Optical imaging in musculoskeletal systems can provide dynamic cellular-resolution analysis of biological tissues and regenerative processes. Here, we advance multiphoton imaging and analysis techniques to explore therapeutic cell homing in the bone, mitochondrial dynamics during regeneration in muscle and bone, and collagen organization in metabolic bone disorders. We develop aberration correction approaches to recover high-resolution imaging of cell dynamics deep in the bone marrow and in muscle. We leverage our imaging techniques to explore single mitochondrial dynamics in skeletal muscle, and the disruptions in mitochondrial order that occur after volumetric muscle loss. Finally, we have developed second harmonic generation imaging using polarized light for analysis of bone collagen assembly into lamella sheet structures and have investigated collagen organization in metabolic bone disorders. Our findings could provide insight into dynamic processes driving musculoskeletal homeostasis and fragility disorders.
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.
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).
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.
KEYWORDS: Luminescence, Prototyping, Data acquisition, In vivo imaging, Signal to noise ratio, Control systems, Flow cytometry, Signal detection, Sensors, Optical spheres
Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×10 3 cells/mL . In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo.
The growing presence of quantum dots (QD) in a variety of biological, medical, and electronics applications means an
increased risk of human exposure in manufacturing, research, and consumer use. However, very few studies have
investigated the susceptibility of skin to penetration of QD - the most common exposure route- and the results of those
that exist are conflicting. This suggests that a technique allowing determination of skin barrier status and prediction of
skin permeability to QD would be of crucial interest as recent findings have provided evidence of in vitro cytotoxicity
and long-term in vivo retention in the body for most QD surface chemistries. Our research focuses on barrier status of the
skin (intact and with ultraviolet radiation induced barrier defect) and its impact on QD skin penetration. These model
studies are particularly relevant to the common application condition of NP containing sunscreen and SPF cosmetics to
UV exposed skin. Herein we present our initial efforts to develop an in vivo model of nanoparticle skin penetration using
the SKH-1 hairless mouse with transepidermal water loss (TEWL) to evaluate skin barrier status and determine its
ability to predict QD penetration. Our results show that ultraviolet radiation increases both TEWL and skin penetration
of QD. Additionally, we demonstrate cytotoxic potential of QD to skin cells using a metastatic melanoma cell line. Our
research suggests future work in specific targeting of nanoparticles, to prevent or enhance penetration. This knowledge
will be used to develop powerful therapeutic agents, decreased penetration cosmetic nanoparticles, and precise skin
cancer imaging modalities.
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