Multiphoton Microscopy Development

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。 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。在已发布的帐户中，我们显示 可以使用增益将扫描速度提高9倍，或将激光功率降低3倍以避免光损坏。 最近，在TEDII上，我们设计了一种可以接近活动物的设备类别。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，以便 以便迅速将技术传播给其他人。 去年，我们专注于与3i(智能成像创新)拥有评估许可的TED的商业合作伙伴合作，开发和测试EPI导向的TED的紧凑型版本CTED。在我们接触到的各种活动物组织中，我们获得了超过2倍(有时是5倍)的亮度增益。随后的出版物出版了。我们还制作了金属版TEDIV的原型，这是一种单片光导，并开始 初步测试。我们正在寻找来自多家公司的平板原型。 我们还开始测试我们的2p器件的改进，以提供双光子磷光寿命成像(用于 细胞内氧气检测)，建立2p和单光子微量磷光计，并表征树枝状氧探针分子。 我们开发了一种基于FRET到O2结合蛋白的新的纳秒氧探测器，我们正在测试第一个探测器，同时对其他探测器进行改造，以获得更大的范围。 除了设备的研制，我们还使用了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其伴侣增强的 、麦克斯。我们的出版物显示了MAX的敲除和siRNA治疗如何揭示更多的C-myc移动性。FCS还可以用来研究整个细胞中蛋白质之间的相互作用。

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.