Multiphoton Microscopy Development

多光子显微镜的发展

• 批准号: 10012682
• 负责人: JAY R KNUTSON
• 金额: $ 69.69万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/10012682
• 关键词: Air; Animals; Anoxia; Arteries; Binding; Binding Proteins; Brain; Buffers; Cell Nucleus; Cells; Chromatin; Collection; Color; Complex; Computers; DNA; Data; Detection; Development; Device or Instrument Development; Devices; Dyes; Environment; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Geometry; Image; Immersion Investigative Technique; Label; Lasers; Learning; Legal patent; Light; Measures; Metabolic; Methods; Microscope; Microscopy; Mitochondria; Molecular Conformation; Morphologic artifacts; Oils; Oxidation-Reduction; Oxygen; Photons; Plasmids; Proteins; Publishing; Rattus; Resolution; Rest; Role; Scanning; Severities; Shapes; Signal Transduction; Spectrum Analysis; Spottings; Surface; Testing; Time; Tissues; Transfection; Water; Work; base; cofactor; density; design; detector; flexibility; improved; intravital imaging; macromolecule; multiphoton microscopy; nanosecond; off-patent; prototype; quantitative imaging; receptor; submicron; tool; transcription factor; two-photon

Multiphoton Microscopy has become the method of choice for intravital imaging at submicron resolution. It works by both temporally and spatially compressing very high numbers of near infrared photons into the focus of a microscope objective. Millimolar photon densities permit the simultaneous absorbtion of two photons by the fluorescent dye, yielding the same excited state one would get with a single bluer photon. This occurs only in a privileged (high photon concentration) zone about a micron tall and 250 nm wide, ellipsoidal in shape, known as the PSF (point spread function). Thus the tiny spot IS the image; one must simply raster it about to get a picture. Importantly, ALL light leaving the dye is useful. In confocal and/or camera based microscopes, only the light coherently imaged onto a detector is of value. In MPM, light can be collected in a "non-imaging" device and the computer reconstructs the picture from raster intensity. Unfortunately, conventional objectives recover only a small portion of the emitted light. The theoretical maximum in clear media is about a third for oil immersion, about a fifth for water objectives and only a tenth in air. In turbid media like tissue, these inefficiencies can double or triple (or more) in severity. We have designed and patented TED ("Total Emission Detection") devices to overcome these signal limits. First, in TEDI, we designed a device for cells and tissue blocks that increases typical signal levels an order of magnitude. More recently, in TEDII, we designed a device class that can approach living animals. In our published accounts, we show that although half the light is necessarily lost in the animal, we efficiently recover the rest, seeing e.g. 2.5x more light from the exposed rat brain. Again, this means we can either scan faster or reduce laser power a third. We also prototyped a planar version of TED, a monolithic lightguide, and began preliminary testing. Using hollow first-surface reflection designs, monolithic TED is currently being used to recover lost light in epi-CARS microscopy, to enhance the sort of data we recovered for water in arteries and to remove certain artifacts of "epi" collection geometry. We had, in previous years, tested dendrimeric oxygen probe moleculesthat phosphoresced. We found this slower than optimal, and probe targeting was tenuous. We instead developed (first in cuvettes, now in cells)a new nanosecond oxygen probe based on FRET to O2 binding proteins, and we are exploiting thesefirst probes while reworking others for greater range and reliability as DNA-based transfections. Cellular tests of probe plasmids were calibrated with known O2 buffers. We have targeted Mb-mCherry, for example, to mitochondria, where we can directly image oxygen levels near their biggest sinks. We have also tested for anoxia within nuclei. Testing of intracellur oxygen levels in differing metabolic conditions are underway. In addition to device development, we can employ the multiphoton microscope to do FCS- Fluorescence Correlation Spectroscopy - of labeled molecules inside living cells. With FCS, we can count a few hundred transcription factors in the cell nucleus and determine their mobility (i.e. are they free or chromatin-bound?) and learn the role of cofactors. For example, we are able to count and learn binding rates for various proteins and their receptors on cells, using RICS (raster FCS), and how transcription factors bundle into large complexes.

多光子显微镜已成为亚微米活体成像的首选方法 解决。它的工作原理是在时间和空间上压缩非常高的数字 近红外光子进入显微镜物镜的焦点。 毫摩尔光子 密度允许荧光染料同时吸收两个光子， 产生与单个蓝色光子相同的激发态。 出现这种情况 仅在约一微米高、约 250 纳米宽的特权（高光子浓度）区域， 椭圆形，称为 PSF（点扩散函数）。 因此这个小点是 图像；人们必须简单地将其光栅化以获得一张照片。 重要的是，离开染料的所有光都是有用的。在共焦和/或基于相机的显微镜中， 只有相干成像到探测器上的光才有价值。 在 MPM 中，可以收集光 在“非成像”设备中，计算机根据光栅强度重建图像。 不幸的是，传统的物镜只能回收一小部分发射的光。 透明介质中的理论最大值约为油浸的三分之一，油浸的理论最大值约为五分之一 水中目标只有十分之一是空中目标。 在组织等混浊介质中，这些低效率 严重程度可以加倍或三倍（或更多）。 我们设计了 TED（“总发射检测”）设备并获得专利来克服这些问题 信号限制。 首先，在 TEDI，我们设计了一种用于细胞和组织块的设备， 将典型信号电平提高一个数量级。最近，在 TEDII 中，我们设计了一个可以接近活体动物的设备类。在 我们发表的报道表明，尽管一半的光线必然在 动物，我们有效地恢复其余部分，例如看到来自暴露的老鼠的光线增加了 2.5 倍 脑。 同样，这意味着我们可以更快地扫描或将激光功率降低三分之一。 我们还制作了 TED 的平面版本（一种单片光导）原型，并开始 初步测试。使用中空第一表面反射设计，单片 TED 目前用于恢复 Epi-CARS 显微镜中丢失的光，以增强我们恢复的动脉中水的数据类型，并消除“epi”收集几何形状的某些伪影。 前几年，我们测试了发磷光的树枝状氧探针分子。我们发现这比最佳状态要慢，并且探针定位很脆弱。 相反，我们开发了（首先在比色皿中，现在在细胞中）一种基于 FRET 与 O2 结合蛋白的新纳秒氧探针，我们正在利用这些第一个探针，同时改造其他探针，以获得更大的范围和可靠性，作为基于 DNA 的转染。探针质粒的细胞测试用已知的 O2 缓冲液进行校准。 例如，我们将 Mb-mCherry 瞄准线粒体，我们可以直接对线粒体最大汇附近的氧气水平进行成像。我们还测试了细胞核内的缺氧情况。 不同代谢条件下细胞内氧水平的测试正在进行中。 除了器件开发之外，我们还可以利用多光子显微镜进行FCS-荧光 相关光谱 - 活细胞内标记分子。 有了FCS，我们就可以计算 细胞核中的数百个转录因子并确定它们的移动性（即 它们是游离的还是染色质结合的？）并了解辅助因子的作用。 例如，我们能够使用 RICS（光栅 FCS）计算和了解细胞上各种蛋白质及其受体的结合率，以及转录因子如何捆绑成大型复合物。