A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Abstract

Image-based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single-cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all-optical ultrafast laser-scanning imaging technique, called free-space angular-chirp-enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s ) 1 to 2 orders of magnitude higher than the camera-based imaging flow cytometers. It also has 2 critical advantages over optical time-stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular-specific cellular assay and (2) it enables high-throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single-cell image analysis which reveals cellular heterogeneity, for example, in the cell-death processes demonstrated in this work-the information generally masked in non-imaging flow cytometry. Therefore, this platform empowers not only efficient large-scale single-cell measurements, but also detailed mechanistic analysis of complex cellular processes.

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

“…1a). The unique feature that enables parallelized 3D imaging in CLAM is motivated by the concept of free-space angular-chirp-enhanced delay (FACED) [23][24][25] , which creates an ultrafast laser linescanning action from multiple reflections of a pulsed laser beam between a pair of angle misaligned plane mirrors. In CLAM, we exploit this unique property to create an array of continuous-wave (CW) light sheets with reconfigurable density and coherency through flexible tuning of the mirror-pair geometry (e.g., the mirror length L, separation S, and misaligned angle α) 24 .…”

Microfluidic Based Optical Microscopes on Chip

“…An additional feature of microfluidic devices is the possibility to optimize the fluidic circuit to maximize the device throughput when combined with the proper optical imaging technique, the sample portability, the automation, or to facilitate the measurement protocols to perform imaging under specific sample conditions, such as under different drug exposures.…”

“…ii) Microfluidic devices allow one to flow the sample through a microscope, providing automatic sample scanning and increasing the imaging throughput. Therefore, in the last years, these devices have been optimized for many fluorescence‐based applications: imaging flow cytometers and parallelized imaging devices have been developed . In these examples, the microscope is not necessarily integrated on the chip and bulk external instrumentations are still required to image the sample.…”

Fast intelligent cell phenotyping for high-throughput optofluidic time-stretch microscopy based on the XGBoost algorithm

“…from abundant cells by capturing cell images rapidly, which is a proper solution for the highly sensitive detection of rare cells. 3 Researchers have explored extensively to further improve the performance of optofluidic time-stretch microscopy, such as having a higher resolution, 4 a lower system cost, 5,6 and an application to broader scenarios. [7][8][9] However, high-throughput time-stretch imaging cytometry still suffers from the analysis of mass amounts of cell images.…”

“….Journal of Biomedical Optics066001-3 June 2020 • Vol. 25(6) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics on 30 Oct 2020 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use…”