Abstract
Coherent anti-stokes Raman scattering (CARS) flow cytometry was demonstrated by combining a laser-scanning CARS microscope with a polydimethylsiloxane (PDMS) based microfluidic device. Line-scanning across the hydrodynamically focused core stream was performed for detection of flowing objects. Parameters were optimized by utilizing polystyrene beads as flowing particles. Population measurements of adipocytes isolated from mouse fat tissues demonstrated the viability of microfluidic CARS cytometry for quantitation of adipocyte size distribution. CARS cytometry could be a new modality for quantitative analysis with vibrational selectivity.
Highlights
With technological advances in the past decades, flow cytometry has been a powerful tool for quantitative analysis of cell populations and intracellular content [1,2]
Two key issues have to be addressed to make coherent anti-Stokes Raman scattering (CARS) flow cytometry a valid technique. Is it possible to minimize the signal fluctuations induced by out-of-focusing of the flowing objects? Second, can the scan speed be sufficient for volumetric detection of each flowing object? In our experiment, PS beads with well-defined size distributions were used to address these issues
The current study reports the development of CARS cytometry by integrating a laser-scanning CARS microscope with a microfluidic device
Summary
With technological advances in the past decades, flow cytometry has been a powerful tool for quantitative analysis of cell populations and intracellular content [1,2]. Signals in flow cytometry could arise from electrical impedance, forward or side light scattering, and fluorescence. Scattering and electrical impedance provide granularity and size information, but limited chemical specificity [2,3]. As a nonlinear optical imaging technique with vibrational selectivity, coherent anti-Stokes Raman scattering (CARS) microscopy has been successfully applied to image cells, tissues, and live animals, with a strong signal from CH2-abundant structures [6,7,8]. Quantitative analysis of cell populations is, limited by the relatively small field of view (< 1 mm2) in CARS microscopy. It is intriguing to combine CARS with flow cytometry for chemically selective cell analysis in a quantitative manner
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