Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization

Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry

Whole-blood sorting, enrichment and in situ immunolabeling of cellular subsets using acoustic microstreaming

Analyzing undiluted whole human blood is a challenge due to its complex composition of hematopoietic cellular populations, nucleic acids, metabolites, and proteins. We present a novel multi-functional microfluidic acoustic streaming platform that enables sorting, enrichment and in situ identification of cellular subsets from whole blood. This single device platform, based on lateral cavity acoustic transducers (LCAT), enables (1) the sorting of undiluted donor whole blood into its cellular subsets (platelets, RBCs, and WBCs), (2) the enrichment and retrieval of breast cancer cells (MCF-7) spiked in donor whole blood at rare cell relevant concentrations (10 mL− 1), and (3) on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns. Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment, to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling

High-throughput continuous dielectrophoretic separation of neural stem cells.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort

An integrated microfluidic platform for size-selective single-cell trapping of monocytes from blood.

Reliable separation and isolation of target single cells from bodily fluids with high purity is of great significance for an accurate and quantitative understanding of the cellular heterogeneity. Here, we describe a fully integrated single-blood-cell analysis platform capable of size-selective cell separation from a population containing a wide distribution of sizes such as diluted blood sample and highly efficient entrapment of single monocytes. The spiked single U937 cells (human monocyte cell line) are separated in sequence by two different-sized microfilters for removing large cell clumps, white blood cells, and red blood cells and then discriminated by dielectrophoretic force and isolated individually by downstream single-cell trapping arrays. When 2% hematocrit blood cells with a final ratio of 1:1000 U937 cells were introduced under the flow rate of 0.2 ml/h, 400 U937 cells were trapped sequentially and deterministically within 40 s with single-cell occupancy of up to 85%. As a proof-of-concept, we also demonstrated single monocyte isolation from diluted blood using the integrated microfluidic device. This size-selective, label-free, and live-cell enrichment microfluidic single blood-cell isolation platform for the processing of cancer and blood cells has a myriad of applications in areas such as single-cell genetic analysis, stem cell biology, point-of-care diagnostics, and cancer diagnostics

A MICROFABRICATION PROCESS FOR POLYMER MICROCHANNEL WITH EMBEDDED VERTICAL ELECTRODES FOR MICROFLUIDIC APPLICATIONS

ABSTRACT In this paper, we will report the use of SU-8 to make an all polymer microchannel with vertical platinum electrodes, which is fabricated by a multilayer SU-8 coating and electroplating process. The first layer is spun to increase the adhesion of the channel and the substrate, the second layer is the microchannel structure. The third layer is spun on another substrate and then capped onto the second channel layer to make a completely sealed SU-8 Channel. The PDMS substrate we used to spin the third layer on is flexible to enable conformably sealing to the microchannel substrate. A process that makes vertical microelectrodes inside the channels is also developed, which makes it a powerful process to make a complicate microfluidic network

Intra-operative point-of-procedure delineation of oral cancer margins using optical coherence tomography.

ObjectivesSurgical margin status is a significant determinant of treatment outcome in oral cancer. Negative surgical margins can decrease the loco-regional recurrence by five-fold. The current standard of care of intraoperative clinical examination supplemented by histological frozen section, can result in a risk of positive margins from 5 to 17 percent. In this study, we attempted to assess the utility of intraoperative optical coherence tomography (OCT) imaging with automated diagnostic algorithm to improve on the current method of clinical evaluation of surgical margin in oral cancer.Materials and methodsWe have used a modified handheld OCT device with automated algorithm based diagnostic platform for imaging. Intraoperatively, images of 125 sites were captured from multiple zones around the tumor of oral cancer patients (n = 14) and compared with the clinical and pathologic diagnosis.ResultsOCT showed sensitivity and specificity of 100%, equivalent to histological diagnosis (kappa, ĸ = 0.922), in detection of malignancy within tumor and tumor margin areas. In comparison, for dysplastic lesions, OCT-based detection showed a sensitivity of 92.5% and specificity of 68.8% and a moderate concordance with histopathology diagnosis (ĸ = 0.59). Additionally, the OCT scores could significantly differentiate squamous cell carcinoma (SCC) from dysplastic lesions (mild/moderate/severe; p ≤ 0.005) as well as the latter from the non-dysplastic lesions (p ≤ 0.05).ConclusionThe current challenges associated with clinical examination-based margin assessment could be improved with intra-operative OCT imaging. OCT is capable of identifying microscopic tumor at the surgical margins and demonstrated the feasibility of mapping of field cancerization around the tumor