"Fluorescence lifetime imaging and fluorescence polarization for elucidation of molecular level events: peptide self-assembly, NP dissassembly in cells, and phage binding."

“荧光寿命成像和荧光偏振用于阐明分子水平事件：肽自组装、细胞中的 NP 分解和噬菌体结合。”

• 批准号: 421864-2012
• 负责人: Unsworth, Larry
• 金额: $ 6.29万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=495672
• 关键词: Fluorescence; lifetime; imaging; fluorescence; polarization

Fluorescence based tools are a cost effective means (compared to pulsed nMR which is the only technique that can provide similar information) of elucidating molecular level events. This is primarily due to the wealth of information that can be obtained from fluorescence emission, which can be quantified in obvious ways like intensity and position, as well as less obvious ways like lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM), for example, can provide detailed information regarding photophysical events otherwise impossible to detect using fluorescence intensity imaging techniques. FLIM technique is relatively insensitive to probe concentration, but susceptible to environmental changes like pH, viscosity, ion or oxygen concentration, refractive index, polarity and quenching via Forster resonance energy transfer (FRET). Fluorescence polarization (anisotropy) may yield information that cannot be obtained from intensity, lifetime or emission spectrum data, including: molecular aggregation, orientation, rotational mobility and the migration of energy between identical fluorophores (homo-FRET). Moreover, fluorescent polarization has been shown to be proportional to molecular size and solution viscosity, whilst being inversely proportional to fluorescence lifetime and solution temperature. Specifically, the Stokes-Einstein equation can be used to relate the rotational diffusion time to molecular size (ie. hydrodynamic volume), viscosity, and solution temperature. This methodology has been used to allow the determination of molecular size and viscosity gradients within lipid membranes or live cells. This equipment will allow for significant advances in the molecular level understanding of how peptide chemistry affects peptide self-assembly, nanofiber formation, and molecular binding. Work considered here is interdisciplinary by nature, facilitating the training of HQP across biomedical, biophysics, pharmacy, medicine, and biochemical engineering.

基于荧光的工具是阐明分子水平事件的一种经济有效的手段(与脉冲核磁共振相比，脉冲核磁共振是唯一可以提供类似信息的技术)。这主要是因为可以从荧光发射中获得丰富的信息，可以用强度和位置等显而易见的方法来量化，也可以用寿命、偏振和波长等不太明显的方法来量化。例如，荧光寿命成像(FLIM)可以提供有关光物理事件的详细信息，否则无法使用荧光强度成像技术检测到。Flim技术对探针浓度相对不敏感，但对环境变化敏感，如pH、粘度、离子或氧浓度、折射率、极性和通过Forster共振能量转移(FRET)猝灭。荧光偏振(各向异性)可能会产生从强度、寿命或发射光谱数据中无法获得的信息，包括：分子聚集、取向、旋转迁移率和相同荧光团之间的能量迁移(Homo-FRET)。此外，荧光偏振已被证明与分子大小和溶液粘度成正比，而与荧光寿命和溶液温度成反比。具体地说，斯托克斯-爱因斯坦方程可以用来关联旋转扩散时间与分子大小(即。流体动力体积)、粘度和溶液温度。这种方法已被用来确定脂膜或活细胞内的分子大小和粘度梯度。该设备将允许在分子水平上取得重大进展，了解多肽化学如何影响多肽自组装、纳米纤维形成和分子结合。这里考虑的工作本质上是跨学科的，促进了HQP在生物医学、生物物理学、药学、医学和生化工程方面的培训。