Instrumentation for optical monitoring of apoptosis in unlabeled cell cultures

描述(申请人提供)：细胞凋亡，“程序性细胞死亡”，是一种表现出明显的生化和形态变化的细胞过程，是细胞水平上正常和疾病过程的关键因素。因此，每年有数以千计的出版物以细胞凋亡为主题，然而，通常用于检测或跟踪细胞凋亡的检测方法没有一种是在活细胞上进行的，因为所有这些都需要破坏培养或组织，通常是通过固定和染色。一种无干扰的细胞凋亡分析将使细胞和细胞培养的实时监测成为可能，极大地促进了这类研究，并通常消除了标准的劳动密集型分析。如果该方法能够揭示和识别特定的凋亡途径，将会产生额外的影响。此外，由于大量细胞凋亡是对大多数类型的抗癌治疗最早的反应指标之一，长期(在该项目之后)在体内应用非侵入性检测可能会影响癌症治疗的管理。弹性散射光谱(ESS)是一种非侵入性的、实时的光学方法，它使用光散射测量来获得关于细胞和组织的微观形态的定量信息。该项目将开发和测试使用ESS的新型仪器，该仪器专门设计用于实现对平板培养和细胞悬液中活细胞培养物中的凋亡进行实时、无干扰的定量监测。这将通过测量选定角度的散射光的波长相关性(光谱)和特定波长的散射光的角度相关性(相函数)来实现，所述特定波长的散射光来自悬浮或常规培养板中的单个细胞或细胞群。光谱ESS系统将包括一个专门的“微型孵化器”光学室，以在一系列测量过程中维护细胞。对于角散射相函数的测量，我们的新型极性浊度计将被改装以便于在平板培养上进行测量。由于单个测量几乎是瞬时的，ESS方法将能够跟踪任何所需时间跨度内微形态的变化。将使用分析和计算方法，应用于所产生的光谱和角度测量，以提取细胞内散射体的尺寸分布，从而提供与细胞超微结构相关的定量测量。将使用众所周知的已建立的生化分析以及环境扫描电子显微镜进行广泛的验证。公共卫生相关性：在研究疾病过程和细胞对治疗的反应时，监测和了解细胞死亡的机制是至关重要的。拟议的仪器将使正在研究细胞对治疗的反应的细胞生物学家能够通过使用基于光的测量而不是当前的化学分析，以更快、更少的劳动强度的方式监测和评估细胞死亡过程。

DESCRIPTION (provided by applicant): Apoptosis, "programmed cell death," is a cellular process exhibiting distinct biochemical and morphological changes, and is a key element of both normal and disease processes at the cellular level. As such, apoptosis is the subject of thousands of publications annually, yet none of the assays that are commonly used to detect or track apoptosis are performed on viable cells, as all require disruption of cultures or tissue, often by fixation and staining. A nondisruptive assay of apoptosis would enable real-time monitoring of cells and cell cultures, dramatically facilitating such studies, and generally eliminating standard labor-intensive assays. Additional impact would obtain if the method could expose and identify specific apoptotic pathways. Moreover, since massive apoptosis is one of the earliest indications of response to most types of anti-cancer treatment, a longer-term (after this project) in-vivo application of a noninvasive assay could impact management of cancer treatment. Elastic scattering spectroscopy (ESS) is a noninvasive, real-time, optical method that uses light scattering measurements to obtain quantitative information about the micromorphology of cells and tissue. This project will develop and test novel instrumentation that uses ESS and is specifically designed to realize nondisruptive, quantitative monitoring of apoptosis in viable cell cultures in real time, for both plated cultures and cell suspensions. This will be effected by measuring the wavelength-dependence (spectrum) of scattered light at selected angles, and the angle-dependence (phase function) of scattered light at specific wavelengths, from individual cells or ensembles of cells in culture, either in suspension or in conventional culture plates. The spectral ESS system will incorporate a specialized "mini-incubator" optical chamber to maintain cells during a sequence of measurements. For measurement of the angular scattering phase function, our novel polar nephelometer will be adapted to facilitate measurements on plated cultures. Since individual measurements are nearly instantaneous, the ESS method will enable the tracking of changes in micromorphology over any desired time span. Analytical and computational methods will be used, applied to the resulting spectra and angle measurements, to extract the size distributions of scatterers within the cells, thus providing quantitative measures related to cellular ultrastructure. Extensive validations will be performed using established biochemical assays that are well understood, as well as environmental scanning electron microscopy. PUBLIC HEALTH RELEVANCE: In studying disease processes and the response of cells to treatments, it is critical to monitor and understand the mechanisms of cell death. The proposed instrumentation will enable cellular biologists, who are studying cellular response to treatment, to monitor and evaluate cell-death processes in a faster and less labor-intensive manner, by using light-based measurements instead of the current chemical assays.