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: 7894810
• 负责人: IRVING J. BIGIO
• 金额: $ 20.31万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894810
• 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化和形态学变化的细胞过程，并且是细胞水平上正常和疾病过程的关键要素。因此，细胞凋亡是每年数以千计的出版物的主题，但通常用于检测或跟踪细胞凋亡的测定均不是在活细胞上进行的，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来破坏。非破坏性的细胞凋亡检测将能够实时监测细胞和细胞培养物，极大地促进此类研究，并通常消除标准的劳动密集型检测。如果该方法能够揭示和识别特定的细胞凋亡途径，则会产生额外的影响。此外，由于大量细胞凋亡是对大多数类型的抗癌治疗产生反应的最早迹象之一，因此长期（在该项目之后）体内应用无创检测可能会影响癌症治疗的管理。弹性散射光谱 (ESS) 是一种无创、实时的光学方法，利用光散射测量来获取有关细胞和组织微形态的定量信息。该项目将开发和测试使用 ESS 的新型仪器，专门用于实时对平板培养物和细胞悬浮液中活细胞培养物中的细胞凋亡进行无破坏性定量监测。这将通过测量来自悬浮液或传统培养板中培养物中的单个细胞或细胞群的选定角度的散射光的波长依赖性（光谱）以及特定波长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“迷你培养箱”光学室，以在一系列测量过程中维持细胞。为了测量角散射相位函数，我们的新型极性浊度计将适合于平板培养物的测量。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的微形态变化。将使用分析和计算方法，应用于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。将使用众所周知的已建立的生化测定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机制至关重要。所提出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、更省力的方式监测和评估细胞死亡过程。