Algorithm for the precise detection of single and cluster cells in microfluidic applications.

Recent advances in imaging flow cytometry and microfluidic applications have led to the development of suitable mathematical algorithms capable of detecting and identifying targeted cells in images. In contrast to currently existing algorithms, we herein proposed the identification and reconstruction of cell edges based on original approaches that overcome frequent detection limitations such as halos, noise, and droplet boundaries in microfluidic applications. Reconstructed cells are then discriminated between single cells and clusters of round-shaped cells, and cell information such as the area and location of a cell in an image is output. Using this method, 76% of cells detected in an image had an error <5% of the cell area size and 41% of the image had an error <1% of the cell area size (n = 1,000). The method developed in the present study is the first image processing algorithm designed to be flexible in use (i.e. independent of the size of an image, using a microfluidic droplet system or not, and able to recognize cell clusters in an image) and provides the scientific community with a very accurate imaging algorithm in the field of microfluidic applications. © 2016 International Society for Advancement of Cytometry.

Size Distribution Analysis with On-Chip Multi-Imaging Cell Sorter for Unlabeled Identification of Circulating Tumor Cells in Blood.

We report a change of the imaging biomarker distribution of circulating tumor cell (CTC) clusters in blood over time using an on-chip multi-imaging flow cytometry system, which can obtain morphometric parameters of cells and those clusters, such as cell number, perimeter, total cross-sectional area, aspect ratio, number of nuclei, and size of nuclei, as "imaging biomarkers". Both bright-field (BF) and fluorescent (FL) images were acquired at 200 frames per second and analyzed within the intervals for real-time cell sorting. A green fluorescent protein-transfected prostate cancer cell line (MAT-LyLu-GFP) was implanted into Copenhagen rats, and the blood samples of these rats were collected 2 to 11 days later and measured using the system. The results showed that cells having BF area of 90 µm² or larger increased in number seven days after the cancer cell implantation, which was specifically detected as a shift of the cell size distribution for blood samples of implanted rats, in comparison with that for control blood. All cells with BF area of 150 µm² or larger were arranged in cell clusters composed of at least two cells, as confirmed by FL nucleus number and area measurements, and they constituted more than 1% of all white blood cells. These results indicate that the mapping of cell size distribution is useful for identifying an increase of irregular cells such as cell clusters in blood, and show that CTC clusters become more abundant in blood over time after malignant tumor formation. The results also reveal that a blood sample of only 50 µL is sufficient to acquire a stable size distribution map of all blood cells to predict the presence of CTC clusters.

On-chip spatiotemporal electrophysiological analysis of human stem cell derived cardiomyocytes enables quantitative assessment of proarrhythmia in drug development.

We examined a simultaneous combined spatiotemporal field potential duration (FPD) and cell-to-cell conduction time (CT) in lined-up shaped human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using an on-chip multielectrode array (MEA) system to evaluate two origins of lethal arrhythmia, repolarization and depolarization. The repolarization index, FPD, was prolonged by E-4031 and astemizole, and shortened by verapamil, flecainide and terfenadine at 10 times higher than therapeutic plasma concentrations of each drug, but it did not change after lidocaine treatment up to 100 µM. CT was increased by astemizol, flecainide, terfenadine, and lidocaine at equivalent concentrations of Nav1.5 IC50, suggesting that CT may be an index of cardiac depolarization because the increase in CT (i.e., decrease in cell-to-cell conduction speed) was relevant to Nav1.5 inhibition. Fluctuations (short-term variability; STV) of FPD and CT, STVFPD and STVCT also discriminated between torsadogenic and non-torsadogenic compounds with significant increases in their fluctuation values, enabling precise prediction of arrhythmogenic risk as potential new indices.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Development of on-chip multi-imaging flow cytometry for identification of imaging biomarkers of clustered circulating tumor cells.

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers", with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm2 and (2) nucleus area larger than 90 µm2 were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm2 were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.