Moreover, we discuss various nonlinear optical microscopy techniques (two-photon excited fluorescence, second and third harmonic generation, and coherent Raman scattering) and their application for label-free non-invasive imaging of embryos in their unperturbed state or post-laser-induced modifications.

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Wide-field broadband CARS microscopy

Coherent anti-Stokes Raman scattering is an extremely powerful non-linear optical (NLO) microscopy technique for label-free vibrational imaging allowing for a detailed study of biological samples in their native state. To overcome the long acquisition times associated with raster sample scanning required in NLO microscopy, which impair real-time investigation of fast biological dynamics, we employ here wide-field signal generation over a large field of view, covering tens of micrometers. To this aim, we exploit an innovative approach based on the use of an amplified femtosecond ytterbium laser source delivering high energy (≈μJ) pulses in the near infrared. This enables the generation of stable broadband Stokes pulses to measure the entire fingerprint region of the molecular vibrational spectrum, the richest in chemical information. Our results pave the way for future translational applications and clinical diagnostics with video-rate imaging capabilities. ©2022 The Authors

Simultaneous label-free autofluorescence-multiharmonic microscopy driven by femtosecond sources based on self-phase modulation enabled spectral selection

Nonlinear optical microscopy technique has unique advantages in tissue imaging, such as enhanced contrast, high resolution, and label-free deep optical sectioning capabilities. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we propose a new wavelength conversion approach—self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. The SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using the suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this work, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with >5-nJ energy and ~84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain a large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources becomes a powerful tool in biomedical imaging.

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Quantum-enhanced stimulated emission detection for label-free microscopy

Nonlinear optical microscopy techniques have emerged as a set of successful tools for biological imaging. Stimulated emission microscopy belongs to a small subset of pump–probe techniques, which can image non-fluorescent samples without requiring fluorescent labeling. However, its sensitivity has been shown to be ultimately limited by the quantum fluctuations in the probe beam. We propose and experimentally implement sub-shot-noise limited stimulated emission microscopy by preparing the probe pulse in an intensity-squeezed state. This technique paves the way for imaging delicate biological samples that have no detectable fluorescence with sensitivity beyond standard quantum fluctuations.

Nonlinear Optical Diagnostics of Thin Polycrystalline Lead Zirconate Titanate Films

Nonlinear optical microscopy techniques have been used to study thin films of single-phase (perovskite) and two-phase (perovskite–pyrochlore) lead zirconate titanate (PZT) deposited on Pt/TiO2/SiO2/Si by means of radio-frequency magnetron sputtering at various target–substrate distances (D = 30–70 mm). Results revealed inhomogeneous distribution of second-harmonic generation in spherulitic perovskite islands, including increased signal intensity at perovskite/pyrochlore interface, which may be related to a nonuniform distribution of mechanical stresses. Factors responsible for the observed strong variation in second-harmonic signal are discussed, including the mutual relationship between changes in the character of spherulitic structure and conditions of PZT film deposition depending on the target–substrate distance.

Monte Carlo simulation of polarization-sensitive second-harmonic generation and propagation in biological tissue.

Polarization-sensitive second harmonic generation (p-SHG) is a nonlinear optical microscopy technique that has shown great promise in biomedicine, such as in detecting changes in the collagen ultrastructure of the tumor microenvironment. However, the complex nature of light-tissue interactions and the heterogeneity of biological samples pose challenges in creating an analytical and experimental quantification platform for tissue characterization via p-SHG. We present a Monte Carlo (MC) p-SHG simulation model based on double Stokes-Mueller polarimetry for the investigation of nonlinear light-tissue interaction. The MC model predictions are compared with experimental measurements of second-order nonlinear susceptibility component ratio and degree of polarization (DOP) in rat-tail collagen. The observed trends in the behavior of these parameters as a function of tissue thickness, as well as the overall extent of agreement between MC and experimental results, are discussed. High sensitivities of the susceptibility ratio and DOP are observed for the varying tissue thickness on the incoming fundamental light propagation pathway.

Label-free Imaging of Myocardial Remodeling in Atrial Fibrillation Using Nonlinear Optical Microscopy: A Feasibility Study.

Atrial fibrillation, characterized by rapid disorganized electrical activation of myocardium, is caused by and accompanied by remodeling of myocardial tissue. We applied nonlinear optical microscopy (NLOM) to visualize typical myocardial features and atrial fibrillation effects in order to test anon-destructive imaging technology that in principle can be applied in vivo.Coherent anti-Stokes Raman scattering, endogenous two-photon excited fluorescence, and second harmonic generation were used to inspect unstained human atrial myocardium from three patients who underwent surgical Cox-MAZE procedure with amputation of left atrial appendage. Using NLOM techniques, we collected detailrich pictures of unstained tissue that enable comprehensive characterization of myocardial characteristics like myocyte structure, collagen and lipofuscin deposition, intercalating disc width, and fatty degradation. Development of in vivo application of the NLOM technique may represent a revolutionary approach in characterizing atrial fibrillation associated myocardial remodeling with important implications for therapy individualization and monitoring.

Viewing Cancer in a New Light: Multimodal Label-Free Specific Chemical Imaging to Distinguish Healthy Cell Tissue from Invasive Carcinoma

Microscopy has always been an important tool when determining whether a biopsy contains invasive carcinomas. For this purpose, light microscopy is often used due to its simplicity in construction and operation. The downside to this is the time lost in preparing biopsies before imaging. Typically, the process of obtaining a biopsy, molding, sectioning, mounting and staining of the biopsy on microscopy glass slides and finally imaging the biopsy takes between 6-10 days. If the preparatory steps could be eliminated, valuable time would be freed. Coherent anti-Stokes Raman Scattering (CARS) microscopy offers a possible solution to this obstacle. CARS is a non-linear optical microscopy technique that allows label-free specific imaging of chemical moieties present in a sample. Specifically, lipids (rich in CH2-moieties) and protein (rich in CH-moieties), as well as water, are easily imaged using CARS. A system capable of performing CARS microscopy will generally also be capable of doing Second-Harmonic Imaging Microscopy (SHIM). This technique uses second-harmonic generation (SHG) to achieve frequency doubling of the incident light. SHIM enables imaging of biological structures, which possess the characteristics of being both highly polarizable and ordered on medium to long length scales. Specifically collagen, which is present especially in the basement membrane, is very capable of producing a second-harmonic signal. By combining CARS, SHIM and multi-photon excitation of auto-fluorescent molecules, it is possible to acquire multimodal micrographs of epithelial tissue samples. The CARS measurements gives a unique insight into cell organization in tissue by clearly visualizing the cell membrane bilayer and lipid distribution, while the SHIM reveals the location of the basement membrane and underlying connecting tissue. Our results show that it is possible, in an unlabeled sample, to differentiate between healthy tissue samples and samples that display varying degrees of dysplasia or invasive carcinomas. This opens up for the possibility for future fast bedside cancer diagnostics using CARS microscopy.

Label-Free Optical Metabolic Imaging in Cells and Tissues.

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.

Abstract 82: Mnsodtg Mice Exhibit Improved Heart and Mitochondrial Function During Chronic Chagas Disease

Background: We observed that mitochondrial reactive oxygen species (mtROS) plays very important roles in the pregression of chagesic disease (CD). In this study, we utilized genetically-modified mice to scavenge mtROS to investigate the impact of improved ROS scavenging capacity on heart function in CD. Methods and Results: C57BL/6 mice (wild-type, MnSODtg, MnSOD+/-) were infected with Trypanosoma cruzi(Tc). Chronically infected mice (≥120dpi) exhibited a substantial decrease in heart tissue MnSOD gene expression, protein level, enzyme activity and antioxidant level; decrease of heart dysfunction via lower of SV, CO, EF, FS and LVPW,s, and increase of ESV/EDS and LVID;s; enhancement of hypertrophy by increase of IVS, LV mass and areas duo to augmentation of collagen expressions. One of our novel observations was that sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) lost its role of maintenance of low cytoplasm free calcium and mediated calcium uptake to intracellular store in Tc-induced chronic chagasic disease. Studies of fresh heart slices using O2K confirmed that Tc diminished heart mitochondrial function like decrease of oxygen flux and respiratory control ratio (RCR), which were caused by enhancements of ROS. Myocardial mitochondrial damage was pronounced and associated with a >x% decline in mitochondrial oxygen flux in chronically infected wild-type and MnSOD transgenic mice. Imaging of intact heart for cardiomyocytes and collagen by the nonlinear optical microscopy techniques showed significant increase in collagen (>x0-fold) in chronically infected wild-type mice; while MnSODtg mice exhibited a basal increase in collagen that did not change during chronic phase. Chronically infected MnSODtg mice exhibited a marginal decline in Tc-induced heart function, heart hypertrophy, mitochondrial dysfunction Conclusions: Overexpression of MnSOD inhibited Tc-induced oxidative damage od heart tissue. , suggesting that enhancing the mitochondrial ROS scavenging capacity was beneficial in controlling the inflammatory and oxidative pathology, and cardiac remodeling responses that are hallmarks of chronic Chagas disease.

In vivo measurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy.

The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ~30-65 μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R² = 0.8895). SFDS melanin distribution thickness is correlated to MPM values (R² = 0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.

Investigating Plasmonic Structure-Dependent Light Amplification and Electronic Dynamics Using Advances in Nonlinear Optical Microscopy

Our group has made several recent advances using single-particle nonlinear optical (NLO) microscopy techniques to examine nanostructure-specific plasmon-mediated interactions with electromagnetic energy. These efforts are directed toward the larger goal of optimizing the structure of photonic nanoparticle assemblies for the use and control of energy at the nanoscale. By combining statistical localization methods with second harmonic generation (SHG) imaging, nonlinear signal hot spots can be located within a plasmonic network with nanometer spatial accuracy. This experimental capability was applied to study electromagnetic lensing effects in plasmonic nanoparticle assemblies, with the efficiency of cascaded energy transfer through the network being determined by the resonantly excited plasmon mode. The polarization and time dependencies of the NLO signals were examined using a sequence of phase-locked broad bandwidth femtosecond laser pulses. The methodologies for generating, characterizing, and incorpora...

Nonlinear optical microscopy: Endogenous signals and exogenous probes

Nonlinear optical microscopy (NLOM) relies on nonlinear light–matter interactions to provide images from larger depths within biological structures compared to conventional confocal fluorescence microscopy. These nonlinear light–matter interactions include multiphoton excitation fluorescence (MPEF), second‐harmonic generation (SHG), coherent anti‐Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS). This review discusses the theories of and instrumentation for various NLOM techniques, with a particular focus on endogenous signals and exogenous probes. These signals and probes expand the breadth of information that optical imaging can provide. We also discuss the application of NLOM in biomedical research, including tissue engineering, drug delivery and clinical diagnostics. Current technological limitations are also discussed. image

Experimental study on multicolor two-photon excited fluorescence microscopy

Two-photon excited fluorescence (TPEF) microscopy is a nonlinear optical microscopy technique. The advantages of TPEF microscopy include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. In traditional TPEF microscopy, a wavelength tunable ultrashort pulsed laser is used as an excitation source. In practical applications, sample usually contains various fluorophores or unknown components. Therefore the excitation wavelength of the ultrafast laser has to be tuned to achieve optimal excitation efficiencies of various fluorophores. In order to acquire the fluorescent signals of different fluorophores simultaneously, we develop a multicolor TPEF microscope system based on a supercontinuum laser source. In experiments, TPEF images of Lily rhizome sample slide stained by two fluorescent dyes with different excitation and emission wavelengths are obtained without tuning the wavelength. Experimental results show that the high-contrast TPEF images of the sample with various fluorophores can be obtained simultaneously by using the multicolor TPEF microscope compared with by using traditional TPEF microscopy. The system is simple in structure, easy in operation, and can provide rich information about the sample, which allows it to be widely used in life and material sciences.

Experimenting Liver Fibrosis Diagnostic by Two Photon Excitation Microscopy and Bag-of-Features Image Classification

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

Shape-Tunable Core–Shell Microparticles

Colloidal polymer particles are an important class of materials finding use in both everyday and basic research applications. Tailoring their composition, shape, and functionality is of key importance. In this article, we describe a new class of shape-tunable core-shell microparticles. They are composed of a cross-linked polystyrene (PS) core and a poly(methyl methacrylate) (PMMA) shell of varying thickness. In the first step, we prepared highly cross-linked PS cores, which are subsequently transferred into a nonpolar dispersant. They serve as the seed dispersion for a nonaqueous dispersion polymerization to generate the PMMA shell. The shape of the particles can subsequently be manipulated. After the shell growth stage, the spherical PS/PMMA core-shell colloids exhibit an uneven and wrinkled surface. An additional tempering procedure allows for smoothing the surface of the core-shell colloids. This results in polymer core-shell particles with a perfectly spherical shape. In addition to this thermal smoothing of the PMMA shell, we generated a selection of shape-anisotropic core-shell particles using a thermomechanical stretching procedure. Because of the unique constitution, we can selectively interrogate molecular vibrations in the PS core or the PMMA shell of the colloids using nonlinear optical microscopy techniques. This is of great interest because no photobleaching occurs, such that the particles can be tracked in real space over long times.