Label-free multichannel spectral cytometry based on stimulated Raman scattering

描述（由申请人提供）：流式细胞术是一种广泛用于细胞群和细胞内内容物高通量定量分析的工具。流式细胞术中的信号来自电阻抗、前向或侧向光散射和荧光。散射和电阻抗提供粒度和尺寸/体积信息，但没有化学特异性。荧光标记作为流式细胞术中细胞分析的主要方法。然而，荧光标签并不适用于所有情况，特别是小分子（如药物），标记可能会显着扰乱其属性。本申请旨在通过开发使用来自固有分子振动的受激拉曼散射（SRS）信号的多通道光谱流式细胞仪来填补这一空白。受激拉曼散射克服了自发拉曼散射信号强度低的缺点。它测量光-物质的能量转移，因此没有相干反斯托克斯拉曼散射中遇到的非共振背景。此外，作为一种非线性光学过程，它是固有的相位匹配，允许弱聚焦共线光束的几何形状，这是兼容的高速检测流动的物体。计划中的仪器有两个具体目标。第一个是建立一个单频SRS流式细胞仪使用飞秒激光源。第二个是建立一个多通道SRS流式细胞仪的光谱分散的SRS信号的多重检测。基于大信号水平，我们预计将达到每秒10，000个细胞的速度。将通过定量脂肪细胞中的脂肪储存和癌细胞的药物摄取来测试无标记光谱细胞仪的性能。

DESCRIPTION (provided by applicant): Flow cytometry is a widely used tool for high-throughput quantitative analysis of cell populations and intracellular content. Signals in flow cytometry arise from electrical impedance, forward or side light scattering, and fluorescence. Scattering and electrical impedance provide granularity and size/volume information, but with no chemical specificity. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, fluorescent tags are not applicable to all cases, especially small molecules (e.g. drugs) for which labeling may significantly perturb their properties. The current application aims to fill this gap through the development of a multichannel spectral flow cytometer using stimulate Raman scattering (SRS) signal from inherent molecular vibration. The stimulated Raman scattering overcomes the low signal level in spontaneous Raman scattering. It measures the light-matter energy transfer and is therefore free of the nonresonant background encountered in coherent anti-Stokes Raman scattering. Moreover, as a nonlinear optical process it is inherently phase matched, permitting a weakly focused collinear beam geometry that is compatible with high-speed detection of flowing objects. The planned instrumentation contains two specific aims. The first is to build a single-frequency SRS flow cytometer using a femtosecond laser source. The second is to build a multichannel SRS flow cytometer by multiplex detection of spectrally dispersed SRS signals. Based on the large signal level, we expect to reach the speed of 10,000 cells per second. Performance of the label-free spectral cytometer will be tested through quantitation of fat storage in adipocytes and of drug uptake by cancer cells.