Abstract
We propose a line-field quantitative phase-imaging flow cytometer for analyzing large populations of label-free cells. Hydrodynamical focusing brings cells into the focus plane of an optical system while diluting the cell suspension, resulting in decreased throughput rate. To overcome the trade-off between throughput rate and in-focus imaging, our cytometer involves digitally extending the depth-of-focus on loosely hydrodynamically focusing cell suspensions. The cells outside the depth-of-focus range in the 70-µm diameter of the core flow were automatically digitally refocused after image acquisition. We verified that refocusing was successful with our cytometer through statistical analysis of image quality before and after digital refocusing.
Highlights
IntroductionImaging flow cytometry [1] has recently emerged as a technique for accessing large populations of cells for research and clinical use from the point of view of the localization of fluorescence-tagged molecules in a cell or the cell’s morphological information provided by fluorescence microcopy, bright-field microscopy including phase-contrast, differential interference contrast (DIC), and quantitative phase microscopy (QPM) [2,3]
Imaging flow cytometry [1] has recently emerged as a technique for accessing large populations of cells for research and clinical use from the point of view of the localization of fluorescence-tagged molecules in a cell or the cell’s morphological information provided by fluorescence microcopy, bright-field microscopy including phase-contrast, differential interference contrast (DIC), and quantitative phase microscopy (QPM) [2,3]. These techniques result in slow imaging speed when compared to conventional non-imaging flow cytometry, which performs at speeds of 1,000 to 100,000 cells per second
We proposed a line-field quantitative phase-imaging flow cytometer and used it to carry out digital refocusing of quantitative phase images and statistical analysis of a large population of cells flowing in a microfluidic channel
Summary
Imaging flow cytometry [1] has recently emerged as a technique for accessing large populations of cells for research and clinical use from the point of view of the localization of fluorescence-tagged molecules in a cell or the cell’s morphological information provided by fluorescence microcopy, bright-field microscopy including phase-contrast, differential interference contrast (DIC), and quantitative phase microscopy (QPM) [2,3]. These techniques result in slow imaging speed when compared to conventional non-imaging flow cytometry, which performs at speeds of 1,000 to 100,000 cells per second. Label-free classification of cells based on physical parameters and subcellular structure [7,26,27,28] requires focused images of cells [29]
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