Microsecond Raman spectroscopy: Assessing single cell metabolism in a vital organism

 描述：实时定量活细胞中的代谢转化对于确定细胞对药物治疗或暴露于危险因素等干预措施的反应是至关重要的。然而，我们关于细胞内容的大部分知识来自于对分离细胞的体外分析或组织匀浆的测量，无论是通过生化分析、组学或测序技术。这一差距突显了开发新技术的必要性，这些技术能够重复评估生命有机体中的同一单个细胞。我们建议开发一种新的拉曼成像平台，以线虫为试验床，重复评估生命有机体中的单细胞代谢。我们的创新是在角度-波长脉冲整形器的傅里叶平面上对飞秒脉冲进行光谱扫描，通过它可以获得每像素20ms尺度的SRS谱。我们研究的长期目标是开发下一代技术，以便能够在3D培养或活动物中对单个活细胞对刺激或治疗的反应进行定量分析。这项R21应用的具体目标是构建一个毫秒时间尺度的光谱成像系统，并纵向评估活体线虫的脂肪代谢。已经成立了一个跨学科的团队。郑继新博士(Pi)是无标记光谱成像的专家。Heidi Tissenbaum博士(co-Pi)是以秀丽线虫为模式生物剖析衰老过程的分子机制的专家。这两位研究人员在开发相干拉曼散射显微镜以研究活体线虫的脂肪代谢方面已经建立了合作关系。在可行性研究中，该团队使用指纹拉曼光谱对成虫体内的脂肪氧化、脂肪去饱和和胆固醇储存进行了高光谱SRS成像。此外，程实验室最近演示了使用角度-波长脉冲整形器的光谱调制SRS成像。这种发展为获取ms时间尺度上的sRS频谱奠定了基础。我们的假设是，拉曼光谱能够纵向评估在衰老、饮食限制或过度喂养期间有生命的线虫的脂肪代谢。为了验证这一假设，我们将设计、建造并测试与波长成形器成角度的微秒拉曼光谱成像。然后，我们将使用该系统纵向评估野生型和突变型线虫单细胞的能量代谢。虽然秀丽线虫被用作试验床，但我们的平台通过评估单个活细胞对干预的反应，包括监测活细胞对风险因素的反应或对刺激的组织再生，预示着对生物医学研究的更广泛影响。

 DESCRIPTION: Quantitation of metabolic conversions in live cells and in real time is essential to determining how a cell responds to an intervention such as drug treatment or exposure to a risk factor. Nevertheless, most of our knowledge about cellular content is derived from in vitro analysis of isolated cells or measurement of tissue homogenates, either by biochemical assays, omics or sequencing technologies. This gap highlights a need of developing new techniques that are able to repetitively assess the same single cell in a vital organism. We propose to develop a new Raman imaging platform to enable repetitive assessment of single cell metabolism in a vital organism, using C.elegans as a test bed. Our innovation is spectral scanning of a femtosecond pulse at the Fourier plane of an angle-to wavelength pulse shaper, through which a SRS spectrum can be acquired on the scale of 20 ms per pixel. The long-term goal of our research is developing next generation technology to enable quantitative analysis of single live cell response to a stimuli or a treatment in 3D cultures or live animals. The specific objectives of this R21 application are constructing a ms time scale spectroscopic imaging system and longitudinally assessing the fat metabolism in vital C.elegans. An interdisciplinary team has been formed. Dr. Ji-Xin Cheng (PI) is an expert in label-free spectroscopic imaging. Dr. Heidi Tissenbaum (co-PI) is an expert in dissecting molecular mechanisms of the aging process using C.elegans as a model organism. The two investigators have an established collaboration in developing coherent Raman scattering microscopy to study lipid metabolism in live C.elegans. In feasibility studies, the team has demonstrated hyperspectral SRS imaging of lipid oxidation, lipid desaturation, and cholesterol storage in adult worms using fingerprint Raman bands. Moreover, the Cheng lab recently demonstrated spectral modulation SRS imaging with an angle-to- wavelength pulse shaper. Such development paves the foundation for acquisition of a SRS spectrum on the ms time scale. Our hypothesis that ms Raman spectroscopy is able to longitudinally assess lipid metabolism in vital C.elegans during aging, diet restriction or overfeeding. To test this hypothesis, we will design and construct, and test a microsecond Raman spectral imaging with an angle to wavelength pulse shaper. We will then use the system to longitudinally assess energy metabolism of single cells in wild-type and mutant C. elegans. Though C.elegans is used as a test bed, our platform heralds a broader impact on biomedical research via assessing single live cell response to an intervention, including monitoring live cell response to a risk factor or tissue regeneration in response to a stimulus.