Biophysical Methods and Models

生物物理方法和模型

• 批准号: 8553833
• 负责人: Ralph Nossal
• 金额: $ 25.93万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553833
• 关键词: Algorithms; Antibiotics; Atomic Force Microscopy; Biocompatible Materials; Biological; Cell physiology; Cells; Charge; Communities; Complex; Computer Simulation; Coupled; Crowding; Data; Development; Diffuse; Diffusion; Dyes; Embryo; Environment; Event; Fluorescence; Fluorescence Microscopy; Fourier Transform; Gel; Goals; Growth Factor; Image; Image Analysis; Label; Light; Link; Measurement; Measures; Membrane Lipids; Methodology; Methods; Microbial Biofilms; Modeling; Molecular; Morphologic artifacts; Motion; Movement; Neutrons; Optics; Paper; Particle Size; Phase; Photons; Physics; Polymers; Polystyrenes; Process; Property; Radial; Sampling; Scheme; Signal Transduction; Solutions; Spectrum Analysis; Structure; Study models; Surface; System; Techniques; Testing; Time; Virus; Water; Work; base; cost; crosslink; fluorescence microscope; imaging modality; insight; instrument; instrumentation; light scattering; liquid crystal; macromolecule; optical imaging; particle; physical property; simulation

The continuing goal of this multifaceted project is to develop and employ physics-based methodologies to investigate complex biological structures and materials. Insights gained from studying model materials may be used in inquiries of specific phenomena of biomedical import. Recently, we collaborated on two projects aimed at developing new, low-cost, optical imaging methods for biological samples. One involved constructing an instrument to perform rapid, full-field polarization fluorescence microscopy. Our scheme utilizes a quarter-wave plate in combination with a liquid crystal variable retarder (LCVR) to provide a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. The efficacy of this method was demonstrated by observing localized molecular orientations of probes attached to lipid membranes. In principle, one can use such instrumentation to study any cellular process that involves changes in molecular orientation, as long as appropriate fluorescently labels can be found. The second activity was undertaken to support the development and use of hyperspectral imaging to characterize the spatial organization of complex, multispecies cellular communities. Microarray phantoms containing multiple dyes were fabricated and used to test image analysis algorithms for this process. Another of our activities involves a study of the effects of multiple scattering on the interpretation of fluorescence correlation spectroscopy (FCS) measurements performed on optically dense samples. A unique power of FCS is that, in principle, it can detect the motions of fluorescent entities while signals from non-fluorescent surroundings can be ignored. For this reason, FCS increasingly is used to study particles moving in complex environments, examples being molecules on the surfaces of or within biological cells, antibiotics and viruses diffusing in biofilms, and growth factors moving within embryos. Using well-defined scattering models, we have investigated the reliability of parameters determined when FCS is used to probe the movement of molecules in such complex environments. For instance, we measured FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads which acted as scatterers. A scattering-linked increase in the illuminated volume, as much as two fold, resulted in only a minimal increase in apparent diffusivity. To analyze the illuminated beam profile, we employed Monte-Carlo simulations, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, our results indicate that multiple scattering does not result in serious measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media. A paper based on this work has been submitted. Finally, we have used FCS to investigate the diffusion of macromolecules and other targets within the water phase of polymer gels and concentrated polymer solutions. Again making use of the ability of FCS to distinguish the movements of fluorescent particles from those of a non-fluorescent background, we have been establishing and studying various physical properties of such systems, including the link between particle size, diffusion, and the degree of crosslinking of the polymer-chain constituents. The ultimate goal of this and related studies (e.g., an earlier project to examine the movement of charged macromolecules through crowded polymer solutions) is to understand the physics underlying transport and viscoelastic properties of complex milieu, such as those found within biological cells.

这个多方面项目的持续目标是开发和采用基于物理的方法来研究复杂的生物结构和材料。从研究模型材料中获得的见解可用于生物医学进口的特定现象的调查。 最近，我们合作了两个项目，旨在开发新的，低成本的，生物样品的光学成像方法。其中一个涉及构建一种仪器来进行快速，全场偏振荧光显微镜。我们的方案利用四分之一波片与液晶可变延迟器（LCVR）相结合，以提供一种可调的方法来旋转光的偏振态之前，它被耦合到荧光显微镜。通过观察附着在脂质膜上的探针的局部分子取向证明了这种方法的有效性。原则上，只要能找到合适的荧光标记，就可以使用这种仪器研究任何涉及分子取向变化的细胞过程。第二项活动是支持开发和使用高光谱成像技术来表征复杂的多物种细胞群落的空间组织。制造含有多种染料的微阵列体模，并用于测试该过程的图像分析算法。 我们的另一项活动涉及多重散射对光学致密样品上荧光相关光谱（FCS）测量结果解释的影响。FCS的一个独特功能是，原则上，它可以检测荧光实体的运动，而来自非荧光环境的信号可以被忽略。因此，FCS越来越多地用于研究在复杂环境中移动的粒子，例如生物细胞表面或内部的分子，生物膜中扩散的抗生素和病毒，以及胚胎内移动的生长因子。 使用定义良好的散射模型，我们已经调查了确定的参数的可靠性时，FCS是用来探测分子在这样复杂的环境中的运动。 例如，我们测量的FCS自相关函数的Atto 488染料分子扩散的聚苯乙烯珠作为散射体的解决方案。一个散射连接的照明体积的增加，多达两倍，导致表观扩散率只有最小的增加。为了分析照明光束轮廓，我们采用了蒙特-卡罗模拟，其表明光束沿着轴向比径向更大的加宽，并且焦点处的入射强度减小。在轴向方向上的体积的加宽对所测量的扩散时间仅具有可忽略的影响，因为在FCS测量中由于径向方向上的扩散事件而导致的强度波动占主导地位。总的来说，我们的研究结果表明，多重散射不会导致严重的测量伪影，因此，当足够的信号强度是可达到的，单光子FCS可以是一个有用的技术，用于测量探针在光学致密介质中的扩散率。已经提交了一份基于这项工作的文件。 最后，我们已经使用FCS来研究聚合物凝胶和浓缩聚合物溶液的水相内的大分子和其他目标的扩散。再次利用FCS的能力，区分荧光颗粒的运动从那些非荧光背景，我们一直在建立和研究这些系统的各种物理性质，包括粒径之间的联系，扩散，和交联度的聚合物链成分。这项研究和相关研究的最终目标（例如，一个较早的研究带电大分子通过拥挤的聚合物溶液的运动的项目）是理解复杂环境（例如在生物细胞内发现的那些）的传输和粘弹性性质的物理基础。