Instrumentation for optical monitoring of apoptosis in unlabeled cell cultures

用于光学监测未标记细胞培养物中细胞凋亡的仪器

• 批准号: 7740280
• 负责人: IRVING J. BIGIO
• 金额: $ 24.38万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740280
• 关键词: Apoptosis; Apoptotic; Biochemical; Biological; Biological Assay; Cell Culture Techniques; Cell Death; Cell Death Process; Cell Surface Receptors; Cell physiology; Cells; Characteristics; Chemicals; Chinese Hamster Ovary Cell; Computing Methodologies; Cultured Cells; Dependence; Disease; Elastic scattering spectroscopy; Electron Microscopy; Elements; Event; Exhibits; Goals; Hormones; Incubators; Individual; Induction of Apoptosis; Measurement; Measures; Methods; Mitochondria; Monitor; Myocardial Infarction; Nuclear; Optical Methods; Optics; Particle Size; Pathway interactions; Phase; Physiological; Pilot Projects; Positioning Attribute; Process; Publications; Receptor Signaling; Reperfusion Injury; Resolution; Sampling; Scanning Electron Microscopy; Signal Transduction; Spectrum Analysis; Staging; Staining method; Stains; Staurosporine; Stimulus; Stroke; Suspension substance; Suspensions; System; Temperature; Testing; Time; Tissues; Validation; base; cancer therapy; cell fixing; cell type; culture plates; design; in vivo; instrument; instrumentation; light scattering; nephelometry; novel; photonics; planetary Atmosphere; public health relevance; response; sample fixation

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.

描述（由申请人提供）：细胞凋亡，“程序性细胞死亡”，是一种表现出独特的生物化学和形态学变化的细胞过程，是细胞水平上正常和疾病过程的关键因素。因此，细胞凋亡是每年数千篇出版物的主题，但通常用于检测或跟踪细胞凋亡的测定都没有在活细胞上进行，因为所有测定都需要破坏培养物或组织，通常通过固定和染色。一个非破坏性的细胞凋亡测定将能够实时监测细胞和细胞培养，大大促进这样的研究，并通常消除标准的劳动密集型测定。如果该方法可以暴露和鉴定特定的凋亡途径，则将获得额外的影响。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，因此非侵入性测定的长期（在该项目之后）体内应用可能会影响癌症治疗的管理。弹性散射光谱（ESS）是一种非侵入性的，实时的，光学方法，使用光散射测量，以获得有关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物和细胞悬浮液中活细胞培养物中细胞凋亡的真实的非破坏性定量监测。这将通过测量来自培养物中的单个细胞或细胞系（在悬浮液中或在常规培养板中）在选定角度下的散射光的波长依赖性（光谱）以及在特定波长下的散射光的角度依赖性（相位函数）来实现。光谱ESS系统将包含一个专门的“微型培养箱”光学室，以在一系列测量过程中保持细胞。对于角散射相函数的测量，我们的新的极性浊度计将被调整，以便于测量平板培养。由于单个测量几乎是瞬时的，ESS方法将能够在任何所需的时间跨度内跟踪微观形态的变化。将使用分析和计算方法，应用到所得的光谱和角度测量，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。将使用已建立的已充分理解的生化试验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至关重要。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测定，以更快和更少劳动密集型的方式监测和评估细胞死亡过程。