Multiphoton Microscopy Development

多光子显微镜的发展

• 批准号: 10262672
• 负责人: JAY R KNUTSON
• 金额: $ 58.76万
• 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/10262672
• 关键词: Air; Animals; Anoxia; Arteries; Binding; Binding Proteins; Brain; Buffers; Cancer Cell Growth; Cell Nucleus; Cells; Chromatin; Collection; Color; Complex; Computers; Correlative Study; DNA; Data; Detection; Development; Device or Instrument Development; Devices; Dyes; Environment; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Geometry; Glycolysis; Image; Immersion; Label; Lasers; Learning; Legal patent; Light; Measures; Metabolic; Metabolism; Methods; Microscope; Microscopy; Mitochondria; Molecular Conformation; Morphologic artifacts; NADH; Oils; Oxidation-Reduction; Oxygen; Oxygen Consumption; 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; neoplastic cell; 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. We have already done correlative studies of oxygen consumption and NADH "redox ratio" indicators of metabolism to examine aggressive vs. passive cancer cell growth and effects on the "OXPHOS to glycolysis" switching done by neoplastic cells. 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 决议。它通过在时间和空间上压缩非常高的数字来工作 近红外光子进入显微镜物镜的焦点。毫米子光子 密度允许荧光染料同时吸收两个光子， 产生与单个更蓝的光子相同的激发态。这种情况会发生 仅在大约一微米高和250纳米宽的特权(高光子密度)区中， 椭球体形状，称为PSF(点扩散函数)。因此，这个微小的斑点是 图像；人们必须简单地对其进行栅格处理才能获得图像。 重要的是，所有离开染料的光都是有用的。在基于共焦和/或照相机的显微镜中， 只有相干成像到探测器上的光才有价值。在MPM中，光可以被收集 在“非成像”设备中，计算机根据栅格强度重建图像。 不幸的是，传统的物镜只能回收一小部分发射的光。 在透明介质中，理论上的最大值约为三分之一(石油浸没)，约五分之一(石油 水上目标和空气中只有十分之一。在像组织这样的混浊介质中，这些低效 严重程度可增加一倍或三倍(或更多)。 我们设计并申请了TED(总排放检测)设备的专利，以克服这些问题 信号极限。首先，在泰迪，我们设计了一种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上，我们设计了一种可以接近活动物的设备。在……里面 我们发表的报告表明，尽管有一半的光线必然会消失在 动物，我们高效地恢复其余部分，例如从暴露的大鼠那里看到2.5倍的光 大脑。同样，这意味着我们要么扫描速度更快，要么减少三分之一的激光功率。 我们还制作了一个平面版本的TED原型，一个单片导光板，并开始 初步测试。使用中空的第一表面反射设计，单片式TED目前正被用于恢复EPI-CARS显微镜中丢失的光，以增强我们为动脉中的水恢复的数据类型，并移除某些“EPI”收集几何图形的人工制品。 在前几年，我们已经测试了磷化的树枝状氧探针分子。我们发现这比最理想的速度慢，探测器定位也很薄弱。 相反，我们(首先是在试管中，现在在细胞中)开发了一种基于FRET到O2结合蛋白的新的纳秒氧探测器，我们正在利用这些第一个探测器，同时重新工作其他探测器，以获得更大的范围和更高的可靠性，作为基于DNA的转染。用已知的O2缓冲液对探针质粒的细胞测试进行了校准。例如，我们已经将Mb-mCherry定位于线粒体，在那里我们可以直接成像它们最大水槽附近的氧气水平。我们还检测了细胞核内的缺氧情况。 在不同代谢条件下对细胞内氧气水平的测试正在进行中。我们已经做了耗氧量和NADH代谢“氧化还原比”指标的相关研究，以检测侵袭性和被动性癌细胞的生长，以及对肿瘤细胞进行的“OXPHOS到糖酵解”转换的影响。 除了设备的开发外，我们还可以使用多光子显微镜来进行FCS荧光 相关光谱学-活细胞内标记分子的研究。有了FCS，我们就可以计算 在细胞核中有数百种转录因子，并决定它们的流动性(即 它们是自由的还是染色质结合的？)学习辅助性因素的作用。例如，我们能够使用RICS(栅格FCS)来计算和了解各种蛋白质及其受体在细胞上的结合率，以及转录因子如何结合成大的复合体。