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公司合作，以提炼和制造TEDII设备，以便 以便迅速将技术传播给其他人。 去年，我们还设计和测试了自适应光学系统(例如可变形反射镜) 以补偿组织的不均质性。就像天文学家必须通过一个 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模糊和闪烁。解决方案是使用可变形的光学元件来补偿 对于已知的扭曲。在天文学中，一个已知的“引导星”点可以提供这一点；在组织中， 我们要么必须建立从组织反射中脱颖而出的导航星，要么使用计算机 进行成功的猜测，以清理图像。对于前者，我们正在合成我们自己的 多层纳米颗粒，在组织内提供可分离的清洁“引导之星”信号。我们还在OCT开始测试这些粒子的对比度电势 (光学相干层析成像)设置。今年投入了更多的努力来反映非理想主义。 我们还评估了双光子磷光寿命成像的可能性 细胞内O2的检测，建立了一个2p和单光子的微量磷光计。 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，我们正在研究 癌基因产物C-myc和了解其染色质亲和力如何被其伴侣增强 、麦克斯。基因敲除和siRNA显示C-myc的流动性更强。FCS还可以用来研究整个细胞中蛋白质之间的相互作用。我们之前发表了关于HIV-1蛋白Nef对细胞表面的混杂亲和力的描述 像CD4(减少重叠感染)和HL A-I(影响免疫监控)这样的蛋白质， 表明亲和力是在几个内部细胞器中保持的。 我们还(与合作者)在FCS中发现了一个非常大的漂白文物 (约100MD)组件和已公布的校正/交叉验证方法。我们继续评估这种人工产物在聚集(例如，斑块形成)蛋白质方面的作用。

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 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 are synthesizing our own multiple-layer nanoparticles to provide a separable clean "guidestar" signal inside tissue. We also begun tests of the contrast potential of these particles in an OCT (Optical Coherence Tomography) setting. More effort this year was devoted to 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. 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 reveal more mobile C-myc. FCS can also be used to study protein-protein interactions throughout the cell. We previously published accounts of the promiscuous avidity of the HIV-1 protein Nef for cell-surface proteins like CD4 (reducing superinfection) and HLA-I (compromising immune surveillance), showing the affinity is maintained in several internal organelles. We have also identified (with collaborators) a bleaching artifact in FCS of very large (ca. 100MD) assemblies and published methods of correction/cross-validation. We continue evaluating this artifact in aggregating (e.g., plaque-forming) proteins.