Multiphoton Microscopy Development

多光子显微镜已成为亚微米活体成像的首选方法 分辨率它的工作原理是在时间和空间上压缩非常高的数字 将近红外光子聚焦到显微镜物镜的焦点。 毫莫耳光子 密度允许荧光染料同时吸收两个光子， 产生与单个蓝光子相同的激发态。 发生这种情况 仅在大约1微米高和250纳米宽的特权（高光子浓度）区域中， 椭圆形，称为PSF（点扩散函数）。 故小者， 图像;人们必须简单地光栅它得到一个图片。 重要的是，所有离开染料的光都是有用的。在基于共焦和/或相机的显微镜中， 只有相干成像到检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，传统的物镜只能恢复一小部分的发射光。 在透明介质中，油浸的理论最大值约为三分之一， 水的目标和只有十分之一的空气。 在像组织这样的混浊介质中， 可以在严重性上加倍或三倍（或更多）。 我们已经设计并申请了TED（“总排放检测”）设备的专利，以克服这些问题。 信号限制。 首先，在TEDI中，我们设计了一种用于细胞和组织块的设备， 将典型的信号电平增加一个数量级。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 我们公布的帐户，我们表明，虽然一半的光是必然失去的， 动物，我们有效地恢复了其余部分，例如，从暴露的大鼠看到2.5倍以上的光线 个脑袋 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。 我们还制作了一个平面版的TED的原型，一个单片光导， 初步测试。使用中空的第一表面反射设计，单片TED目前被用于恢复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，并去除某些“epi”收集几何形状的伪影。 在前几年，我们测试了树枝状氧探针分子，我们发现这比最佳速度要慢，探测器的目标也很脆弱。 相反，我们开发了一种新的纳秒氧探针（首先在试管中，现在在细胞中），该探针基于FRET到O2结合蛋白，我们正在利用这些第一探针，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。用已知的O2缓冲液校准探针质粒的细胞测试。 例如，我们已经将Mb-mCherry靶向线粒体，在那里我们可以直接成像其最大汇附近的氧水平。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。 除了设备开发，我们可以使用多光子显微镜进行FCS-荧光 相关光谱学-活细胞内的标记分子。 有了FCS，我们可以 细胞核中的几百个转录因子，并决定它们的移动性（即， 它们是自由的还是染色质结合的？）了解辅助因子的作用。 例如，我们能够使用RICS（光栅FCS）计算和学习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的复合物。

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.