An optofluidic system with integrated microlens arrays for parallel imaging flow cytometry

Lab on a Chip ◽  
2018 ◽  
Vol 18 (23) ◽  
pp. 3631-3637 ◽  
Author(s):  
Gregor Holzner ◽  
Ying Du ◽  
Xiaobao Cao ◽  
Jaebum Choo ◽  
Andrew J. deMello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Speed ◽  
Parallel Imaging ◽  
Single Cells ◽  
Rapid Analysis ◽  
High Speed Imaging ◽  
Microlens Arrays ◽  
Imaging Flow Cytometry

In recent years, high-speed imaging has become increasingly effective for the rapid analysis of single cells in flowing environments.

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

2019 ◽  
Vol 116 (32) ◽  
pp. 15842-15848 ◽  
Author(s):  
Yuta Suzuki ◽  
Koya Kobayashi ◽  
Yoshifumi Wakisaka ◽  
Dinghuan Deng ◽  
Shunji Tanaka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Raman Scattering ◽  
Cancer Biology ◽  
High Speed ◽  
Stimulated Raman Scattering ◽  
Chemical Imaging ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Imaging Flow Cytometry ◽  
Stimulated Raman

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.

scholarly journals Virtual-freezing fluorescence imaging flow cytometry

2020 ◽  
Author(s):  
Hideharu Mikami ◽  
Makoto Kawaguchi ◽  
Chun-Jung Huang ◽  
Hiroki Matsumura ◽  
Takeaki Sugimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Spatial Resolution ◽  
High Throughput ◽  
Cancer Biology ◽  
Single Cells ◽  
Image Sensor ◽  
Imaging Method ◽  
Microscopy Imaging ◽  
Trade Off ◽  
Imaging Flow Cytometry

ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. For example, at high flow speed (i.e., high throughput), the integration time of the image sensor becomes short, resulting in reduced sensitivity or pixel resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually “freezing” the motion of flowing cells on the image sensor to effectively achieve 1,000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells/s without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.

Deformability Measurement of Single-Cells at High-Throughput With Imaging Flow Cytometry

2015 ◽  
Vol 33 (16) ◽  
pp. 3475-3480 ◽  
Author(s):  
Gangadhar Eluru ◽  
Rajesh Srinivasan ◽  
Sai Siva Gorthi
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Single Cells ◽  
Imaging Flow Cytometry

Deep learning‐enabled imaging flow cytometry for high‐speed Cryptosporidium and Giardia detection

2021 ◽  
Author(s):  
Shaobo Luo ◽  
Kim Truc Nguyen ◽  
Binh T. T. Nguyen ◽  
Shilun Feng ◽  
Yuzhi Shi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
High Speed ◽  
Imaging Flow Cytometry

Contribution of Imaging Flow Cytometry to Storage Lesion Assessment: Identification of a Sub-Population of Morphologically Altered Erythrocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2343-2343
Author(s):  
Pascal Amireault ◽  
Camille A Roussel ◽  
Michaël Dussiot ◽  
Mickael Marin ◽  
Alexandre Morel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multispectral Imaging ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Storage Period ◽  
Morphological Alteration ◽  
Storage Lesion ◽  
Imaging Flow Cytometry ◽  
Duration Of Storage

Abstract The storage lesion encompasses a series of biochemical and molecular modifications that alter erythrocytes during hypothermic storage, reducing transfusion yield. Indeed, in humans, up to 25% of transfused erythrocytes are cleared from the circulation in a few hours (Luten et al., 2008). The mechanisms underlying this rapid clearance are not fully elucidated but it is reasonable to assume that these erythrocyte alterations are sensed by the spleen, resulting in retention. Among those, membrane shedding may have a major impact on post-transfusion clearance of erythrocytes since it causes a progressive decrease in the surface-volume ratio of the cell, leading to the loss of its flexible biconcave shape. The proportion of "damaged" erythrocytes cleared in the hours following transfusion increases with the duration of storage while the deformability measured by ektacytometry (Frank et al., 2013) progressively decreases during this period. To characterize the morphological alteration of stored erythrocytes, we used imaging flow cytometry (Imagestream X Mark II, AMNIS°). This technology allows a simultaneous high-speed multispectral imaging of cells in brightfield, darkfield, and 9 channels of fluorescence. It combines the ability of conventional flow cytometry to analyze a very high number of events with a powerful exploration of cell morphology. We analyzed the morphological, biochemical, metabolic, and bio-mechanical characteristics of erythrocytes stored in optimal blood bank conditions for 6 donors, at Day 3, 21, 28, 35 and 42 of the storage period. This longitudinal study of parameters such as mean corpuscular volume, intracellular ATP level, hemolysis, osmotic fragility, deformability and the plasma levels of ions and metabolites has highlighted a great inter-donor variability in the storage lesion process. Moreover, Imagestream analysis of front views of sharp, single cells revealed a subpopulation of small erythrocytes. The "projected surface area" distribution on normalized frequency plots was bimodal in 5 of 6 concentrates, showing a well-demarcated subpopulation of less than 62 µm2. The proportion of this sub-population increased with storage from 0.5-3.4% at D3 to 4-23.5% at D42 (p<0.05). These cells displayed a low fluorescence staining in the EMA-binding test, a diagnostic test for hereditary spherocytosis. With a more detailed morphological analysis we could determine that this sub-population corresponds to a mix of echinocytes III, spherocytes and sphero-echinocytes (Bessis classification). These results have been confirmed by differential interference phase contrast microscopy (DIC) observations, carried out in parallel, as a gold standard of our imaging flow cytometry study. Indeed, we found a very good correlation between the proportion of small erythrocytes detected in imaging flow cytometry and the echinocytes III, sphero- and spheroechino-cytes detected by DIC (correlation coefficient=0.84). These morphological alterations have been considered irreversible (Berezina et al., 2002) and are reminiscent of those associated with mechanical clearance of erythrocytes in the spleen of patients with hereditary spherocytosis (Mohandas et al., 2008). We hypothesize that these storage-induced small erythrocytes correspond to the subpopulation of "damaged" erythrocytes that are rapidly cleared after transfusion. Confirmation of these findings using ex-vivo perfusion of human spleens and observational studies in transfused patients would support the use of imaging flow cytometry to predict transfusion yield. Disclosures No relevant conflicts of interest to declare.

The rapid analysis of single marine cells by flow cytometry

1990 ◽  
Vol 333 (1628) ◽  
pp. 99-112 ◽  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Rapid Analysis ◽  
Light Scatter ◽  
Fluorescent Labelling ◽  
Photosynthetic Organisms ◽  
Novel Technique ◽  
Multiple Measurements ◽  
Fluorescence Light ◽  
Near Future

Analytical flow cytometry (AFC) is a novel technique for the rapid (more than 103 s-1) analysis and sorting of single cells based upon simultaneous, multiple measurements of laser-induced particle fluorescence, light scatter and impedance. Originally developed for biomedical use, AFC is now being adapted to analyse single-celled organisms such as phytoplankton and bacteria which are present as trace but functionally important components in seawater. Marine AFC has been used to analytically differentiate and sort these organisms from the heterogeneous assemblage of particles present in seawater. Chlorophyll autofluorescence is an unique biomarker for photosynthetic organisms and has been used to analyse phytoplankton cytometrically both in the laboratory and at sea. A theoretical and practical framework for the cytometric quantitation of cellular chlorophyll in phytoplankton based on autofluorescence is presented. Other subcellular constituents such as enzymes, lipids, nucleic acids and toxins in phytoplankton have recently been analysed by AFC using immuno-, induced or applied fluorescent labelling techniques. Examples are presented together with novel developments in fringe areas of cytometry that are likely to influence AFC of single marine cells in the near future.

scholarly journals Biophysical mechanism of ultrafast helical twisting contraction in the giant unicellular ciliate Spirostomum ambiguum

2019 ◽  
Author(s):  
L. X. Xu ◽  
M. S. Bhamla
Keyword(s):  
Physical Model ◽  
High Speed ◽  
Structural Changes ◽  
Single Cells ◽  
Cellular Level ◽  
Cell Biophysics ◽  
High Speed Imaging ◽  
Simple Physical Model ◽  
Biophysical Mechanism ◽  
Spirostomum Ambiguum

The biophysical mechanism of cytoskeletal structures has been fundamental to understanding of cellular dynamics. Here, we present a mechanism for the ultrafast contraction exhibited by the unicellular ciliate Spirostomum ambiguum. Powered by a Ca2+ binding myoneme mesh architecture, Spirostomum is able to twist its two ends in the same direction and fully contract to 75% of its body length within five milliseconds, followed by a slow elongation mechanism driven by the uncoiling of the microtubules. To elucidate the principles of this rapid contraction and slow elongation cycle, we used high-speed imaging to examine the same-direction coiling of the two ends of the cell and immunofluorescence techniques to visualize and quantify the structural changes in the myoneme mesh, microtubule arrays, and the cell membrane. Lastly, we provide support for our hypotheses using a simple physical model that captures key features of Spirostomum’s ultrafast twisting contraction.SIGNIFICANCEUltrafast movements are ubiquitous in nature, and some of the most fascinating ultrafast biophysical systems are found on the cellular level. Quantitative studies and models are key to understand the biophysics of these fast movements. In this work, we study Spirostomum’s ultrafast contraction by using high-speed imaging, labeling relevant cytoskeletal structures, and building a physical model to provide a biophysical mechanism especially of the helical same direction twisting of this extremely large single cell organism. Deeper understanding of how single cells can execute extreme shape changes hold potential for advancing basic cell biophysics and also inspire new cellular inspired actuators for engineering applications.

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