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， 严重程度会增加一倍或三倍 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。在公开的报道中，我们展示了 增益可用于扫描快9倍或减少激光功率3倍以避免光损伤。 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。 我们目前正在与一家小型显微镜公司合作，以改进和制造TEDII设备， 迅速将技术传播给其他人。 在过去的一年里，我们还设计和测试了自适应光学（例如变形镜） 以补偿组织的不均匀性。 就像天文学家必须观察 不均匀的、移动的大气，我们必须在半透明的组织中产生焦点。 这两个问题都会导致图像模糊和闪烁。 解决方案是使用可变形光学元件来补偿 对于已知的失真。 在天文学中，一个已知的“导航星”点可以提供;在组织中， 我们必须建立从组织反射中突出的导航星， 来进行猜测以清理图像。 对于前者，我们已经建立了自己的 多层纳米颗粒，以在组织内提供可分离的干净的“引导星”信号。 然而，今年的大部分工作都是由我们在LCE中的合作者致力于开环优化启动;额外的努力也进入了镜像非理想性。 我们还评估了双光子磷光寿命成像的可能性， 细胞内O2检测，构建2 p和单光子显微磷光计，并表征树枝状氧探针分子。 除了设备开发，我们还采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，我们正在研究 癌基因产物C-myc，并了解其配偶体如何增强其染色质亲和力 、MAX. MAX的敲除和siRNA处理揭示了更微妙的移动的C-myc。 FCS也可用于研究整个细胞中的蛋白质-蛋白质相互作用。

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 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. In published accounts, we show the gain could be used to scan 9x faster or reduce laser power 3x to avoid photodamage. Most 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 are currently collaborating with a small microscopy company to refine and manufacture TEDII devices , in order to quickly disseminate the technology to others. In the last year, we have also designed and tested adaptive optics (e.g. deformable mirrors) to compensate for the inhomogeneity of tissue. Just as astronomers must look though an inhomogeneous, moving atmosphere, we must generate the focal spot in translucent tissue. Both problems cause blur and twinkling. The solution is to use a deformable optic to compensate for the known distortion. In astronomy, a known "guidestar" point can provide that; in tissue, we must either build guidestars that stand out from the tissue reflection or use the computer to make succesive guesses to clean up the image. For the former, we have built our own multiple-layer nanoparticles to provide a separable clean "guidestar" signal inside tissue. Most effort this year, however, was devoted to open-loop optimization startegies by our collaborators in LCE; additional effort also went into mirror nonidealities. We have also evaluated the possibility of two-photon phosphorescence lifetime imaging for intracellular O2 detection, building a 2p and single photon microphosphorimeter, and characterizing dendrimeric oxygen probe molecules. In addition to device development, we 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 studying the oncogene product C-myc and learning how its chromatin affinity is potentiated by its partner , MAX. Knockdowns and siRNA treatment of MAX reveal subtly more mobile C-myc. FCS can also be used to study protein-protein interactions throughout the cell.