MSGM: An Advanced Deep Multi-Size Guiding Matching Network for Whole Slide Histopathology Images Addressing Staining Variation and Low Visibility Challenges.

Matching whole slide histopathology images to provide comprehensive information on homologous tissues is beneficial for cancer diagnosis. However, the challenge arises with the Giga-pixel whole slide images (WSIs) when aiming for high-accuracy matching. Learning-based methods are difficult to generalize well with large-size WSIs, necessitating the integration of traditional matching methods to enhance accuracy as the size increases. In this paper, we propose a multi-size guiding matching method applicable high-accuracy requirements. Specifically, we design learning multiscale texture to train deep descriptors, called TDescNet, that trains 64 ×64×256 and 256 ×256×128 size convolution layer as C64 and C256 descriptors to overcome staining variation and low visibility challenges. Furthermore, we develop the 3D-ring descriptor using sparse keypoints to support the description of large-size WSIs. Finally, we employ C64, C256, and 3D-ring descriptors to progressively guide refined local matching, utilizing geometric consistency to identify correct matching results. Experiments show that when matching WSIs of size 4096×4096 pixels, our average matching error is 123.48 [Formula: see text] and the success rate is 93.02 % in 43 cases. Notably, our method achieves an average improvement of 65.52 [Formula: see text] in matching accuracy compared to recent state-of-the-art methods, with enhancements ranging from 36.27 [Formula: see text] to 131.66 [Formula: see text]. Therefore, we achieve high-fidelity whole-slice image matching, and overcome staining variation and low visibility challenges, enabling assistance in comprehensive cancer diagnosis through matched WSIs.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Microenvironmental Analysis and Control for Local Cells under Confluent Conditions via a Capillary-Based Microfluidic Device.

Sophisticated functions of biological tissues are supported by small biological units of cells that are localized within a region of 100 µm scale. The cells in these units secrete molecules to form their microenvironment to play a vital role in biological functions. Various microfluidic devices have been developed to analyze the microenvironment but were not designed for cells in a culture dish in a confluent condition, a typical setup for cell and tissue cultivation. This study presents a novel glass capillary-based microfluidic device for studying confluent cells in a culture dish. The multiple capillaries allow the device to confine the local flow in 100 µm or smaller scale to form two adjacent regions with different chemical properties; it can simultaneously perform local cell stimulation and collect secreted molecules from the stimulated cells. Cell removal was achieved upon trypsin stimulation from a limited area (3.8 × 10-3 ± 1.0 × 10-3 mm2), which corresponded to 7.6 ± 2.0 cells, using the mouse skeletal myoblast cell line (C2C12 cells) in a confluent condition. Microenvironmental analysis was demonstrated by measuring the secreted tumor necrosis factor alpha (TNF-α) collected from the microenvironment of the stimulated and unstimulated mouse leukemic monocyte cell line (RAW264 cells) to track temporal changes in the TNF-α production. The TNF-α secreted from stimulated cells was approximately four-fold higher than that from unstimulated cells in 90 min. This device enables local cell stimulation and the collection of secreted molecules for cells under confluent conditions, which contributes to the analysis of the cellular microenvironment.

A pressure driven electric energy generator exploiting a micro- to nano-scale glass porous filter with ion flow originating from water.

We demonstrated a pressure driven energy harvesting device using water and that features a glass filter with porous channels. We employed powder sintering to fabricate the glass filter (2 cm diameter, 3 mm thickness) by packing a powder of borosilicate glass particles into a carbon mold and then thermally fusing this at 700°C under pressure. In constant flow rate experiment, the optimum average pore radius of the filter for power generation was 12 µm. Using this filter, power of 3.8 mW (27 V, 0.14 mA, 0.021% energy efficiency) was generated at a water flow speed of 50 mm/s. In constant pressure experiment, a power generator was equipped with a foot press unit with a 60 kg weight (830 kPa) and 50 mL of water. The optimum average pore radius for power generation in this experiment was 12 µm and power of 4.8 mW (18 V, 0.26 mA, 0.017% energy efficiency) was generated with 1.7 s duration. This was enough power for direct LED lighting and the capacitors could store enough energy to rotate a fan and operate a wireless communicator. Our pressure driven device is suitable for energy harvesting from slow movements like certain human physiological functions, e.g. walking.

Anhydrobiotic chironomid larval motion-based multi-sensing microdevice for the exploration of survivable locations.

African chironomid (Polypedilum vanderplanki) larvae can suspend their metabolism by undergoing severe desiccation and then resume this activity by simple rehydration. We present a microdevice using interdigital comb electrodes to detect the larval motion using the natural surface charge of the living larvae in water. The larvae were most active 2 h after soaking them in water at 30°C; they exhibited motions with 2 Hz frequency. This was comparable to the signal obtained from the microdevice via fast Fourier transform (FFT) processing. The amplitude of the voltage and current were 0.11 mV and 730 nA, respectively. They would be enough to be detected by a low power consumption microcomputer. Temperature and pH sensing were demonstrated by detecting the vital motions of the revived larvae under different conditions. This multi-functional biosensor will be a useful microdevice to search for survivable locations under extreme environmental conditions like those on other planets.

Specific capture and intact release of breast cancer cells using a twin-layer vein-shaped microchip with a self-assembled surface.

Breast cancer is the most fatal disease among female cancers yet its detection still relies on needle biopsy. The unique physical and immune characteristics of breast cancer cells different from blood cells make them suitable to be employed as excellent biomarkers in liquid biopsy, through which breast cancer cells are collected from peripheral blood for further cancer diagnosis, medical treatment monitoring, and drug screening. Although the separation and enrichment of breast cancer cells from peripheral blood have been studied for years, there are still two problems to be solved in these methods: the low efficiency of on-chip immunologic capture in the flow state and the influence of the conjugated antibodies for the following analyses during cell release. In this paper, a vein-shaped microchip with self-assembled surface was developed for the specific and robust capture (91.2%) of breast cancer cells in the flow state. A protein-recovery process was proposed, in which trypsin served as a mild release reagent, releasing 92% of cells with high viability (96%), normal adherent proliferation, and complete proteins on the cell membrane, avoiding disturbance of the conjugated chemical molecules in the following clinical study. The excellent performance demonstrated in isolating free breast cancer cells from real peripheral blood sample, originating from the orthotopic 4T1 breast cancer metastatic models, suggest the microchip could be utilized as a multiple circulating tumor cell capture and release platform that could allow providing more reliable information in liquid biopsies.

Hydrodynamic particle focusing enhanced by femtosecond laser deep grooving at low Reynolds numbers.

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re < 1. Moreover, we achieved continuous focusing of live cells with different sizes ranging from 10 to 15 µm, i.e. human T-cell lymphoma Jurkat cells, rat adrenal pheochromocytoma PC12 cells and dog kidney MDCK cells. The glass grooves fabricated by fs laser are expected to be integrated with on-chip detection components, such as contact imaging and fluorescence lifetime-resolved imaging, for various biological and biomedical applications, where particle focusing at a relatively low flow rate is desirable.

Sheathless Inertial Focusing Chip Combining a Spiral Channel with Periodic Expansion Structures for Efficient and Stable Particle Sorting.

Efficient and reliable manipulation of biological particles is crucial in medical diagnosis and chemical synthesis. Inertial microfluidic devices utilizing passive hydrodynamic forces in the secondary flow have drawn considerable attention for their high throughputs, low costs, and harmless particle manipulation. However, as the dominant mechanism, the inertial lift force is difficult to quantitatively analyze because of the uncertainties of its magnitude and direction. The equilibrium position of particles varies along the migration process, thus inducing the instabilities of particle separation. Herein, we present a designable inertial microfluidic chip combining a spiral channel with periodic expansion structures for the sheathless separation of particles with different sizes. The stable vortex-induced lift force arising from the periodic expansion and the Dean drag force significantly enhanced the focusing process and determined the final equilibrium position. The experimental results showed that over 99% of target particles could be isolated with the high target sample purity of 86.12%. In the biological experiment, 93.5% of the MCF-7, 89.5% of the Hela, and 88.6% of the A549 cells were steadily recovered with excellent viabilities to verify the potential of the device in dealing with biological particles over a broad range of throughputs. The device presented in this study can further serve as a lab-on-chip platform for liquid biopsy and diagnostic analysis.

Intelligent whole-blood imaging flow cytometry for simple, rapid, and cost-effective drug-susceptibility testing of leukemia.

Drug susceptibility (also called chemosensitivity) is an important criterion for developing a therapeutic strategy for various cancer types such as breast cancer and leukemia. Recently, functional assays such as high-content screening together with genomic analysis have been shown to be effective for predicting drug susceptibility, but their clinical applicability is poor since they are time-consuming (several days long), labor-intensive, and costly. Here we present a highly simple, rapid, and cost-effective liquid biopsy for ex vivo drug-susceptibility testing of leukemia. The method is based on an extreme-throughput (>1 million cells per second), label-free, whole-blood imaging flow cytometer with a deep convolutional autoencoder, enabling image-based identification of the drug susceptibility of every single white blood cell in whole blood within 24 hours by simply flowing a drug-treated whole blood sample as little as 500 µL into the imaging flow cytometer without labeling. Our results show that the method accurately evaluates the drug susceptibility of white blood cells from untreated patients with acute lymphoblastic leukemia. Our method holds promise for affordable precision medicine.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Time Sequential Single-Cell Patterning with High Efficiency and High Density.

Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm², which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.