Nonlinear Microscopy Techniques Research Articles

Investigation of Artists Pigments with a Nonlinear Microscopy Technique

Investigation of Artists Pigments with a Nonlinear Microscopy Technique

Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques – a short review including new research

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or ‘label-free’ imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive. Computational imaging denotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques, with an emphasis on Fourier ptychographic microscopy (FPM) and advanced x-ray microscopies. We next demonstrate with our own results computational imaging through FPM and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are explored. Furthermore, X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

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.

Collagen organization and structure in FBLN5-/- mice using label-free microscopy: implications for pelvic organ prolapse.

Pelvic organ prolapse (POP) is a gynecological disorder described by the descent of superior pelvic organs into or out of the vagina as a consequence of disrupted muscles and tissue. A thorough understanding of the etiology of POP is limited by the availability of clinically relevant samples, restricting longitudinal POP studies on soft-tissue biomechanics and structure to POP-induced models such as fibulin-5 knockout (FBLN5-/- ) mice. Despite being a principal constituent in the extracellular matrix, little is known about structural perturbations to collagen networks in the FBLN5-/- mouse cervix. We identify significantly different collagen network populations in normal and prolapsed cervical cross-sections using two label-free, nonlinear microscopy techniques. Collagen in the prolapsed mouse cervix tends to be more isotropic, and displays reduced alignment persistence via 2-D Fourier transform analysis of images acquired using second harmonic generation microscopy. Furthermore, coherent Raman hyperspectral imaging revealed elevated disorder in the secondary structure of collagen in prolapsed tissues. Our results underscore the need for in situ multimodal monitoring of collagen organization to improve POP predictive capabilities.

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.

Expanding the applicability of multiphoton fluorescence recovery after photobleaching by incorporating shear stress in laminar flow.

Multi-photon fluorescence recovery after photobleaching (MPFRAP) is a nonlinear microscopy technique used to measure the diffusion coefficient of fluorescently tagged molecules in solution. Previous MPFRAP fitting models calculate the diffusion coefficient in systems with diffusion or diffusion in laminar flow. We propose an MPFRAP fitting model that accounts for shear stress in laminar flow, making it a more applicable technique for in vitro and in vivo studies involving diffusion. Fluorescence recovery curves are generated using high-throughput molecular dynamics simulations and then fit to all three models (diffusion, diffusion and flow, and diffusion and shear flow) to define the limits within which accurate diffusion coefficients are produced. Diffusion is simulated as a random walk with a variable horizontal bias to account for shear flow. Contour maps of the accuracy of the fitted diffusion coefficient as a function of scaled velocity and scaled shear rate show the parameter space within which each model produces accurate diffusion coefficients; the shear-flow model covers a larger area than the previous models. The shear-flow model allows MPFRAP to be a viable optical tool for studying more biophysical systems than previous models.

Non-invasive monitoring of microbial triterpenoid production using nonlinear microscopy techniques.

Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals, pharmaceutics, and cosmetic ingredients. So far, there is no direct method for detecting triterpenoids under physiological conditions on a cellular level, information yet highly relevant to rationalizing microbial engineering. Methods: Here, we show in a proof-of-concept study, that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques. We applied CARS and SHG microscopy measurements, and for comparison classical Nile Red staining, on immobilized and growing triterpenoid-producing, and non-producing reference Saccharomyces cerevisiae strains. Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by Nile red staining. In growing cultures, both CARS and SHG signals showed changes over time, enabling new insights into the dynamics of triterpenoid production and storage inside cells.

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.

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.

Experimental determination of shift-less aberration bases for sensorless adaptive optics in nonlinear microscopy.

Adaptive optics can improve the performance of optical systems and devices by correcting phase aberrations. While in most applications wavefront sensing is employed to drive the adaptive optics correction, some microscopy methods may require sensorless optimization of the wavefront. In these cases, the correction is performed by describing the aberration as a linear combination of a base of influence functions, optimizing an image quality metric as a function of the coefficients. The influence functions base is generally chosen to either efficiently represent the adaptive device used or to describe generic wavefronts in an orthogonal fashion. A rarely discussed problem is that most correction bases have elements which introduce, together with a correction of the aberration, a shift of the imaging field of view in three dimensions. While simple methods to solve the problem are available for linear microscopy methods, nonlinear microscopy techniques such as multiphoton or second harmonic generation microscopy require non-trivial base determination. In this paper, we discuss the problem, and we present a method for calibrating a shift-less base on a spatial light modulator for two-photon microscopy.

Energy-time-entangled two-photon molecular absorption

Nonlinear spectroscopy and microscopy techniques are ubiquitous in a wide range of applications across physics and biology. However, these usually rely on high-powered pulsed laser systems. A promising alternative is to exploit entangled two-photon absorption (ETPA), which can lead to tens of orders of magnitude lower incident flux rates than in conventional two-photon absorption (TPA) schemes. However, the role of different entangled degrees of freedom in ETPA was unclear following recent experimental studies, when compared to earlier theoretical works. Here, we first demonstrate a linear dependence of the ETPA rate with the photon-pair flux, a clear signature of ETPA, and estimate the first values for the concentration-dependent ETPA cross-section for Rhodamine 6G.We then investigate the signature of energy-time entanglement and polarization dependence in the ETPA fluorescence rate and demonstrate a strong dependence of the signal on the inter-photon delay that reflects the coherence time of the entangled two-photon wave-packet.

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.

Self validating Mueller matrix Micro-Mesoscope (SAMMM) for the characterization of biological media.

Reflectance Mueller matrix (MM) polarimetry is being used to characterize biological media in multiple clinical applications. The origin of the reflectance polarimetric data is often unclear due to the impact of multiple scattering and tissue heterogeneity. We have developed a new, to the best of our knowledge, multimodal imaging technique combining MM reflectance, MM digital confocal imaging, and co-registered nonlinear microscopy techniques. The instrument unveils the origin of reflectance polarimetric signature in terms of confocal reflectance data. The reconstructed reflected MM demonstrates the capability of our method to provide depth-resolved 3D polarization response from complex biological media in terms of depolarization, retardance, and orientation parameters.

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.

Non-linear label-free imaging through a multimode graded index optical fibre

In recent years, great advances have been made in developing minimal footprint micro-endoscopes using multimode optical fibres (MMF) [1]. By employing wavefront shaping methods the seemingly random speckle pattern resulting from the guiding of coherent light through an MMF can be formed into a diffraction limited spot. This enables the implementation of multiple laser scanning techniques. In this work we show that this approach can be employed to realize label-free non-linear microscopy techniques such as coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG). Backscattered light makes epi-detection possible even though these processes preferably emit light in the direction of beam propagation [2].

Femtosecond Stimulated Raman microscopy: home-built realization and a case study of biological imaging

The progresses of femtosecond laser physics have had a significant impact on research and development of nonlinear microscopy techniques, such as Stimulated Raman scattering microscopy. The main advantages of this technique, based on vibrational spectroscopy, is its abilities to perform label-free imaging with high sensitivity, high spatial and spectral resolution, 3D sectioning capability and fast time of image acquisition (typically a few seconds). The main aim of this paper is to demonstrates the home-built realization of an imaging system based on Stimulated Raman Scattering (SRS) powered by femtosecond laser sources, using a National Instrument data acquisition (NI-DAQ) card. The SRS microscope is obtained by the integration of an SRS experimental set-up with a commercial laser scanning microscope. The successful implementation of the imaging system is demonstrated reporting label free images of polystyrene beads with diameter of 3 microns and evaluating their signal to noise ratio. Finally, the reliability of our system in a biological environment is tested. Taking advantage of SRS microscopy, subcellular localizations of lipid droplets inside adipocyte cells are demonstrated.

Terahertz Field Confinement in Nonlinear Metamaterials and Near-Field Imaging

This article reviews recent advances in terahertz science and technology that rely on confining the energy of incident terahertz radiation to small, very sub-wavelength sized regions. We focus on two broad areas of application for such field confinement: metamaterial-based nonlinear terahertz devices and terahertz near-field microscopy and spectroscopy techniques. In particular, we focus on field confinement in: terahertz nonlinear absorbers, metamaterial enhanced nonlinear terahertz spectroscopy, and in sub-wavelength terahertz imaging systems.

Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications.

Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. Ex vivo models are of great value for translating in vitro tissue engineered approaches into clinically relevant conditions. Our aim is to obtain a viable human osteochondral (OC) model to test hydrogel-based materials for cartilage repair. Here we describe a detailed step-by-step framework for the generation of human OC plugs, their culture in a perfusion device and the processing procedures for histological and advanced microscopy imaging. Our ex vivo OC model fulfils the following requirements: the model is metabolically stable for a relevant culture period of 4 weeks in a perfusion bioreactor, the processing procedures allowed for the analysis of 3 different tissues or materials (cartilage, bone and hydrogel) without compromising their integrity. We determined a protocol and the settings for a non-linear microscopy technique on label free sections. Furthermore, we established a clearing protocol to perform light sheet-based observations on the cartilage layer without the need for tedious and destructive histological procedures. Finally, we showed that our OC system is a clinically relevant in terms of cartilage regeneration potential. In conclusion, this OC model represents a valuable preclinical ex vivo tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for in vivo testing.