Speaker
Description
Light-sheet fluorescence microscopy (LSFM) uses planar illumination to selectively excite a thin optical section of the sample, enabling three-dimensional imaging with reduced photobleaching and phototoxicity [1]. Imaging flow cytometry (IFC) enables high-throughput analysis of large cell populations but is limited to two-dimensional images and relatively low spatial resolution [2]. The combination of LSFM with flow-based imaging provides a possible approach for achieving high-throughput single-cell analysis while preserving optical sectioning and high resolution [3]. The system incorporates multi-wavelength laser excitation via single-mode fiber delivery and multiband detection, enabling multicolor imaging and co-localization analysis of cells and bioparticles in flow.
We present a single-objective imaging system that integrates LSFM and IFC to enable automatic sample scanning with effective background suppression through optical sectioning. This system is designed to acquire high, near-isotropic images of biological samples in flow. The system exploits a microfluidic chip incorporating a 500 µm right-angled prism that generates a thin, focused light sheet orthogonal to the flow direction, while samples flow axially toward the detection objective. Near-isotropic resolution is achieved through axially scanned light-sheet excitation, synchronized with the rolling shutter of a scientific CMOS camera, via a tunable lens.
System performance was evaluated using fluorescent microbeads and biological samples spanning sizes from hundreds of nanometers to tens of microns, including single particles and cells such as macrophage. We present volumetric imaging of fixed and live cells under continuous flow with effective optical sectioning.
We further apply the system to the study of bioparticles, including extracellular vesicles (EVs). Conventional flow cytometry lacks spatial information, hindering unambiguous assignment of fluorescence signals to individual bioparticles, while imaging flow cytometry is limited by strong fluorescence background fluorescence given by the out of focus particles, particularly when studying EVs at high concentrations. This limits the study to one single particle at a time. The proposed light-sheet imaging flow cytometer enables parallel acquisition of multiple particles per frame, targeting 100 samples/frame.
References
[1] J. Huisken, J. Swoger, F. Del Bene, et al. Science. 2004;305(5686):1007–1009
[2] McGrath et al., Cytometry Part A, 2010
[3] F. Sala, M. Castriotta, P. Paiè, et al. Optics Express. 2020;28(9):13039–13051