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Precise label-free leukocyte subpopulation separation using hybrid acoustic-optical chip
摘要： Leukocyte subpopulations contain crucial physiological information; hence, precise and specific leukocyte separation is very important for leukemia diagnosis and analysis. However, conventional centrifugation and immunofluorescence-based separation methods are inaccurate and inconvenient due to the overlapping cell size and density or complex marking processes. Herein, we report a new label-free technology for precise leukocyte subpopulation separation by synergy of acoustic and optical technologies. Standing surface acoustic wave (SSAW) solved the problem of gentle and precise focusing of cells in optical systems. In addition, SSAW was used for the separation of granulocytes, which have evident size distinction from other components. In case of lymphocytes and monocytes, which have overlap in size/density, optical force could distinguish them accurately based on the RI difference, with the convenience of acoustic pre-focusing. In this experiment, separation of three types of leukocyte subtypes with considerable throughput and purity was conducted, through which we obtained 99% pure lymphocytes, 98% pure monocytes, and 95% pure granulocytes. Experimental results prove that the device has robust ability in separating leukocyte phenotypes and have the advantages of being non-invasive, label-free and precise. In the future, this convenient hybrid method will be a potential powerful tool for auxiliary clinical diagnosis and analysis.
关键词： label-free,cell sorting,microfluidics,acoustic-optical chip,leukocyte separation
更新于2025-11-25 10:30:42
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Enhanced In‐vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57Bl/6 Mice using a Highly Bright Near‐Infrared BODIPY Dye
摘要： Delving deeper is possible in whole body in vivo imaging using a super-bright membrane targeting BODIPY dye (BD). The dye was employed to monitor homing of ex vivo, fluorescently labelled neutrophils to an injured liver of dark pigmented C57BL/6 mice. In Vivo Imaging System (IVIS) data conclusively showed an enhanced signal intensity and a higher signal-to-noise ratio in mice receiving neutrophils labelled with the BD dye compared to those labelled with a gold standard dye at 2 hr post in vivo administration of fluorescently labelled cells. Fluorescence-activated cell sorting (FACS) confirmed that BD was non-toxic, and an exceptional cell labelling dye that opens up precision deep organ in vivo imaging of inflammation in mice routinely used for biomedical research. The origin of enhanced performance is identified with the molecular structure, and the distinct localisation of the dye within cells that enable remarkable changes in its optical parameters.
关键词： In-vivo imaging,Cell Sorting,Bodipy,Liver,Fluorescence
更新于2025-11-19 16:46:39
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Additional Stability Testing of Cryopreserved Intestinal Biopsies for Downstream Flow Cytometric Analysis
摘要： The use of flow cytometry (FC) in therapeutic clinical trials involving patients with gastrointestinal (GI) diseases may accelerate drug development by providing insights into pharmacokinetic/pharmacodynamic relationships and target engagement, as well as by identifying responder populations. The application of FC in these trials, however, has been constrained by the practical limitations of storing and shipping intestinal biopsy samples prior to cell extraction and FC analysis, and the potential impact of handling and storage conditions on viable cell yield. Furthermore, there has been a lack of standardization for these processes. We have undertaken research to attempt to address these constraints to the inclusion of translational science in multi-center clinical trials. Our previous work, Wildenberg et al. (2018, 459:50-54), published in this journal, demonstrated that it was feasible to store intestinal biopsies from patients with Crohn's disease under conditions that allow for subsequent processing with preservation of adequate numbers of viable cells for valid FC analyses. This study demonstrated that storage of intestinal tissue biopsies at ?20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. Although these preliminary findings provide support for the shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials, some practical limitations remain. For example, in the clinical trial setting, where patient enrolment and study procedures occur in an ongoing fashion and frequently over an extended period of time, it is impracticable from a resource perspective for a central laboratory to process study samples as they are collected from individual patients. Furthermore, contemporary clinical trials in the inflammatory bowel diseases are most often conducted on a global scale. Shipping and processing of biopsy samples by a central laboratory within a 48 h timeframe in this context would compound costs and add logistical hurdles that might discourage industry support for the inclusion of translational science in the multi-center clinical trial setting. In this regard, longer-term storage, and batch processing of study biopsy samples would be a preferred method. This approach would reduce both the variability and costs associated with more frequent assays and expansive resource utilization. To that end, we developed an addendum to our original study protocol to examine the effect of longer-term storage condition prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. Consistent with the original protocol, the study was performed at Tytgat Institute (Academic Medical Center, Amsterdam, the Netherlands) under the original ethics approval, and with informed consent. Biopsies (N = 180) were procured from surgically resected inflamed or non-inflamed ileal and/or colonic tissue from four patients undergoing Crohn's disease-related surgeries (colectomy [n = 2], subtotal colectomy, ileo-cecal resection). Two biopsies were pooled and processed as a single sample, resulting in 90 samples per surgical specimen and 18 samples per condition tested. The effects of 5 different storage conditions on cell viability and subsequent FC analyses were compared in this study addendum; immediate (< 1 h from sample receipt) processing, storage at ?20 °C and ?80 °C for 24 h with subsequent overnight storage on dry ice (to mimic real life shipping conditions) followed by long-term (2–3 months) storage at ?20 °C and ? 80 °C, and immediate long-term storage at ?20 °C and ? 80 °C. Subsequent methods for cell isolation, staining, and FACS analysis were as previously described (Wildenberg et al. 2018, 459:50–54). Analysis of freshly processed biopsy samples resulted in a mean (standard deviation, SD) yield of 36916.7 (59194.7) live immune cells as identified by CD45+ staining. Storage of biopsies for greater than or equal to two months at either ?20° or ? 80 °C significantly reduced the mean [standard deviation] number of live CD45+ cells compared to immediate processing (372.0 [607.1] and 4065.3 [4489.7], respectively, p < 0.001 for both comparisons with immediate processing). Sufficient cell yield (≥ 300 live CD45+ cells) for subsequent FC analysis was obtained from 77.8% (14/18) of samples processed immediately compared to 22.2% (4/18) and 27.8% (5/18) of samples stored at ?20 °C (with and without simulated shipment; p < 0.001 chi square test) and 77.8% (14/18) and 94.4% (17/18) of samples stored at ?80 °C (with and without simulated shipping; p = 0.439). We further evaluated the effect of storage on the composition of specific cell types in the samples and observed a significant decrease in the proportion of CD3+ and CD4+ cells after storage at ?20 °C compared to samples that were immediately processed (Table 1 and Fig. 1, panels A and B). The proportion of CD8+ and CD14+ cells was unaffected by storage temperature, although the standard deviations for the proportions of these cells in samples stored at ?20 °C were larger than for those observed for samples stored at ?80 °C (Table 1 and Fig. 1, panels C and D). In conclusion, our previous work demonstrated that it is feasible to store mucosal biopsies at ?20 °C for 48 h in DMSO/citrate buffer before further processing with preservation of adequate numbers of viable cells for valid FC analyses suggesting that centralized FC is possible. These data provided a basis for performing additional stability testing (including shipping and longer storage). Results from this protocol addendum suggest that lower temperatures (?80 °C) may be required for longer-term storage of mucosal biopsies, and that long-term storage at ?20 °C may both reduce viable cell yield, and alter the proportion of certain immune cells detected on subsequent FC analysis. We cannot, however, exclude the possibility that our results were confounded by selection bias and/or that the effects that we observed on cell proportion were the result of significantly fewer samples with ≥300 live CD45+ cells for analysis after storage at ?20 °C (albeit a result of decreased viability). Overnight storage of biopsies on dry ice to mimic real life shipping conditions to a central laboratory did not appear to affect live cell yield or immune cell proportions for the markers studied (CD3+, CD4+, CD8+, CD14+).
关键词： Biopsy,Long-term storage,Viability,Inflammatory bowel disease,Fluorescence activated cell sorting
更新于2025-09-23 15:23:52
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[Methods in Molecular Biology] Immunological Tolerance Volume 1899 (Methods and Protocols) || Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting
摘要： Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4+CD25+CD127lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25+ PBMC, which reduces the duration of the subsequent FACS isolation.
关键词： FACS,Fluorescence-activated cell sorting,Regulatory T cells,Cell isolation,Treg
更新于2025-09-23 15:22:29
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Examining Storm Asymmetries in Hurricane Irma (2017) Using Polarimetric Radar Observations
摘要： Dual-polarization radar observations of Hurricane Irma (2017) provide new insight into the microphysical structure of a mature tropical cyclone that can be tied to the cyclone dynamics. The primary eyewall exhibited a radar signature of hydrometeor size sorting, which implied that large drops fell out near persistent upward motion in the front-right quadrant of the storm, while smaller drops were advected downstream. In the outer rainbands, convective initiation was also preferred in the front-right quadrant, whereas stratiform precipitation was predominant downwind. For both the primary eyewall and outer rainbands, the preferred quadrant for convective initiation was consistent with the expected kinematic asymmetry of a tropical cyclone in weak environmental wind shear but with moderate translation speed. The developing secondary eyewall exhibited a different asymmetry that indicated a stratiform-to-convective transition associated with heavy precipitation in the rear quadrants. This transition is consistent with hypothesized dynamical theories for secondary eyewall formation.
关键词： hydrometeor size sorting,stratiform precipitation,microphysical structure,Hurricane Irma,tropical cyclone,convective initiation,dual-polarization radar,secondary eyewall formation
更新于2025-09-23 15:19:57
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Sorting of micron-sized particles using holographic optical Raman tweezers in aqueous medium
摘要： We have constructed a holographic optical tweezers system combined with Raman spectroscopy to sort trapped particles. Our software automatically moves the trapped objects to the measurement positions to obtain individual Raman signals from multiple trapped particles. We performed the sorting by comparing their spectra with the previously measured training dataset using the correlation coefficients. We used yeast cells and polystyrene beads as test particles. This study aims to show that biological particles can be separated using single cell analysis with combined holographic optical tweezers and Raman spectroscopy system.
关键词： Holographic optical tweezers,multivariate statistical analysis,particle sorting,Raman spectroscopy
更新于2025-09-19 17:15:36
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[IEEE 2018 11th Biomedical Engineering International Conference (BMEiCON) - Chiang Mai, Thailand (2018.11.21-2018.11.24)] 2018 11th Biomedical Engineering International Conference (BMEiCON) - Implementation of Asymmetric Kernel Median Filtering for Real-Time Ultrasound Imaging
摘要： Ultrasound images contains speckles that could be considered as salt-and-pepper noise. Median filtering can efficiently remove the noise without blurring the edges. However, median filtering is computationally intensive and not suitable for real-time imaging. In this paper, median filtering on the intermediate ultrasound data using asymmetric median kernel sizes is proposed. To reduce the computational time, the median selection networks with compare-and-swap stages are applied instead of reducing the redundancy of the median calculation among the adjacent image pixels. Moreover, the modification of the compare-and-swap stages to suit for the GPU is also proposed. The computational times using CPU and GPU programs are compared and CNRs of the ultrasound images are also reported.
关键词： ultrasound imaging,median filtering,GPU,sorting network,median selection network
更新于2025-09-19 17:15:36
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[IEEE 2019 44th International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz) - Paris, France (2019.9.1-2019.9.6)] 2019 44th International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz) - Full-field THz polarimetric imaging with THz quantum cascade laser and THz imager
摘要： Model checking is a powerful approach for the formal verification of hardware and software systems. However, this approach suffers from the state space explosion problem, which limits its application to large-scale systems due to space shortage. To overcome this drawback, one of the most effective solutions is to use external memory algorithms. In this paper, we propose an I/O efficient model checking algorithm for large-scale systems. To lower I/O complexity and improve time efficiency, we combine three new techniques: 1) a linear hash-sorting technique; 2) a cached duplicate detection technique; and 3) a dynamic path management technique. We show that the new algorithm has a lower I/O complexity than state-of-the-art I/O efficient model checking algorithms, including detect accepting cycle, maximal accepting predecessors, and iterative-deepening depth-first search. In addition, the experiments show that our algorithm obviously outperforms these three algorithms on the selected representative benchmarks in terms of performance.
关键词： state space explosion,model checking,Duplicate detection,linear hash-sorting,dynamic search path management
更新于2025-09-19 17:13:59
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Compressive Holography from Poisson Noise plagued Holograms using Expectation-Maximization
摘要： We present the concept of an acoustic rake receiver—a microphone beamformer that uses echoes to improve the noise and interference suppression. The rake idea is well-known in wireless communications; it involves constructively combining different multipath components that arrive at the receiver antennas. Unlike spread-spectrum signals used in wireless communications, speech signals are not orthogonal to their shifts. Therefore, we focus on the spatial structure, rather than the temporal. Instead of explicitly estimating the channel, we create correspondences between early echoes in time and image sources in space. These multiple sources of the desired and the interfering signal offer additional spatial diversity that we can exploit in the beamformer design. We present several “intuitive” and optimal formulations of acoustic rake receivers, and show theoretically and numerically that the rake formulation of the maximum signal-to-interference-and-noise ratio beamformer offers significant performance boosts in terms of noise and interference suppression. Beyond signal-to-noise ratio, we observe gains in terms of the perceptual evaluation of speech quality (PESQ) metric for the speech quality. We accompany the paper by the complete simulation and processing chain written in Python. The code and the sound samples are available online at http://lcav.github.io/AcousticRake-Receiver/.
关键词： echo sorting,noise suppression,interference cancellation,room impulse response,beamforming,Acoustic rake receiver,perceptual evaluation of speech quality (PESQ)
更新于2025-09-19 17:13:59
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[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Online Detection and Sorting of Single-Unit Recording Signal for Closed Loop Optogenetics Controlling
摘要： Electrical brain stimulation provides therapeutic benefits for patients with drug-resistant neurological disorders. But it has restricted access to cell-type selectivity. Optogenetics, in contrast, enables precise targeting of a specific cell type which can address the issue with electrical brain stimulation. Optogenetics is a light-based stimulation method, in which the target cells are transfected with light-sensitive ion channels called opsin. This technique can be used for both excitation and inhibition of cells based on light wavelength and opsin properties. To online modulation of neurons need a closed loop controlling. Closed-loop optogenetics system compares the neural signals with a predefined value at every moment and then decides what to do to achieve the desired value by optical stimulation [1]. One of the important brain signal recordings is single-unit recording (SUR) [2] whiles the most of signal processing method is offline. In this paper, an algorithm was proposed to online spike detection and sorting them based on wavelet transform which was programmed by Labview software. The algorithm consists of three main steps such as detecting spikes, extracting the features of every detected spike and assigning similar spikes to one group. The background noise in multi-unit recording has Gaussian distribution and it contains spikes which the mean value of the signal was considered in signal processing. Time of calculating the main value is about 6.04 μs. If the mean value of a dataset changes noticeably from the estimated value, a candidate spike has occurred. Detected spikes are a discrete sample of the real spikes which the waveforms should be interpolated to improve the detection spike [3]. With detecting a spike, its features must be extracted as the input of spike sorting algorithm. Spikes were sorted in different clusters based on their shapes. Wavelet transform compares the input signal with a predefined function known as a wavelet in different scales. In this algorithm was used 20 scales and Mexican hat wavelet to transform the SUR signal [4]. Total time for interpolation and applying wavelet transform takes about 5.3 ms. The final step of the algorithm is sorting the detected spikes using the extracted features based on applying the wavelet transform and compared all wavelet coefficients of a detected spike with the corresponding coefficients of all the clusters. If the wavelet coefficient in the clusters was within 30% of the corresponding coefficient of the detected spike in all scales, spike put in one group. This part of the algorithm takes about 3.1 ms. Schematic diagram of spike sorting and a photo of the front panel of designed software was shown in Fig.1. In conclusion, designed software has a high processing speed and can be used for online and fully automatic spike sorting for studying and controlling the neural network system with closed loop optogenetics technique.
关键词： Wavelet transform,Spike sorting,Optogenetics,Spike detection,Single-unit recording,Labview
更新于2025-09-16 10:30:52