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TED设备， 迅速将技术传播给其他人。 在过去的一年里，我们专注于开发和测试一个紧凑版的epi-directed TED，cTED，与拥有TED评估许可的3 i（智能成像创新）的商业合作伙伴合作。我们在我们接近的各种活动物组织中实现了超过2倍（有时5倍）的亮度增益。 随后的出版物出版了。我们还制作了一个金属版的TEDIV的原型，一个单片光导， 初步测试。我们正在寻求来自多家公司的平板原型。 我们也已经开始测试我们的2 p器件的修改，以提供双光子磷光寿命成像（用于 细胞内O2检测），构建2 p和单光子微磷光计，并表征树枝状氧探针分子。 我们开发了一种新的纳秒氧探针的基础上FRET到O2结合蛋白，我们正在测试第一个探针，同时改造其他更大的范围。 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，我们研究了癌基因产物C-myc，并了解了它的染色质亲和力是如何被它的伴侣增强的。 、MAX. 我们的出版物显示了MAX的敲除和siRNA处理如何揭示更多的移动的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 TED devices , in order to quickly disseminate the technology to others. In the last year, we focused on the development and testing of a compact version of the epi-directed TED, cTED, in collaboration with commercial partners at 3i (Intelligent Imaging Innovations) who have evaluation-licensed TED. We achieved over 2-fold (sometimes 5) brightness gain in a variety of live animal tissues we approached. The subsequent publication was published. We also prototyped a metal version of TEDIV, a monolithic lightguide, and began preliminary testing. We are seeking slab prototypes from multiple companies. We have also begun testing modifications of our 2p devices to provide for two-photon phosphorescence lifetime imaging (for intracellular O2 detection), building both a 2p and single photon microphosphorimeter, and characterizing dendrimeric oxygen probe molecules. We developed a new nanosecond oxygen probe based on FRET to O2 binding proteins, and we are testing the first probes while reworking others for greater range. 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 studied the oncogene product C-myc and learning how its chromatin affinity is potentiated by its partner , MAX. Our publication showed how knockdowns and siRNA treatment of MAX reveal more mobile C-myc. FCS can also be used to study protein-protein interactions throughout the cell.