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Skin is the largest organ and also the first barrier of body. Skin diseases are common, and cutaneous microcirculation is relative to various diseases. Researchers attempt to develop novel imaging techniques to obtain the complex structure, components, and functions of skin. Modern optical techniques provide a powerful tool with non-invasiveness, but the imaging performance suffers from the turbid character of skin. In vivo skin optical clearing technique has been proposed to reduce tissue scattering and enhance penetration depth of light and became a hot topic of research.
Aim
The aim of this review is to provide a comprehensive overview of recent development of in vivo skin optical clearing methods, how in vivo skin optical clearing enhances imaging performance, and its applications in study and light therapy of various diseases.
Approach
Based on the references published over the last decade, the important milestones on the mechanism, methods, and its fundamental and clinical applications of in vivo skin optical clearing technique are provided.
Results
With the deepening understanding of skin optical clearing mechanisms, efficient in vivo skin optical clearing methods were constantly screened out. These methods have been combined with various optical imaging techniques to improve imaging performances and acquire deeper and finer skin-related information. In addition, in vivo skin optical clearing technique has been widely applied in assisting study of diseases as well as achieving safe, high-efficiency light-induced therapy.
Conclusions
In the last decade, in vivo skin optical clearing technique has developed rapidly and played an important role in skin-related studies.
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Perturbation and differential Monte Carlo (pMC/dMC) methods, used in conjunction with nonlinear optimization methods, have been successfully applied to solve inverse problems in diffuse optics. Application of pMC to systems over a large range of optical properties requires optimal “placement” of baseline conventional Monte Carlo (cMC) simulations to minimize the pMC variance. The inability to predict the growth in pMC solution uncertainty with perturbation size limits the application of pMC, especially for multispectral datasets where the variation of optical properties can be substantial.
Aim
We aim to predict the variation of pMC variance with perturbation size without explicit computation of perturbed photon weights. Our proposed method can be used to determine the range of optical properties over which pMC predictions provide sufficient accuracy. This method can be used to specify the optical properties for the reference cMC simulations that pMC utilizes to provide accurate predictions over a desired optical property range.
Approach
We utilize a conventional error propagation methodology to calculate changes in pMC relative error for Monte Carlo simulations. We demonstrate this methodology for spatially resolved diffuse reflectance measurements with ±20% scattering perturbations. We examine the performance of our method for reference simulations spanning a broad range of optical properties relevant for diffuse optical imaging of biological tissues. Our predictions are computed using the variance, covariance, and skewness of the photon weight, path length, and collision distributions generated by the reference simulation.
Results
We find that our methodology performs best when used in conjunction with reference cMC simulations that utilize Russian Roulette (RR) method. Specifically, we demonstrate that for a proximal detector placed immediately adjacent to the source, we can estimate the pMC relative error within 5% of the true value for scattering perturbations in the range of [ − 15 % , + 20 % ] . For a distal detector placed at ∼3 transport mean free paths relative to the source, our method provides relative error estimates within 20% for scattering perturbations in the range of [ − 8 % , + 15 % ] . Moreover, reference simulations performed at lower (μs′/μa) values showed better performance for both proximal and distal detectors.
Conclusions
These findings indicate that reference simulations utilizing continuous absorption weighting (CAW) with the Russian Roulette method and executed using optical properties with a low (μs′/μa) ratio spanning the desired range of μs values, are highly advantageous for the deployment of pMC to obtain radiative transport estimates over a wide range of optical properties.
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Blood lipid levels (i.e., triglycerides (TGs) and cholesterol) are a strong predictor of cardiovascular disease (CVD) risk. Current methods for measuring blood lipids require invasive blood draws and traditional lab testing, limiting their practicality for frequent monitoring. Optical measurements of lipoproteins, which carry TG and cholesterol in blood, may lead to simpler invasive or non-invasive methods for more frequent and rapid blood lipid measurements.
Aim
To investigate the effect of lipoproteins on optical properties of blood before and after a high-fat meal (i.e., the pre- and post-prandial state).
Approach
Simulations were performed using Mie theory to estimate lipoprotein scattering properties. A literature review was conducted to identify key simulation parameters including lipoprotein size distributions and number density. Experimental validation of ex-vivo blood samples was conducted using spatial frequency domain imaging.
Results
Our results indicated that lipoproteins in blood, particularly very low-density lipoproteins and chylomicrons, are highly scattering in the visible and near-infrared wavelength region. Estimates of the increase in the reduced scattering coefficient (μs′) of blood at 730 nm after a high-fat meal ranged from 4% for a healthy individual, to 15% for those with type 2 diabetes, to up to 64% for those suffering from hypertriglyceridemia. A reduction in blood scattering anisotropy (g) also occurred as a function of TG concentration increase.
Conclusion
These findings lay the foundation for future research in the development of optical methods for invasive and non-invasive optical measure of blood lipoproteins, which could improve early detection and management of CVD risk.
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Daniel Anaya, Gautam Batra, Peter Bracewell, Ryan Catoen, Dev Chakraborty, Mark Chevillet, Pradeep Damodara, Alvin Dominguez, Laurence Emms, Zifan Jiang, Ealgoo Kim, Keith Klumb, Frances Lau, Rosemary Le, Jamie Li, Brett Mateo, Laura Matloff, Asha Mehta, Emily M. Mugler, Akansh Murthy, Sho Nakagome, Ryan Orendorff, E-Fann Saung, Roland Schwarz, Ruben Sethi, Rudy Sevile, Ajay Srivastava, John Sundberg, Ying Yang, Allen Yin
We present a fiberless, portable, and modular continuous wave-functional near-infrared spectroscopy system, Spotlight, consisting of multiple palm-sized modules—each containing high-density light-emitting diode and silicon photomultiplier detector arrays embedded in a flexible membrane that facilitates optode coupling to scalp curvature.
Aim
Spotlight’s goal is to be a more portable, accessible, and powerful functional near-infrared spectroscopy (fNIRS) device for neuroscience and brain–computer interface (BCI) applications. We hope that the Spotlight designs we share here can spur more advances in fNIRS technology and better enable future non-invasive neuroscience and BCI research.
Approach
We report sensor characteristics in system validation on phantoms and motor cortical hemodynamic responses in a human finger-tapping experiment, where subjects wore custom 3D-printed caps with two sensor modules.
Results
The task conditions can be decoded offline with a median accuracy of 69.6%, reaching 94.7% for the best subject, and at a comparable accuracy in real time for a subset of subjects. We quantified how well the custom caps fitted to each subject and observed that better fit leads to more observed task-dependent hemodynamic response and better decoding accuracy.
Conclusions
The advances presented here should serve to make fNIRS more accessible for BCI applications.
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Collagen and lipid are important components of tumor microenvironments (TME) and participates in tumor development and invasion. It has been reported that collagen and lipid can be used as a hallmark to diagnosis and differentiate tumors.
Aim
We aim to introduce photoacoustic spectral analysis (PASA) method that can provide both the content and structure distribution of endogenous chromophores in biological tissues to characterize the tumor-related features for identifying different types of tumors.
Approach
Ex vivo human tissues with suspected squamous cell carcinoma (SCC), suspected basal cell carcinoma (BCC), and normal tissue were used in this study. The relative lipid and collagen contents in the TME were assessed based on the PASA parameters and compared with histology. Support vector machine (SVM), one of the simplest machine learning tools, was applied for automatic skin cancer type detection.
Results
The PASA results showed that the lipid and collagen levels of the tumors were significantly lower than those of the normal tissue, and there was a statistical difference between SCC and BCC (p < 0.05), consistent with the histopathological results. The SVM-based categorization achieved diagnostic accuracies of 91.7% (normal), 93.3% (SCC), and 91.7% (BCC).
Conclusions
We verified the potential use of collagen and lipid in the TME as biomarkers of tumor diversity and achieved accurate tumor classification based on the collagen and lipid content using PASA. The proposed method provides a new way to diagnose tumors.
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Near-infrared (NIR) diffuse reflectance spectroscopy has been widely used for non-invasive glucose measurement in humans, as glucose can induce a significant and detectable optical signal change in tissue. However, the scattering-dominated glucose spectrum in the range of 1000 to 1700 nm is easily confused with many other scattering factors, such as particle density, particle size, and tissue refractive index.
Aim
Our aim is to identify the subtle distinctions between glucose and these factors through theoretical analysis and experimental verification, in order to employ suitable methods for eliminating these interferences, thus increasing the accuracy of non-invasive glucose measurement.
Approach
We present a theoretical analysis of the spectra of 1000 to 1700 nm for glucose and some scattering factors, which is then verified by an experiment on a 3% Intralipid solution.
Results
We found that both the theoretical and experimental results show that the effective attenuation coefficient of glucose has distinct spectral characteristics, which are distinct from the spectra caused by particle density and refractive index, particularly in the range of 1400 to 1700 nm.
Conclusions
Our findings can offer a theoretical foundation for eliminating these interferences in non-invasive glucose measurement, aiding mathematical methods to model appropriately and enhance the accuracy of glucose prediction.
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TOPICS: Laser irradiation, Fluorescence, Monte Carlo methods, Fluorescence intensity, Diffusion, Molecules, Data modeling, Gold nanoparticles, Time metrology, Pulsed laser operation
Resealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation.
Aim
To establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells.
Approach
Voltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis.
Results
The measured resealing time after perforation varied from 28.6 to 163.8 s on Hela cells when the irradiation fluence was increased, with a correlation coefficient (R2) of 0.9938. This result is in agreement with the resealing time (1-2 min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size.
Conclusion
The method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.
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Parametric imaging of the attenuation coefficient μOCT using optical coherence tomography (OCT) is a promising approach for evaluating abnormalities in tissue. To date, a standardized measure of accuracy and precision of μOCT by the depth-resolved estimation (DRE) method, as an alternative to least squares fitting, is missing.
Aim
We present a robust theoretical framework to determine accuracy and precision of the DRE of μOCT.
Approach
We derive and validate analytical expressions for the accuracy and precision of μOCT determination by the DRE using simulated OCT signals in absence and presence of noise. We compare the theoretically achievable precisions of the DRE method and the least-squares fitting approach.
Results
Our analytical expressions agree with the numerical simulations for high signal-to-noise ratios and qualitatively describe the dependence on noise otherwise. A commonly used simplification of the DRE method results in a systematic overestimation of the attenuation coefficient in the order of μOCT2×Δ, where Δ is the pixel stepsize. When μOCT · | AFR | ≲ 1.8, μOCT is reconstructed with higher precision by the depth-resolved method compared to fitting over the length of an axial fitting range | AFR | .
Conclusions
We derived and validated expressions for the accuracy and precision of DRE of μOCT. A commonly used simplification of this method is not recommended as being used for OCT-attenuation reconstruction. We give a rule of thumb providing guidance in the choice of estimation method.
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When developing a new quantitative optoacoustic computed tomography (OAT) system for diagnostic imaging of breast cancer, objective assessments of various system designs through human trials are infeasible due to cost and ethical concerns. In prototype stages, however, different system designs can be cost-efficiently assessed via virtual imaging trials (VITs) employing ensembles of digital breast phantoms, i.e., numerical breast phantoms (NBPs), that convey clinically relevant variability in anatomy and optoacoustic tissue properties.
Aim
The aim is to develop a framework for generating ensembles of realistic three-dimensional (3D) anatomical, functional, optical, and acoustic NBPs and numerical lesion phantoms (NLPs) for use in VITs of OAT applications in the diagnostic imaging of breast cancer.
Approach
The generation of the anatomical NBPs was accomplished by extending existing NBPs developed by the U.S. Food and Drug Administration. As these were designed for use in mammography applications, substantial modifications were made to improve blood vasculature modeling for use in OAT. The NLPs were modeled to include viable tumor cells only or a combination of viable tumor cells, necrotic core, and peripheral angiogenesis region. Realistic optoacoustic tissue properties were stochastically assigned in the NBPs and NLPs.
Results
To advance optoacoustic and optical imaging research, 84 datasets have been released; these consist of anatomical, functional, optical, and acoustic NBPs and the corresponding simulated multi-wavelength optical fluence, initial pressure, and OAT measurements. The generated NBPs were compared with clinical data with respect to the volume of breast blood vessels and spatially averaged effective optical attenuation. The usefulness of the proposed framework was demonstrated through a case study to investigate the impact of acoustic heterogeneity on OAT images of the breast.
Conclusions
The proposed framework will enhance the authenticity of virtual OAT studies and can be widely employed for the investigation and development of advanced image reconstruction and machine learning-based methods, as well as the objective evaluation and optimization of the OAT system designs.
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Cholesteatoma is an expansile destructive lesion of the middle ear and mastoid, which can result in significant complications by eroding adjacent bony structures. Currently, there is an inability to accurately distinguish cholesteatoma tissue margins from middle ear mucosa tissue, causing a high recidivism rate. Accurately differentiating cholesteatoma and mucosa will enable a more complete removal of the tissue.
Aim
Develop an imaging system to enhance the visibility of cholesteatoma tissue and margins during surgery.
Approach
Cholesteatoma and mucosa tissue samples were excised from the inner ear of patients and illuminated with 405, 450, and 520 nm narrowband lights. Measurements were made with a spectroradiometer equipped with a series of different longpass filters. Images were obtained using a red-green-blue (RGB) digital camera equipped with a long pass filter to block reflected light.
Results
Cholesteatoma tissue fluoresced under 405 and 450 nm illumination. Middle ear mucosa tissue did not fluoresce under the same illumination and measurement conditions. All measurements were negligible under 520 nm illumination conditions. All spectroradiometric measurements of cholesteatoma tissue fluorescence can be predicted by a linear combination of emissions from keratin and flavin adenine dinucleotide. We built a prototype of a fluorescence imaging system using a 495 nm longpass filter in combination with an RGB camera. The system was used to capture calibrated digital camera images of cholesteatoma and mucosa tissue samples. The results confirm that cholesteatoma emits light when it is illuminated with 405 and 450 nm, whereas mucosa tissue does not.
Conclusions
We prototyped an imaging system that is capable of measuring cholesteatoma tissue autofluorescence.
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Conventional diagnosis of laryngeal cancer is normally made by a combination of endoscopic examination, a subsequent biopsy, and histopathology, but this requires several days and unnecessary biopsies can increase pathologist workload. Nonlinear imaging implemented through endoscopy can shorten this diagnosis time, and localize the margin of the cancerous area with high resolution.
Aim
Develop a rigid endomicroscope for the head and neck region, aiming for in-vivo multimodal imaging with a large field of view (FOV) and tissue ablation.
Approach
Three nonlinear imaging modalities, which are coherent anti-Stokes Raman scattering, two-photon excitation fluorescence, and second harmonic generation, as well as the single photon fluorescence of indocyanine green, are applied for multimodal endomicroscopic imaging. High-energy femtosecond laser pulses are transmitted for tissue ablation.
Results
This endomicroscopic system consists of two major parts, one is the rigid endomicroscopic tube 250 mm in length and 6 mm in diameter, and the other is the scan-head (10 × 12 × 6 cm3 in size) for quasi-static scanning imaging. The final multimodal image accomplishes a maximum FOV up to 650 μm, and a resolution of 1 μm is achieved over 560 μm FOV. The optics can easily guide sub-picosecond pulses for ablation.
Conclusions
The system exhibits large potential for helping real-time tissue diagnosis in surgery, by providing histological tissue information with a large FOV and high resolution, label-free. By guiding high-energy fs laser pulses, the system is even able to remove suspicious tissue areas, as has been shown for thin tissue sections in this study.
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Fluorescence molecular tomography (FMT) is a promising imaging modality, which has played a key role in disease progression and treatment response. However, the quality of FMT reconstruction is limited by the strong scattering and inadequate surface measurements, which makes it a highly ill-posed problem. Improving the quality of FMT reconstruction is crucial to meet the actual clinical application requirements.
Aim
We propose an algorithm, neighbor-based adaptive sparsity orthogonal least square (NASOLS), to improve the quality of FMT reconstruction.
Approach
The proposed NASOLS does not require sparsity prior information and is designed to efficiently establish a support set using a neighbor expansion strategy based on the orthogonal least squares algorithm. The performance of the algorithm was tested through numerical simulations, physical phantom experiments, and small animal experiments.
Results
The results of the experiments demonstrated that the NASOLS significantly improves the reconstruction of images according to indicators, especially for double-target reconstruction.
Conclusion
NASOLS can recover the fluorescence target with a good location error according to simulation experiments, phantom experiments and small mice experiments. This method is suitable for sparsity target reconstruction, and it would be applied to early detection of tumors.
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Adaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method.
Aim
Provide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples.
Approach
Development of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget.
Results
Fast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO.
Conclusion
We developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.
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Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.
Aim
We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.
Approach
A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.
Results
An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.
Conclusions
FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
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Bilirubin forms by the breakdown of heme proteins in the liver, but a newborn’s sluggish liver can lead to elevated serum bilirubin levels that cross the blood–brain barrier and result in kernicterus. Earlier studies have used the 400 to 500 nm optical wavelength range to characterize the bilirubin content. There is not a universally established correlation among other wavelengths and the amount of bilirubin in clinical whole blood samples.
Aim
We demonstrated that the amount of bilirubin could be quantified with ∼82 % accuracy in a label-free, self-referenced manner using only a few wavelengths, viz. 468, 492, 500, 560, 605, 645, 660, and 675 nm, wherein band-averaged absorption measurements are used.
Approach
We addressed the above problem by conducting a preliminary study containing 50 neonates through an absorption spectrum measurement of whole blood in 3 to 5 μl samples from the neonates. We constructed a hierarchical decision method that first grossly divides the 30 neonates of the training set into <10 mg / dl and ≥10 mg / dl bilirubin level cohorts. A subsequent boundary condition further divides the ≥10 mg / dl group into two >15 mg / dl and ≤15 mg / dl bilirubin level cohorts. A finer measure later predicted the bilirubin content of each of these groups as low (<10 mg / dl), medium (10 to 15 mg / dl), and high (>15 mg / dl).
Results
Using this hierarchical decision model statistical approach, we quantified the amount of bilirubin in the 20 testing set samples with 82% accuracy.
Conclusions
We formulated a biostatistical model in which we automated the spectrometric determination of total bilirubin in the whole blood for patients of neonatal hyperbilirubinemia.
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