Nonlinear Microscopy Research Articles

Микроструктура сферолитовых тонких пленок цирконата-титаната свинца

The features of the microstructure of radiant spherulites in lead zirconate titanate thin films are studied using scanning electron and nonlinear optical microscopy by varying the blocks size of the perovskite structure. It is shown that with an increase in the radius of the spherulite (or the size of blocks), the angle of rotation of the growth axis increases, the speed of the angle of rotation, and the magnitude of the signal of the second optical harmonic It is assumed that the reasons determining specifics of the structure and signal of the second optical harmonic are associated with a change in the magnitude of mechanical stresses in the films that arise due to a change in the phase density at perovskite phase crystallization from low-temperature pyrochlore phase or at perovskite phase recrystallization

Quantitative analysis of ovarian cancer pathology using nonlinear optical imaging and lifetime microscopy

Quantitative analysis of ovarian cancer pathology using nonlinear optical imaging and lifetime microscopy

Nonlinear optical microscopy for skinin vivo: Basics, development and applications

Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis.

Second harmonic generation from aluminum plasmonic nanocavities: from scanning to imaging.

Metamaterials and plasmonic structures made from aluminum (Al) have attracted significant interest due to their low cost, long-term stability, and the relative abundance of aluminum compared to the rare metals. Also, aluminum displays distinct dielectric properties allowing for the excitation of surface plasmons in the ultraviolet region with minimal non-radiative losses. Despite these clear advantages, most of the research has been focused on either gold or silver, probably due to difficulties in forming smooth thin films of aluminum. In the present work, we detect and characterize second harmonic generation (SHG) in the optical regime, emanating from triangular hole arrays milled into thin aluminum films in reflection mode, at normal incidence. We report intense nonlinear responses, year-long stability, and overall superior performances with respect to gold. The robustness of the Al structures and high reproducibility of the measured SHG responses allowed us to investigate changes in the directional emission upon tiny modifications in the structure's symmetry. We also demonstrate large-field instantaneous SHG imaging over areas containing several hole arrays using a recent, non-linear single-spinning disk microscope. Such high spatio-temporal resolution imaging has important applications, e.g., when studying chemical transformations occurring at electrode surfaces during charging and discharging cycles, as well as ageing.

Nonlinear Microscopy Imaging of Living Cells

蛍光分子の2光子励起に代表される非線形光学現象には，それ以外にも様々な種類がある．これらは生細胞イメージングに適した特長を共有しつつ，それぞれ独自の情報を観測者に与える．近年の技術発展により応用性が拡大する種々の非線形光学顕微鏡の活用により，生細胞の可視化解析に新たな展開がもたらされると期待される．

Quantitative tissue perfusion imaging using nonlinear ultrasound localization microscopy

Ultrasound localization microscopy (ULM) is a recent advancement in ultrasound imaging that uses microbubble contrast agents to yield vascular images that break the classical diffraction limit on spatial resolution. Current approaches cannot image blood flow at the tissue perfusion level since they rely solely on differences in velocity to separate tissue and microbubble signals; lower velocity microbubble echoes are removed during high pass wall filtering. To visualize blood flow in the entire vascular tree, we have developed nonlinear ULM, which combines nonlinear pulsing sequences with plane-wave imaging to segment microbubble signals independent of their velocity. Bubble localization and inter-frame tracking produces super-resolved images and, with parameters derived from the bubble tracks, a rich quantitative feature set that can describe the relative quality of microcirculatory flow. Using the rat spinal cord as a model system, we showed that nonlinear ULM better resolves some smaller branching vasculature compared to conventional ULM. Following contusion injury, both gold-standard histological techniques and nonlinear ULM depicted reduced in-plane vessel length between the penumbra and contralateral gray matter (−16.7% vs. −20.5%, respectively). Here, we demonstrate that nonlinear ULM uniquely enables investigation and potential quantification of tissue perfusion, arguably the most important component of blood flow.

In-depth polarisation resolved SHG microscopy in biological tissues using iterative wavefront optimisation.

Polarised nonlinear microscopy has been extensively developed to study molecular organisation in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarisation. Polarisation Second harmonic Generation (PSHG) in particular is a powerful tool to decipher sub-microscopic modifications of fibrillar collagen organisation in type I and III collagen-rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometres in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarisation control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimisation methods to recover a high-quality linear polarisation and investigate how this approach can be applied to in-depth PSHG imaging in tissues, namely tendon and mouse cranial bone.

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae.

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models.

Optical diffraction tomography of second-order nonlinear structures in weak scattering media: theoretical analysis and experimental consideration.

Computed tomography (CT) allows for high lateral and axial resolution imaging of the endogenous structure of matter thanks to its large spatial frequency support and has been realized in X-ray and linear optical domain known as optical diffraction tomography (ODT). Here, we present the theoretical basis and experimental considerations for ODT of second-order nonlinear structures in weak scattering media. We have derived the relation between second harmonic wave and the anisotropic nonlinear tensor in spatial frequency domain under first-order Born approximation. Our results show that, under a plane wave illumination, the two dimensional (2D) spatial spectra of generated second harmonic complex field relates to the inverse lattice of nonlinear structure on Ewald sphere shells. The centers of the Ewald spheres are determined by 2 times wavevector of the incident fundamental wave and the radii are determined by the modulus of the second harmonic wavevector. More importantly, it shows that the 2D spatial spectra is a superposition of the Ewald spheres of different components of the anisotropic nonlinear tensor. We propose to solve the inverse problem by controlling the polarizations of the fundamental and second harmonic signal. We tested the feasibility of the proposed method using a numerical phantom and make some discussions on practical implementations, including angular scanning schemes, polarization detection and illumination profile for optimizing reconstruction region. Possessing high resolution, wide-field imaging and polarization-sensitive property, we believe that the proposed scheme would have important applications in nonlinear microscopy.

Label-free imaging of engineered human myocardium (EHM) overtime for comprehensive structural characterization using non-linear optical microscopy (NLOM)

Label-free imaging of engineered human myocardium (EHM) overtime for comprehensive structural characterization using non-linear optical microscopy (NLOM)

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.

Rapid multiplex ultrafast nonlinear microscopy for material characterization.

We demonstrate rapid imaging based on four-wave mixing (FWM) by assessing the quality of advanced materials through measurement of their nonlinear response, exciton dephasing, and exciton lifetimes. We use a WSe2 monolayer grown by chemical vapor deposition as a canonical example to demonstrate these capabilities. By comparison, we show that extracting material parameters such as FWM intensity, dephasing times, excited state lifetimes, and distribution of dark/localized states allows for a more accurate assessment of the quality of a sample than current prevalent techniques, including white light microscopy and linear micro-reflectance spectroscopy. We further discuss future improvements of the ultrafast FWM techniques by modeling the robustness of exponential decay fits to different spacing of the sampling points. Employing ultrafast nonlinear imaging in real-time at room temperature bears the potential for rapid in-situ sample characterization of advanced materials and beyond.

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions.

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression

Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

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.

Emerging photonic technologies for cultural heritage studies: the examples of non-linear optical microscopy and photoacoustic imaging

The availability of non-invasive technologies, which can be used separately or in combination for obtaining chemical composition data and structural information of Cultural Heritage (CH) materials, is of prime importance for improving the understanding the environmental or ageing impact on monuments and artefacts and defining optimal strategies for their conservation. This paper overviews and assesses the potential of two emerging photonic technologies, the Non-linear Optical Microscopy (NLOM) and Photoacoustic (PA) imaging modalities, for a variety of diagnostic applications in preservation science. These techniques, which are well-established in biomedical research, during the last few years have been also investigated as non-invasive tools for the in-depth, high-resolution analysis of various CH objects, including paintings, documents and murals. We discuss on the applicability of these diagnostic optical methods to obtain precise stratigraphic information in artefacts, evaluating additionally the presence and the extent of potential morphological or chemical changes in several CH materials due to ageing. Furthermore, we demonstrate that the contrast complementarity of NLOM and PA imaging provides invaluable insights into the structural integrity of an artwork, which can be subsequently utilized for the early and accurate detection of depth degradation effects.

Dual wavelength generation and wavelength tunability in Yb-doped mode-locked laser using few-mode fiber as a saturable absorber

In this research, we demonstrate wavelength tunability and dual wavelength generation in all-fiber ytterbium (Yb)-doped mode-locked laser using a single mode–few-mode–single mode fiber (SMF–FMF–SMF) combination act as a saturable absorber (SA). The all-fiber SA exhibits high modulation depth of 34.53% and a saturation intensity of 9.17 MW/cm2. The SMF–FMF–SMF structure encompasses an inherent filtering along with SA property. By combining the tunable intra-cavity birefringence of the laser with the SA features result in dual-wavelength generation and wavelength tunability. Thus, the tuning of center wavelength of output spectrum from 1028.88 through 1076.18 nm, with the compressed pulse widths from 319 to 324 fs respectively, is achieved. Besides, the generated dual-wavelength is switched from 1033.32/1058.2 nm to 1049.7/1075.7 nm, respectively. These results indicate that an all-fiber SMF–FMF–SMF structure is a potential SA element for wavelength tunability and dual-wavelength mode-locking in Yb-doped fiber lasers, which can be used for nonlinear microscopy, bio-degradable material processing and terahertz spectroscopy.

Multidimensional quantitative characterization of the tumor microenvironment by multicontrast nonlinear microscopy.

Characterization of the microenvironment features of tumors, such as its microstructures, biomolecular metabolism, and functional dynamics, may provide essential pathologic information about the tumor, tumor margin, and adjacent normal tissue for early and intraoperative diagnosis. However, it can be particularly challenging to obtain faithful and comprehensive pathological information simultaneously from unperturbed tissues due to the complexity of the microenvironment in organisms. Super-multiplex nonlinear optical imaging system emerged and matured as an attractive tool for acquisition and elucidation of the nonlinear properties correlated with tumor microenvironment. Here, we introduced a nonlinear effects-based multidimensional optical imaging platform and methodology to simultaneously and efficiently capture contrasting and complementary nonlinear optical signatures of freshly excised human skin tissues. The qualitative and quantitative analysis of autofluorescence (FAD), collagen fiber, and intracellular components (lipids and proteins) illustrated the differences about morphological changes and biomolecular metabolic processes of the epidermis and dermis in different skin carcinogenic types. Interpretation of multi-parameter stain-free histological findings complements conventional H&E-stained slides for investigating basal cell carcinoma and pigmented nevus, validates the platform's versatility and efficiency for classifying subtypes of skin carcinoma, and provides the potential to translate endogenous molecule into biomarker for assisting in rapid cancer screening and diagnosis.

In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study.

.SignificanceThe creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring.AimWe aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids.ApproachA benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention.ResultsFocused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation.ConclusionsWe successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Deep low-excitation fluorescence imaging enhancement

In this work, to the best of our knowledge, we provide the first deep low-excitation fluorescence imaging enhancement solution to reconstruct optimized-excitation fluorescence images from captured low-excitation ones aimed at reducing photobleaching and phototoxicity due to strong excitation. In such a solution, a new framework named Kindred-Nets is designed aimed at improving the effective feature utilization rate; and additionally, a mixed fine-tuning tactic is employed to significantly reduce the required number of fluorescence images for training but still to increase the effective feature density. Proved in applications, the proposed solution can obtain optimized-excitation fluorescence images in high contrast and avoid the dimming effect due to negative optimization from the ineffective features on the neural networks. This work can be employed in fluorescence imaging with reduced excitation as well as extended to nonlinear optical microscopy especially in conditions with low output nonlinear signals. Furthermore, this work is open source available at https://github.com/GuYuanjie/KindredNets.