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Traditional pathology workflow suffers from limitations including biopsy invasiveness, small fraction of large tissue samples being analyzed, and complex and time-consuming processing.
Aim
We address limitations of conventional pathology workflow through development of a laser microbiopsy device for minimally invasive harvest of sub-microliter tissue volumes. Laser microbiopsy combined with rapid diagnostic methods, such as virtual hematoxylin and eosin (H&E) imaging has potential to provide rapid minimally invasive tissue diagnosis.
Approach
Laser microbiopsies were harvested using an annular shaped Ho:YAG laser beam focused onto the tissue surface. As the annulus was ablated, the tissue section in the center of the annulus was ejected and collected directly onto a glass slide for analysis. Cryogen spray cooling was used before and after laser harvest to limit thermal damage. Microbiopsies were collected from porcine skin and kidney. Harvested microbiopsies were imaged with confocal microscopy and digitally false colored to provide virtual H&E images.
Results
Microbiopsies were successfully harvested from porcine skin and kidney. Computational and experimental results show the benefit of cryogen pre- and post-cooling to limit thermal damage. Virtual H&E images of microbiopsies retained observable cellular features including cell nuclei.
Conclusions
Laser microbiopsy with virtual H&E imaging shows promise as a potential rapid and minimally invasive tool for biopsy and diagnosis.
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TOPICS: Wound healing, Arteries, Data acquisition, Diseases and disorders, Vascular diseases, Lab on a chip, Blood, Optical spectroscopy, Animal model studies, Optical sensing
Due to the persistence of chronic wounds, a second surgical intervention is often necessary for patients with peripheral arterial disease (PAD) within a year of the first intervention. The dynamic vascular optical spectroscopy system (DVOS) may assist physicians in determining patient prognosis only a month after the first surgical intervention.
Aim
We aim to assess the DVOS utility in characterizing wound healing in PAD patients after endovascular intervention.
Approach
The DVOS used near-infrared light (670 < λ < 850 nm) to record hemodynamic response to a cuff inflation in 14 PAD patients with lower limb ulcers immediately before, immediately after, and at a first follow-up 3 to 4 weeks after intervention. Ankle-brachial index (ABI) and arterial duplex ultrasound (A-DUS) measurements were obtained when possible.
Results
The total hemoglobin plateau time differed significantly between patients with ulcers that reduced in size (N = 9) and patients with ulcers that did not (N = 5) 3 to 4 weeks after intervention (p value < 0.001). Data correlated strongly (89% sensitivity, 100% specificity, and AUC = 0.96) with long-term wound healing. ABI and A-DUS measurements were not statistically associated with wound healing.
Conclusions
This pilot study demonstrates the potential of the DVOS to aid physicians in giving accurate long-term wound healing prognoses 1 month after intervention.
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Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons.
Aim
The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry.
Approach
The sensitivity functions were simulated with a Monte Carlo method. A compressed-sensing-like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method.
Results
3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object.
Conclusions
Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
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The capillaries are the smallest blood vessels in the body, typically imaged using video capillaroscopy to aid diagnosis of connective tissue diseases, such as systemic sclerosis. Video capillaroscopy allows visualization of morphological changes in the nailfold capillaries but does not provide any physiological information about the blood contained within the capillary network. Extracting parameters such as hemoglobin oxygenation could increase sensitivity for diagnosis and measurement of microvascular disease progression.
Aim
To design, construct, and test a low-cost multispectral imaging (MSI) system using light-emitting diode (LED) illumination to assess relative hemoglobin oxygenation in the nailfold capillaries.
Approach
An LED ring light was first designed and modeled. The ring light was fabricated using four commercially available LED colors and a custom-designed printed circuit board. The experimental system was characterized and results compared with the illumination model. A blood phantom with variable oxygenation was used to determine the feasibility of using the illumination-based MSI system for oximetry. Nailfold capillaries were then imaged in a healthy subject.
Results
The illumination modeling results were in close agreement with the constructed system. Imaging of the blood phantom demonstrated sensitivity to changing hemoglobin oxygenation, which was in line with the spectral modeling of reflection. The morphological properties of the volunteer capillaries were comparable to those measured in current gold standard systems.
Conclusions
LED-based illumination could be used as a low-cost approach to enable MSI of the nailfold capillaries to provide insight into the oxygenation of the blood contained within the capillary network.
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Mueller matrix polarimetry (MMP) and spatial frequency domain imaging (SFDI) are wide-field optical imaging modalities that differentiate tissue primarily by structure alignment and photon transport coefficient, respectively. Because these effects can be related, combining MMP and SFDI may enhance tissue differentiation beyond the capability of each modality alone.
Aim
An instrument was developed to combine MMP and SFDI with the goal of testing whether it enhances contrast of features in reflection mode.
Approach
The instrument was constructed using liquid crystal elements for polarization control, a digital light processing projector for generating sinusoidal illumination patterns, and a digital camera for imaging. A theoretical analysis shows that the SFD Mueller matrix is complex-valued and does not follow the same behavior as a regular Mueller matrix. Images were acquired from an anisotropic tissue phantom, an optical fiber bundle, and cerebellum, thalamus, and cerebrum tissues.
Results
The measurement results suggest that singly scattered, few scattered, and diffusely scattered photon paths can be distinguished in some of the samples investigated. The combined imaging modality yields additional spatial frequency phase information, which highlights paths having only a few scattering events.
Conclusions
The combination of MMP and SFDI offers contrast mechanisms inaccessible by each modality used alone.
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An unmet need is recognized for early detection and diagnosis of neurological diseases. Many psychological markers emerge years after disease onset. Mitochondrial dysfunction and corresponding neurodegeneration occur before onset of large-scale cell and tissue pathology. Early detection of subcellular morphology changes could serve as a beacon for early detection of neurological diseases. This study is on bacterial colonies, Bacillus subtilis, which are similar in size to mitochondria.
Aim
This study investigates whether morphological changes can be detected in Bacillus subtilis using scattering angle resolved optical coherence tomography (SAR-OCT).
Approach
The SAR-OCT was applied to detect scattering angle distribution changes in Bacillus subtilis. The rod-to-coccus shape transition of the bacteria was imaged, and the backscattering angle was analyzed by recording the distribution of the ratio of low- to medium angle scattering (L/M ratio). Bacillus orientation at different locations in colonies was analytically modeled and compared with SAR-OCT results.
Results
Significant differences in the distribution of backscattering angle were observed in Bacillus subtilis transitioning from rod-to-coccus shapes. In Bacillus subtilis, the C-parameter of the Burr distribution of the SAR-OCT-derived L/M ratio was significantly smaller in coccus compared with rod-shaped bacteria. SAR-OCT-derived L/M ratio varied with bacterial position in the colony and is consistent with predicted orientations from previous studies.
Conclusions
Study results support the potential of utilizing SAR-OCT to detect bacterial morphological changes.
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Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth (z) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field.
Aim
To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system.
Approach
We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker.
Results
Bead localization precision was <25 nm in the transverse dimensions and ≤70 nm along the axial dimension over an axial range of 6 μm. Higher axial precision (≤50 nm) was achieved over a shallower focal depth of 2.9 μm. 3D diffusion constants of chromatin matched expected values.
Conclusions
Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.
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