High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry.

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevents accurate quantification. We report a high-throughput, inexpensive, reliable and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.

An image cytometer based on angular spatial frequency processing and its validation for rapid detection and quantification of waterborne microorganisms.

We introduce a new image cytometer design for the detection of very small particulates and demonstrate its capability in water analysis. The device is a compact microscope composed of off-the-shelf components, such as a light emitting diode (LED) source, a complementary metal-oxide-semiconductor (CMOS) image sensor, and a specific combination of optical lenses that allow, through an appropriate software, Fourier transform processing of the sample volume. Waterborne microorganisms, such as Escherichia coli (E. coli), Legionella pneumophila (L. pneumophila) and phytoplankton, are detected by interrogating the volume sample either in a fluorescent or label-free mode, i.e. with or without fluorescein isothiocyanate (FITC) molecules attached to the micro-organisms, respectively. We achieve a sensitivity of 50 cells per ml, which can be further increased to 0.2 cells per ml by pre-concentrating an initial sample volume of 500 ml with an ad hoc fluidic system. We also prove the capability of the proposed image cytometer of differentiating microbiological populations by size with a resolution of 3 µm and operating in real contaminated water.

A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.

Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis. Currently, fluorescence-based cell death assays have not been demonstrated by the Cellometer system, which can potentially provide a quick, simple, and inexpensive alternative method for smaller biomedical research laboratories. In this study, we demonstrate for the first time the use of Cellometer imaging cytometry for necrosis/apoptosis detection by studying the dose-response effect of heat and drug-induced cell death in Jurkat cells labeled with annexin V-FITC (apoptotic) and propidium iodide (necrotic). The experimental results were evaluated to validate the imaging cytometric capabilities of the Cellometer system as compared to the conventional flow cytometry. Similar cell population results were obtained from the two methods. The ability of Cellometer to rapidly and cost-effectively perform fluorescent cell-based assays has the potential of improving research efficiency, especially where a flow or laser scanning cytometer is not available or in situations where a rapid analysis of data is desired.

CD4 and CD8 enumeration for HIV monitoring in resource-constrained settings.

BACKGROUND: We developed a volumetric single platform image cytometer (SP ICM) that is dedicated to count CD4(+) and CD8(+) T lymphocytes for HIV monitoring in resource-constrained settings. The instrument was designed to be low-cost, yet reliable, easy-to-use, and robust. METHODS: Whole blood is incubated with CD3-magnetic nanoparticles, CD4-phycoerythrin (PE), and CD8-peridinin-chlorophyll-protein complex (PerCP). The CD3 cells are immunomagnetically attracted to an analysis surface, where fluorescence images of CD4(+) and CD8(+) T lymphocytes are recorded and analyzed, respectively. We compared CD4, CD8 counts, and CD4/CD8 ratio obtained by the SP ICM with those from a SP flow cytometer (FCM) tetraCXP method on blood samples from 145 patients. RESULTS: Good correlations were obtained (R: 0.96-0.99) between the SP ICM and the SP FCM. There was approximately 10% CD8 undercount in the SP ICM, which could be partly caused by CD8(+dim) T lymphocytes that were not detected by the instrument or not counted by the image analysis due to the cross-talk from the CD4-PE signal in the CD8-PerCP image. CONCLUSIONS: The SP ICM is a good candidate for HIV monitoring in point-of-care settings of resource-constrained countries.

CD4+ T lymphocytes enumeration by an easy-to-use single platform image cytometer for HIV monitoring in resource-constrained settings.

BACKGROUND: HIV monitoring in resource-constrained settings demands affordable and reliable CD4(+) T lymphocytes enumeration methods. We developed a simple single platform image cytometer (SP ICM), which is a dedicated volumetric CD4(+) T lymphocytes enumeration system that uses immunomagnetic and immunofluorescent technologies. The instrument was designed to be a low-cost, yet reliable and robust one. In this article we test the instrument and the immunochemical procedures used on blood from HIV negative and HIV positive patients. METHODS: After CD4 immunomagnetic labeling in whole blood, CD4(+) T lymphocytes, CD4(+dim) monocytes and some nonspecifically labeled cells are magnetically attracted to an analysis surface. Combining with CD3-Phycoerythrin (PE) labeling, only CD3(+)CD4(+) T lymphocytes are fluorescently labeled and visible in a fluorescent image of the analysis surface. The number of CD4(+) T lymphocytes is obtained by image analysis. Alternatively, CD3 immunomagnetic selection in combination with CD4 immunofluorescent labeling can also be applied for CD4(+) T lymphocytes enumeration. RESULTS: The SP ICM system was compared with two single platform flow cytometer (SP FCM) methods: tetraCXP and TruCount methods. The SP ICM system has excellent precision, accuracy and linearity for CD4(+) T lymphocytes enumeration. Good correlations were obtained between the SP ICM and the SP FCM methods for blood specimens of 44 HIV(-) patients, and of 63 HIV(+) patients. Bland-Altman plots showed interchangeability between the SP ICM and the SP FCM methods. CONCLUSIONS: The immunolabeling methods and the instrumentation are simple and easy-to-handle for less-trained operators. The SP ICM system is a good candidate for CD4(+) T lymphocytes enumeration in point-of-care settings of resource-constrained countries.

Personal cytometers: slow flow or no flow?

BACKGROUND: Although some manufacturers have optimistically described instruments with prices in the 40,000 US dollars range as "personal cytometers", analogy with the personal computer suggests that the target price for a true "personal" cytometer should be under 5,000 US dollars. Since such an apparatus could find a wide range of applications in cytomics in both developing and developed countries, it seemed desirable to consider its technical and economic feasibility. METHODS: Using resolution targets and a variety of fluorescent bead standards immobilized on filters and/or slides, we evaluated high-intensity LEDs as fluorescence excitation sources, relatively inexpensive CCD cameras as detectors, and 35 mm camera lenses and plastic low-power microscope optics for light collection in a simple, inexpensive low-resolution imaging cytometer. RESULTS: The components tested could be combined toproduce an instrument capable of detecting fewer than 10,000 molecules of cell-associated fluorescent label, and thus applicable to a broad range of cytometric tasks. CONCLUSIONS: Given the requirements for light sources, detectors, optics, mechanics, electronics and data analysis hardware and software, and the components presently available, it should be easier to reach the desired 5,000 US dollars price point with an image cytometer than with a flow cytometer.

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.

RIKEN integrated sequence analysis (RISA) system--384-format sequencing pipeline with 384 multicapillary sequencer.

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.