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The current classical blood smear technique to observe the morphology of single red blood cells (RBCs) for classification is a laborious and error-prone process. To objectively evaluate the morphology of blood cells, we established a method of computational imaging based on a programmable light emitting diode array. By using quantitative differential phase contrast (qDPC), we characterized the morphology of unlabeled RBCs as well as blood smears. By focusing on comparing the difference of imaging between unlabeled RBCs and stained RBCs under multimode microscopic imaging technology, we demonstrated that qDPC could clearly differentiate discocytes and spherocytes in both unlabeled RBCs and blood smears. The phase map provided by quantitative phase imaging further enhanced the classification accuracy. According to statistical analysis from morphological indexes, the qDPC imaging has a significantly improvement in non-circularity, texture inhomogeneity and equivalent diameters of cells. Thus, this method has a significant superiority in the capability to analyze the morphology of RBCs and could be applied to clinical assays for determining morphological, functional, and structural deterioration of RBCs.
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Eritrócitos , Contagem de Eritrócitos , Microscopia de Contraste de FaseRESUMO
Point-of-care testing (POCT) plays an increasingly important role in biomedical research and health care. Quantitative phase microscopes (QPMs) with good contrast, no invasion, no labeling, high speed and automation could be effectively applied for POCT. However, most QPMs are fixed on the optical platform with bulky size, lack of timeliness, which remained challenging in POCT solutions. In this paper, we proposed a plug-and-play QPM with multimode imaging based on the quantitative differential phase contrast (qDPC) method. The system employs a programmable LED array as the light source and uses the GPU to accelerate the calculation, which can realize multi-contrast imaging with six modes. Accurate phase measurement and real-time phase imaging are implemented by the proposed qDPC algorithms for quantitative phase targets and biomedical samples. A 3D electric control platform is designed for mechanical control of field of view and focusing without manual operations. The experimental results verify the robustness and high performance of the setup. Even a rookie could finish the POCT scheme for biomedical applications at the scene using the QPM with a compact size of 140 × 165 × 250 mm3.
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BACKGROUND AND OBJECTIVE: The quality of quantitative differential phase contrast reconstruction (qDPC) can be severely degenerated by the mismatch of the background of two oblique illuminated images, yielding problematic phase recovery results. These background mismatches may result from illumination patterns, inhomogeneous media distribution, or other defocusing layers. In previous reports, the background is manually calibrated which is time-consuming, and unstable, since new calibrations are needed if any modification to the optical system was made. It is also impossible to calibrate the background from the defocusing layers, or for high dynamic observation as the background changes over time. The background mismatch reduces the experimental robustness of qDPC and largely limits its applications. To tackle the mismatch of background and increases the experimental robustness, we propose the Retinex-qDPC. METHODS: In Retinex-qDPC, we replace the data fidelity term of the previous cost function for qDPC inverse problem, by the images' edge features yielding L2-Retinex-qDPC and L1-Retinex-qDPC for high background-robustness qDPC reconstruction. The split Bregman method is used to solve the L1-Retinex DPC. We compare both Retinex-qDPC models against state-of-the-art DPC reconstruction algorithms including total-variation regularized qDPC, and isotropic-qDPC using both simulated and experimental data. RESULTS: Retinex qDPC can significantly improve the phase recovery quality by suppressing the impact of mismatch background. Within, the L1-Retinex-qDPC is better than L2-Retinex and other state-of-the-art qDPC algorithms. CONCLUSIONS: The Retinex-qDPC increases the experimental robustness against background illumination without any modification of the optical system, which will benefit all qDPC applications.
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Algoritmos , IluminaçãoRESUMO
It is important to measure the deformability of red blood cells (RBCs) before transfusion, which is a key factor in the gas transport ability of RBCs and changes during storage of RBCs in vitro. Moreover, the morphology of RBCs also changes during storage. It is proposed that the change in morphology is related to the change in deformability. However, the efficiency of typical methods that use particles as handles is low, especially in the deformability measurement of echinocyte and spherocytes. Therefore, the deformability of RBCs with different morphologies is hard to be measured and compared in the same experiment. In this study, we developed a cost-effective and efficient rotating-glass-plate-based scanning optical tweezers device for the measurement of deformability of RBCs. The performance of this device was evaluated, and the deformability of three types of RBCs was measured using this device. Our results clearly show that the change of erythrocyte morphology from discocyte to echinocyte and spherocyte during storage in vitro is accompanied by a decrease in deformability.
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Measurement of the sample temperature in biophysics research is challenging, as the samples are commonly placed in a miniature sample chamber under a microscope. In this study, we proposed a method to measure the temperature of an aqueous solution in miniature sample chambers in a microscopic system. Existing studies show that the absorption coefficient spectrum of water shifts with temperature, especially in the near-infrared (NIR) band. We measured the absorption spectra of water with different temperatures and analyzed them, to build a mathematical model relating the temperature and the spectrum. A setup for temperature measurement in a microscopic system was designed and implemented by coupling a spectrometer and a light source to a microscope. The temperature could be calculated by the spectral data and the mathematical model while simultaneously observing the micro-image of the sample. A series of liquid samples at different temperatures were tested using the setup, and the root mean square error of the calculated temperature is less than 0.5 °C. The results demonstrate that the method based on the NIR spectrum can be used for noncontact and quick measurement of the liquid sample temperature in a microscopic system.
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Modelos Teóricos , Água , TemperaturaRESUMO
Distributive shock is considered to be a condition of microvascular hypoperfusion, which can be fatal in severe cases. However, traditional therapeutic methods to restore the macro blood flow are difficult to accurately control the blood perfusion of microvessels, and the currently developed manipulation techniques are inevitably incompatible with biological systems. In our approach, infrared optical tweezers are used to dynamically control the microvascular reperfusion within subdermal capillaries in the pinna of mice. Furthermore, we estimate the effect of different optical trap positions on reperfusion at branch and investigate the effect of the laser power on reperfusion. The results demonstrate the ability of optical tweezers to control microvascular reperfusion. This strategy allows near-noninvasive reperfusion of the microvascular hypoperfusion in vivo. Hence, our work is expected to provide unprecedented insights into the treatment of distributive shock.
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Motility is one of the most critical features to evaluate sperm quality. As longitudinal rolling of human sperm has long been ignored until recently, its detailed dynamics and cellular biological mechanisms are still largely unknown. Here we report an optical-tweezers-based method to evaluate the chirality and frequency of sperm rotation. According to the intensity distribution patterns of off-focus micron-size particles, we established a method to judge the orientation of the sperm head along the optical axis in the optical trap. Together with the rotation direction of the projection of the sperm head, the chirality of longitudinal rolling of sperm can be measured without the application of three-dimensional tracking techniques or complex optical design. By video tracking optically trapped sperm cells from different patients, both rolling chirality and rolling frequency were analyzed. In this study, all the vertically trapped human sperm cells adopt a right-hand longitudinal rolling. The orientation and rolling frequency but not the rolling chirality of sperm in the optical trap are affected by the trap height. The rotation analysis method developed in this study may have clinical potential for sperm quality evaluation.