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

Abstract

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.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

“…Studies using vibrational microspectroscopy (Raman and CARS) have pointed out high lipid levels in cancer cells e.g. breast 24 , or colorectal 25 cancers, as well as in circulating tumor cells 26,27 that could be detected using the Stimulated Raman Scattering imaging flow cytometer, a marker-free detection technology 28 . However, none of these studies focused on the origin of this increased lipid content.…”

Raman image-activated cell sorting

“…This image construction is continuously repeated for multiple events. The primary difference between this ultrafast multicolor SRS microscope and our earlier SRS microscope 19 lies in their image acquisition scheme; the present SRS microscope has a parallel detection system that allows imaging at a higher flow speed than the earlier SRS microscope that has a point-scanning serial detection system.…”

High-speed particle detection and tracking in microfluidic devices using event-based sensing

“…In the presence of mixed populations, as often occurs in microfluidic systems, detecting and differentiating particles without fluorescent signals can be a challenge. New approaches are emerging to unlock the potential of high-speed data acquisition; [37][38][39][40][41] however, micro-particle image velocimetry (μPIV) remains the most widely accessible technique for high-speed fluorescence imaging. In μPIV, the illumination is provided by high-power, pulsed lasers to record pairs of images with a short time delay.…”