Instrumentation for optical monitoring of apoptosis in unlabeled cell cultures

描述（由申请人提供）：细胞凋亡，“程序性细胞死亡”，是一种细胞过程，表现出不同的生化和形态变化，是细胞水平上正常和疾病过程的关键因素。因此，细胞凋亡是每年数千篇出版物的主题，然而，没有一种常用的检测或跟踪细胞凋亡的方法是在活细胞上进行的，因为所有这些方法都需要破坏培养物或组织，通常是通过固定和染色。一种无干扰的细胞凋亡检测方法能够实时监测细胞和细胞培养，极大地促进了这类研究，并且通常消除了标准的劳动密集型检测方法。如果该方法可以揭示和识别特定的凋亡途径，将获得额外的影响。此外，由于大量细胞凋亡是对大多数类型的抗癌治疗反应的最早迹象之一，因此（在该项目之后）长期的无创检测在体内应用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、实时的光学方法，它利用光散射测量来获得有关细胞和组织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，该仪器是专门设计用于实现无干扰、实时定量监测活细胞培养中细胞凋亡的，适用于电镀培养和细胞悬浮液。这将通过测量特定角度散射光的波长依赖性（光谱）和特定波长散射光的角度依赖性（相位函数）来实现，这些散射光来自培养中的单个细胞或细胞群，无论是悬浮还是传统培养板。光谱ESS系统将包含一个专门的“迷你培养箱”光学室，用于在一系列测量期间维持细胞。为了测量角散射相函数，我们的新极浊度计将适应于镀培养物的测量。由于单个测量几乎是瞬时的，ESS方法将能够在任何期望的时间跨度内跟踪微观形貌的变化。将使用分析和计算方法，将结果应用于光谱和角度测量，以提取细胞内散射体的大小分布，从而提供与细胞超微结构相关的定量测量。广泛的验证将使用已建立的生化分析，以及环境扫描电子显微镜进行。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至关重要。该仪器将使研究细胞对治疗反应的细胞生物学家能够以更快、更少劳动密集型的方式监测和评估细胞死亡过程，使用基于光的测量代替目前的化学分析。

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.