Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-µFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-µFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-µFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-µFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-µFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-µFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

Quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity.

This study presented a quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity. Droplets encapsulating single cells and even distribution of fluorescein labeled antibodies removed from targeted cells deformed through the constriction microchannel where the excited fluorescent signals were sampled and interpreted into numbers of proteins based on volume equivalence in measurement of droplets and calibration of fluorescence. To improve the detection reliability, a comprehensive analysis and comparison of multiple stripping agents such as proteinase K, guanidine hydrochloride, and urea was conducted. To improve the detection sensitivity, light modulation was used to address electrical noises and quartz microchannels were fabricated to address optical noises. As a demonstration, based on this quantitative flow cytometry of droplet microfluidics, (1) mutant p53 expressions of single cells were quantified as 1.95 ± 0.60 × 105 (ncell  = 2918 of A431) and 1.30 ± 0.70 × 105 (ncell  = 3954 of T47D); (2) single-cell expressions of Ras, c-Myc, and ß-tubulin were quantified as 1.90 ± 0.59 × 105 , 4.39 ± 1.44 × 105 , and 2.97 ± 0.81 × 105 (ncell  = 3298 of CAL 27), 1.83 ± 0.58 × 105 , 2.08 ± 0.13 × 106 , and 1.96 ± 0.74 × 105 (ncell  = 5459 of WSU-HN6). As a microfluidic tool capable of quantitatively estimating single-cell protein expressions, this methodology may provide a new quantitative perspective for the field of flow cytometry.

Multiplex fluorescence detection of single-cell droplet microfluidics and its application in quantifying protein expression levels.

This study presented a platform of multiplex fluorescence detection of single-cell droplet microfluidics with demonstrative applications in quantifying protein expression levels. The platform of multiplex fluorescence detection mainly included optical paths adopted from conventional microscopy enabling the generation of three optical spots from three laser sources for multiple fluorescence excitation and capture of multiple fluorescence signals by four photomultiplier tubes. As to platform characterization, microscopic images of three optical spots were obtained where clear Gaussian distributions of intensities without skewness confirmed the functionality of the scanning lens, while the controllable distances among three optical spots validated the functionality of fiber collimators and the reflector lens. As to demonstration, this platform was used to quantify single-cell protein expression within droplets where four-type protein expression of α-tubulin, Ras, c-Myc, and ß-tubulin of CAL 27 (Ncell = 1921) vs WSU-HN6 (Ncell = 1881) were quantitatively estimated, which were (2.85 ± 0.72) × 105 vs (4.83 ± 1.58) × 105, (3.69 ± 1.41) × 104 vs (5.07 ± 2.13) × 104, (5.90 ± 1.45) × 104 vs (9.57 ± 2.85) × 104, and (3.84 ± 1.28) × 105 vs (3.30 ± 1.10) × 105, respectively. Neural pattern recognition was utilized for the classification of cell types, achieving successful rates of 69.0% (α-tubulin), 75.4% (Ras), 89.1% (c-Myc), 65.8% (ß-tubulin), and 99.1% in combination, validating the capability of this platform of multiplex fluorescence detection to quantify various types of single-cell proteins, which could provide comprehensive evaluations on cell status.

Personal exposure to black carbon during commuting in peak and off-peak hours in Shanghai.

A study on a commuter's exposure to black carbon (BC) in five different traffic modes (taxi, bus, subway, cycling and walking) was conducted in Xuhui District, Shanghai. A commuter's real-time exposure concentrations were recorded by MicroAeth AE51 BC monitors, and the average BC exposure concentration and inhalation dose were analyzed. Data collected by cyclist was applied to characterize the micro-variability in relation to traffic density and street topology. The distance to the traffic and the street topology as well as the volume of heavy diesel trucks were the dominant factors influencing the BC concentrations. In this study, a high variability of BC concentrations between streets and even within streets was observed, and also between days and hour of the day. The average BC exposure concentrations were 5.59±1.02 µg/m(3), 6.58±1.78 µg/m(3), 7.28±1.87 µg/m(3), 8.62±4.13 µg/m(3) and 9.43±2.89 µg/m(3) for walking, cycling, bus, taxi and subway trips, respectively. Exposure levels of in-vehicle microenvironments were 8.66±3.66 µg/m(3), 9.39±6.98 µg/m(3) and 10.96±2.72 µg/m(3) for bus, taxi and subway, respectively. While inhalation doses were 0.68±0.33 µg, 0.95±0.29 µg, 1.36±0.37 µg, 1.50±0.39 µg and 1.58±0.29 µg for taxi, subway, cycling, bus and walking, respectively. BC exposure level of walking was the lowest among all the traffic modes, but its inhalation dose was the highest.