Melanopsin, a tri-stable photopigment found in intrinsically-photosensitive retinal ganglion cells (ipRGCs), drives circadian rhythms and other non-image forming functions in the nervous system. Despite increased understanding of the biomolecular and spectroscopic properties of melanopsin, its multiphoton and ultrafast optical absorption properties remain underexplored. We demonstrate the effects of two-photon absorption of melanopsin using 900-1160 nm optical stimulation. Excitation in this bandwidth causes consistent increases in calcium levels in transfected HEK293T cells. Our results demonstrate the first reported nonlinear optical properties and corresponding functional responses of two-photon excitation of melanopsin in vitro, along with the effects of spectral-phase modulation on activation.
Nonlinear microscopy encompasses a range of imaging techniques that leverage laser technology to reveal the chemical composition and structure of a sample. Nonlinear microscopes exploit femtosecond laser pulses to target intrinsic biomolecules of cells and tissues. Fiber lasers have limited bandwidth and reduced wavelength tunability, leading to long pulse durations and limited molecular applications. Supercontinuum generation solves this problem, enabling <50 fs pulses and a larger range of molecular excitation. This paper presents nonlinear microscopy with supercontinuum generation from a Yttrium Aluminum Garnet (YAG) crystal enabling simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy with high resolution and specificity in biological tissues.
Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy is a nonlinear multimodal optical imaging technique with sub-micron spatial resolution, enabling 3-D visualization and analysis of live cells, complex in vitro models, and tissues. SLAM microscopy detects NAD(P)H and FAD autofluorescence as well as second and third harmonic generation signals simultaneously from biological samples. It can be used for a wide range of applications in cell-to-clinic pharmaceutical research. To run proof-of-concept, longitudinal, and clinical studies of interest to GSK project teams, the GSK Center for Optical Molecular Imaging (COMI) was established in 2015. Based on promising results from these studies, GSK contracted with spin-out start-up, LiveBx, to design and develop the first portable SLAM microscope, and is currently being used for studies on-site at GSK. In this presentation, major milestones and challenges in translating the SLAM technology from academia to industry and key learnings from this process will be shared from multiple perspectives.
Multiphoton microscopy applied in bone tissue is susceptible to optical aberrations caused by heterogeneity in refractive index. Optical clearing can be applied to alleviate some of these aberrations, but it is invasive and causes deviations from normal tissue biology. We recover diffraction limited imaging by means of a high spatial frequency digital micromirror device (DMD), and binary wavefront modulation. A genetic algorithm optimizes the DMD pattern by evaluating the intensity of the Second Harmonic Generation point spread function measured in the bone sample. We present a five-fold GFP intensity improvement, and a 29% spatial resolution increase within an ex vivo mouse sample.
Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. In this study, we create a next-generation two-photon fluorescence microscope that leverages low-order wavefront correction by Shack-Hartmann wavefront sensor based on different metrics to achieve fast imaging of subcellular organelles of highly scattering living mice. Metrics include maximum intensity, minimum full width at half maximum (FWHM), and maximum energy of the point spread function (PSF), enabling accuracy and robustness of sensorless correction of the system. Using AO increases the fluorescence intensity and FWHM of the PSF and achieves fast imaging of subcellular organelles with 400nm resolution through 85 μm of highly scattering tissue. This study demonstrates a promising tool for imaging mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.
We propose a versatile pulse shaper using a 2D spatial light modulator to perform simultaneous and independent control of the spectral amplitude and phase of a fiber-based supercontinuum source. Different spectral optimizations will be demonstrated in a multimodal optical imaging system, combining optical coherence tomography (OCT), multiphoton microscopy (MPM), and nonlinear Raman imaging. This custom pulse shaper enables fast wavelength-tunable excitation for Raman spectroscopy with high spectral resolution. Single beam simultaneous MPM and OCT can also be enabled by compressing a selected multiphoton excitation band while chirping the whole spectrum for ultra-high-resolution OCT.
This research presents our discovery of two new nonlinear optical biomarkers of Alzheimer’s disease (AD), namely 3-photon autofluorescence (3PAF), and Third Harmonic Generation (THG). A hallmark of AD is the aggregation of the Amyloid-Beta (Aβ) protein and Tau protein. Identification of these plaques and analysis of the surrounding cells and tissue is most often done using immunohistochemistry, often with inconsistent results. Using label-free nonlinear optical microscopy, new optical biomarkers were found for identifying a plaque. We present longitudinal imaging of AD progression in mice ranging from 8 to 52 weeks in age, in the hippocampal and cortical regions.
KEYWORDS: Signal to noise ratio, Microscopy, Heterodyning, Harmonic generation, Tissues, Signal processing, Interference (communication), In vivo imaging, Ultrafast lasers, Tissue optics
Nonlinear label-free microscopy techniques have shown great promise for analyzing biological tissues with an unmatched level of information. These techniques are capable of identifying intrinsic optical signals generated in response to ultrafast laser pulses. A roadblock in fast label-free imaging is the limited signal-to-noise ratio (SNR), due to the low number of photons close to the noise level. We implemented heterodyne detection of the third-order signals to overcome this limit and surpass the 1/f noise. Our method increases the SNR by a factor of 2.3, leading to a factor of 5 reduction in imaging time.
Hematoxylin and eosin (H&E) staining in histology, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and detect disorders such as cancer. Even in the modern era, several new label-free imaging techniques have been developed which provide many more precise layers of information, but they have yet to translate to the clinic, mainly due to a lack of direct comparison between these techniques. Here we present the use of laser ablation to produce fiduciary markers to enable the correlation of nonlinear microscopy with the gold-standard tool of histology.
Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the low imaging speed and the accompanying photodamage risk hinder further clinical applications. To reduce these adverse effects, in this study, we constructed a new generation of simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy, featuring high-speed, multimodal imaging, monitorable-photodamage, and tunable ultrashort pulses. By using birefringent photonic crystal fiber and a pulse shaper, this system has the ability to allow users to independently adjust repetition rate, pulse width, and average power without overlapping interference, and can realize multiphoton generation in each channel from a single excitation pulse. These outstanding advantages represent a powerful and user-friendly imaging platform.
Image Scanning Microscopy (ISM) super-resolution microscopy has gained momentum for its almost instantaneous improved resolution capabilities. Multiphoton ISM utilizes the shorter emission wavelength and confocal acquisition by exploiting each pixel on the camera as a pinhole, and numerical enhancement to achieve sub-diffraction-limit resolution. We present the use of a multiplexed approach for signal acquisition using a regular EMCCD camera. A spatiotemporal modulation scheme is employed to direct the ultrafast laser pulses to select foci within a field-of-view. Combined with a novel image acquisition method, we show that only 49 images are required to achieve a resolution of 100 nm.
Scattering is a major obstacle on the way of imaging deeper than a few mean-free-paths through bone. The high density of mineralization and collagen fibers deposition make bone a very inhomogeneous tissue that produces severe scattering. Although long wavelength excitation extends the mean-free-path for multi-photon microscopy, however imaging more than 150 microns through bone suffers from loss of resolution and intensity. We previously simulated the wavefront distortions caused by bone using phase accumulation ray tracing (PART) method. Our findings show that some low-order optical aberrations can be corrected using traditional adaptive optics systems such as a deformable mirror, however, a significant amount of high order aberrations are remaining, which require a secondary correction method to restore the point spread function at depth. In this work, we use a high-speed binary wavefront correction method using a digital light processor (DLP) to correct the wavefront in a hostile environment such as bone. We use the PART method to produce an initial estimate of the wavefront, and use a genetic algorithm to evolve it to an optimum using maximum intensity metric. The binary wavefront correction produces a factor of 21 enhancement and the initialization using PART method increases the enhancement 2.5 times.
More than 54 million Americans have or are at high risk of developing a metabolic bone disease; disorders of bone strength that leave individuals with fragile bones and disabilities. The gold standard to evaluate these diseases is dual energy x-ray absorptiometry, but this only measures mineral content. These diseases, however, impact collagen and mineral integrity which impede the bone’s ability to store hormones, proteoglycans, and glycoproteins imperative to homeostasis. We have established a second harmonic generation (SHG) polarimetric assay that describes bone collagen organization. To further our analysis, we propose multimodal optical evaluation of bone quality with third harmonic generation (THG) to measure osteocyte dendritic processes. This method of analysis could be used to evaluate the disease state of bone and response to therapy.
Near infrared and infrared multi-photon imaging through or inside bone is an emerging field that promises to help answer many biological questions that require minimally invasive intravital imaging. Neuroscience researchers especially have begun to take advantage of long wavelength imaging to overcome multiple scattering and image deep inside the brain through intact or partially intact bone. Since the murine model is used in many biological experiments, here we investigate the optical aberrations caused by mouse cranial bone, and their effects on light propagation. We previously developed a ray tracing model that uses second harmonic generation in collagen fibers of bone to estimate the refractive index structure of the sample. This technique is able to rapidly provide initial information for a closed loop adaptive optics system. However, the ray tracing method does not account for refraction or scattering. Here, we extend our work to investigate the wavefront aberrations in bone using a full electromagnetic model. We used Finite-Difference Time-Domain modeling of light propagation in refractive index bone datasets acquired with second harmonic generation imaging. In this paper we show modeled wavefront phase from different originating points across the field of view.
Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.
Bone growth and strength is severely impacted by Hypophosphatasia (HPP). It is a genetic disease that affects the mineralization of the bone. We hypothesize that it impacts overall organization, density, and porosity of collagen fibers. Lower density of fibers and higher porosity cause less absorption and scattering of light, and therefore a different regime of transport mean free path. To find a cure for this disease, a metric for the evaluation of bone is required. Here we present an evaluation method based on our Phase Accumulation Ray Tracing (PART) method. This method uses second harmonic generation (SHG) in bone collagen fiber to model bone indices of refraction, which is used to calculate phase retardation on the propagation path of light in bone. The calculated phase is then expanded using Zernike polynomials up to 15th order, to make a quantitative analysis of tissue anomalies. Because the Zernike modes are a complete set of orthogonal polynomials, we can compare low and high order modes in HPP, compare them with healthy wild type mice, to identify the differences between their geometry and structure. Larger coefficients of low order modes show more uniform fiber density and less porosity, whereas the opposite is shown with larger coefficients of higher order modes. Our analyses show significant difference between Zernike modes in different types of bone evidenced by Principal Components Analysis (PCA).
Superresolution microscopy is rapidly becoming an essential tool in the biological sciences allowing imaging biological structure at length scales below 250 nm. Currently, superresolution microscopy has been applied successfully on single cells achieving resolutions of 100nm down to 20nm over a few microns of depth. When superresolution microscopy is applied in thicker samples the resolution rapidly degrades. Optical aberrations and scattering distort and reduce the point spread function causing different superresolution techniques to fail in different ways. I will discuss our work on combining structured illumination microscopy and stochastic optical reconstruction microscopy with adaptive optics to achieve sub-diffraction resolution in thick tissue.
Optical aberrations significantly affect the resolution and signal-to-noise ratio of deep tissue microscopy. As multiphoton microscopy is applied deeper into tissue, the loss of resolution and signal due to propagation of light in a medium with heterogeneous refractive index becomes more serious. Efforts in imaging through the intact skull of mice cannot typically reach past the bone marrow (∼150 μm of depth) and have limited resolution and penetration depth. Mechanical bone thinning or optical ablation of bone enables deeper imaging, but these methods are highly invasive and may impact tissue biology. Adaptive optics is a promising noninvasive alternative for restoring optical resolution. We characterize the aberrations present in bone using second-harmonic generation imaging of collagen. We simulate light propagation through highly scattering bone and evaluate the effect of aberrations on the point spread function. We then calculate the wavefront and expand it in Zernike orthogonal polynomials to determine the strength of different optical aberrations. We further compare the corrected wavefront and the residual wavefront error, and suggest a correction element with high number of elements or multiconjugate wavefront correction for this highly scattering environment.
Multiphoton imaging through the bone to image into the bone marrow or the brain is an emerging need in the scientific community. Due to the highly scattering nature of bone, bone thinning or removal is typically required to enhance the resolution and signal intensity at the imaging plane. The optical aberrations and scattering in the bone significantly affect the resolution and signal to noise ratio of deep tissue microscopy. Multiphoton microscopy uses long wavelength (nearinfrared and infrared) excitation light to reduce the effects of scattering. However, it is still susceptible to optical aberrations and scattering since the light propagates through several layers of media with inhomogeneous indices of refraction. Mechanical removal of bone is highly invasive, laborious, and cannot be applied in experiments where imaging inside of the bone is desired. Adaptive optics technology can compensate for these optical aberrations and potentially restore the diffraction limited point spread function of the system even in deep tissue. To design an adaptive optics system, a priori knowledge of the sample structure assists selection of the proper correction element and sensing methods. In this work we present the characterization of optical aberrations caused by mouse cranial bone, using second harmonic generation imaging of bone collagen. We simulate light propagation through the bone, calculate aberrations and determine the correction that can be achieved using a deformable mirror.
Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acousto-optics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electro-wetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning.
Osteogenesis imperfecta (OI) is a currently uncurable disease where a mutation in collagen type I yields brittle bones. One potential therapy is transplantation of mesenchymal stem cells (MSCs), but controlling and enhancing transplanted cell survival has proven challenging. Therefore, we use a 2- photon imaging system to study individual transplanted cells in the living bone marrow. We ablated cells deep in the bone marrow and observed minimal collateral damage to surrounding tissue. Future work will evaluate the local impact of transplanted MSCs on bone deposition in vivo.
Wavefront sensorless schemes for correction of aberrations induced by biological specimens require a time invariant property of an image as a measure of fitness. Image intensity cannot be used as a metric for Single Molecule Localization (SML) microscopy because the intensity of blinking fluorophores follows exponential statistics. Therefore a robust intensity-independent metric is required. We previously reported a Fourier Metric (FM) that is relatively intensity independent. The Fourier metric has been successfully tested on two machine learning algorithms, a Genetic Algorithm and Particle Swarm Optimization, for wavefront correction about 50 μm deep inside the Central Nervous System (CNS) of Drosophila. However, since the spatial frequencies that need to be optimized fall into regions of the Optical Transfer Function (OTF) that are more susceptible to noise, adding a level of denoising can improve performance. Here we present wavelet-based approaches to lower the noise level and produce a more consistent metric. We compare performance of different wavelets such as Daubechies, Bi-Orthogonal, and reverse Bi-orthogonal of different degrees and orders for pre-processing of images.
Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.
Although Single Molecule Localization (SML) techniques have pushed the resolution of fluorescence microscopy beyond the diffraction limit, the accuracy of SML has been limited by the brightness of the fluorophores. The introduction of Quantum Dots (QD) for SML promises to overcome this barrier, and the QD Blueing technique provides a novel approach to SML microscopy. QDs have a higher quantum yield and absorption cross-section, making them brighter, thereby providing a higher accuracy of localization. The blueing technique is also faster and more quantitative than other SML techniques such as dSTORM. The initial bleaching step required by dSTORM is not necessary and each QD is imaged only once as its emission spectrum moves through the blueing window in contrast to dSTORM where the same molecule might be imaged multiple times. Single color QD Blueing has been demonstrated. However in biological imaging, multi-color imaging is essential for understanding the samples under study. Here we introduce two color superresolution microscopy using QD Blueing on biological samples. We demonstrate simultaneous imaging of microtubules and mitochondria in HepG2 cells with a localization accuracy of 40nm. We further show how QD Blueing can be optimized through the control of the sample mounting medium.
Stochastic Optical Reconstruction Microscopy (STORM) requires a high Strehl ratio point spread function (PSF) to
achieve high resolution, especially in the presence of background fluorescence. The PSF is degraded by aberrations
caused by imperfections in the optics, the refractive index mismatch between the sample and coverslip, and the refractive
index variations of the sample. These aberrations distort the shape of the PSF and increase the PSF width directly
reducing the resolution of STORM. Here we discuss the use of Adaptive Optics (AO) to correct aberrations, maintaining
a high Strehl ratio even in thick tissue. Because the intensity fluctuates strongly from frame to frame, image intensity is
not a reliable measure of PSF quality, and the choice of a robust optimization metric is critical. We demonstrate the use
of genetic algorithms with single molecule imaging for optimization of the wavefront and introduce a metric that is
relatively insensitive to image intensity. We demonstrate the correction of the wavefront from measurements of single
quantum dots.
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