Biological functions rely on local microvasculature for delivering oxygen and nutrients and carrying away metabolic waste. Alterations to local oxygenation level are manifested in diseases including cancer, diabetes mellitus, etc. The ability to in vivo quantify oxygen saturation (sO2) of single vessels down to capillary level to assess local tissue oxygenation and metabolic function is highly sought after. Visible light optical coherence tomography (vis-OCT) has shown promise in reaching this goal. However, to achieve reliable measurement in small vessels are challenging due to the reduced signal and requires data averaging to improve the spectral data quality. Therefore, a method to quality control the vis-OCT data from small vessels becomes essential to reject unreliable readings. In this work, we present a generalized method with several quantitative metrics to evaluate the spectral data for reliable sO2 measurements. Parameters of the scanning protocol and the statistical data cleaning can be flexibly adjusted according to different applications and system performances. We used this method to measure sO2 of C57BL/6J mice lower extremity microvasculature and validated it via introducing hyperoxia for expected sO2 changes. After validation, we applied this method on C57BL/J mouse ear microvasculature to conduct in vivo single capillary OCT oximetry. This work intends to standardize the data processing method for in vivo oximetry in small vessels by vis-OCT.
Fluorescence photo-switching of native, unmodified DNA using visible light enables label-free, nanoscale, single-molecule photon localization microscopy (PLM) of chromatin structure. Compared with conventional label-based super resolution imaging techniques, the label-free DNA-PLM has the advantage of faithfully resolving the native nucleotides under non-perturbing conditions, thus allowing a reliable analysis of the chromatin organization. Recently, we have developed an algorithm to quantify the chromatin spatial distribution based on label-free DNA-PLM images by calculating the fractal dimension from the chromatin cluster size and the number of photon emission events. For demonstration, we employed label-free DNA-PLM with TIRF illumination, and imaged the nuclei of ovarian cancer cells with three descending chromatin heterogeneities: the P53 mutation (M248), the wild type (A2780), and the wild type treated with a commonly-used chemotherapeutic drug celecoxib (Cele). Using the algorithm, we extracted the fractal dimensions for nuclear chromatin. We found that the fractal dimension is between 2 to 3 for all cells, which lies in the range of reported values from other techniques (e.g., TEM). We also observed that M248 has the highest fractal dimension while Cele has the lowest, a perfect match with the experimental expectations. We believe this study can provide a new approach to quantify label-free super-resolution imaging of macromolecular structures and could contribute to our knowledge of native in-vitro nuclear chromatin configurations.
Oxygen saturation (sO2) of RBCs in capillaries can indirectly assess local tissue oxygenation and metabolic function. For example, the altered retinal oxygenation in diabetic retinopathy and local hypoxia during tumor development in cancer are reflected by abnormal sO2 of local capillary networks. However, it is far from clear whether accurate label-free optical oximetry (i.e. measuring hemoglobin sO2) is feasible from dispersed red blood cells (RBCs) at the single-capillary level. The sO2-dependent hemoglobin absorption contrast present in optical scattering signal is complicated by geometry-dependent scattering from RBCs. Here we provide a theoretical model to calculate the backscattering spectra of single RBCs based on the first-order Born approximation, considering the orientation, size variation, and deformation of RBCs. We show that the oscillatory spectral behavior of RBC geometries is smoothed by variations in cell size and orientation, resulting in clear sO2-dependent spectral contrast. In addition, this spectral contrast persists with different deformations of RBCs, allowing the sO2 of individual RBCs in capillaries to be characterized. The theoretical model is verified by Mie theory and experiments using visible light optical coherence tomography (vis-OCT). Thus, this study shows for the first time the feasibility of, and provides a theoretical model for, label-free optical oximetry at the single-capillary level by backscattering-based imaging modalities, challenging the popular view that such measurements are impossible at the single-capillary level. This is promising for in vivo backscattering-based optical oximetry at the single-capillary level, to measure local capillary sO2 for early diagnosis, progression monitoring, and treatment evaluation of diabetic retinopathy and cancer.
Oxygen saturation (sO2) of red blood cells (RBCs) in capillaries can indirectly assess local tissue oxygenation and metabolic function. For example, the altered retinal oxygenation in diabetic retinopathy and local hypoxia during tumor development in cancer are reflected by abnormal sO2 of local capillary networks. However, it is far from clear whether accurate label-free optical oximetry (i.e., measuring hemoglobin sO2) is feasible from dispersed RBCs at the single capillary level. The sO2-dependent hemoglobin absorption contrast present in optical scattering signal is complicated by geometry-dependent scattering from RBCs. We present a numerical study of backscattering spectra from single RBCs based on the first-order Born approximation, considering practical factors: RBC orientations, size variation, and deformations. We show that the oscillatory spectral behavior of RBC geometries is smoothed by variations in cell size and orientation, resulting in clear sO2-dependent spectral contrast. In addition, this spectral contrast persists with different mean cellular hemoglobin content and different deformations of RBCs. This study shows for the first time the feasibility of, and provides a theoretical model for, label-free optical oximetry at the single capillary level using backscattering-based imaging modalities, challenging the popular view that such measurements are impossible at the single capillary level.
The oxygen-dependent absorption of hemoglobin provides the fundamental contrast for all label-free techniques measuring blood oxygenation. When hemoglobin is packaged into red blood cells (RBCs), the structure of the cells creates light scattering which also depends on the absorption based on the Kramers-Kronig relationship. Thus a proper characterization of the optical behaviors of blood has been a key to any accurate measurement of blood oxygenation, particularly at the capillary level where RBCs are dispersed individually in contrast to a densely packed whole blood. Here we provided a theoretical model under Born Approximation to characterize the oxygen dependent backscattering spectroscopic contrast from single RBCs. Using this theoretical model, we conducted simulations on both oxygenated and deoxygenated single RBCs with different sizes for standard and possible deformed cell geometries in blood flow, all which suggested similar backscattering spectroscopic contrast and were confirmed by Mie Theory and experiments using visible Optical Coherence Tomography (visOCT). As long as the cell size satisfies Gaussian distribution with a coefficient variance (C.V.) large enough, there is clear absorption contrast between the backscattering spectra of oxygenated and deoxygenated single RBCs calculated by this model, so oxygen saturation can then be characterized. Thus, this theoretical model can be extended to extract absorption features of other scattering particles as long as they satisfy Born Approximation.
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