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使用的工具，用于高通量定量分析细胞群和细胞内含量。流式细胞术中的信号来自电阻抗、正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。荧光标记是流式细胞术中细胞分析的主要方法。然而，荧光标记并不适用于所有情况，特别是小分子（如药物），因为标记可能会显著干扰其性质。当前的应用旨在通过利用来自固有分子振动的刺激拉曼散射（SRS）信号的多通道光谱流式细胞仪的开发来填补这一空白。受激拉曼散射克服了自发拉曼散射中的低信号电平。它测量光-物质能量传递，因此不存在相干反斯托克斯拉曼散射中遇到的非共振背景。此外，作为一个非线性光学过程，它本质上是相位匹配的，允许弱聚焦共线光束几何形状，与流动物体的高速检测兼容。计划中的仪器仪表包含两个具体目标。首先是利用飞秒激光源构建单频SRS流式细胞仪。二是通过对频谱分散的SRS信号进行多重检测，构建多通道SRS流式细胞仪。基于大信号水平，我们期望达到每秒10,000个细胞的速度。无标记光谱细胞仪的性能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。

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.