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。我们之前开发了TED（“总排放检测”）设备来克服这些信号限制。 在前几年，我们测试了能发出磷光的树枝状氧探针分子。我们发现这比最佳速度慢，难以瞄准，而且往往有毒。 相反，我们开发了（首先在比色皿中，现在在细胞中）一种新的纳秒氧探针，该探针基于FRET到O2结合蛋白，我们正在利用这些第一个探针，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。 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。我们还发表了细胞核内轻度缺氧的条件。 在正常细胞和癌细胞中，已经完成了在不同代谢条件下的细胞内氧水平的测试。 我们已经发表了耗氧量和代谢的NADH "氧化还原比"指标的相关研究，以检查侵袭性与被动性癌细胞生长以及对肿瘤细胞进行的"OXPHOS至糖酵解"转换的影响。 今年，我们展示了感知"活性氧"的能力（例如，自由基）通过切换到黄色荧光蛋白，其寿命报告肌红蛋白的"met"形式。 我们已经发表了报告如何光敏化和一氧化氮产生试剂诱导高铁肌红蛋白今年。 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。 同样的原理也适用于线粒体的流动性。 今年，我们专注于将该方法传播到多个合作项目，特别是肌细胞和癌细胞，并改进图像的全球分析，以获得更精确的测量结果。

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. We previously developed TED ("Total Emission Detection") devices to overcome these signal limits. We had, in previous years, tested dendrimeric oxygen probe molecules that phosphoresced. We found this slower than optimal, hard to target, and often toxic. 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 these first probes while reworking others for greater range and reliability as DNA-based transfections. We have targeted Mb-mCherry, for example, to mitochondria, where we can directly image oxygen levels near their biggest sinks. We have also published conditions for mild hypoxia within nuclei. Testing of intracellular oxygen levels in differing metabolic conditions have been done, in normal and cancer cells. We have published correlative studies of oxygen consumption and NADH "redox ratio" indicators of metabolism to examine aggressive vs. passive cancer cell growth and effects on the "OXPHOS to glycolysis" switching done by neoplastic cells. This year, we showcased the ability to sense "Reactive Oxygen Species" (e.g., free radicals) by switching to a yellow fluorescent protein whose lifetime reports on the "met" form of myoglobin. We have published reports how both photosensitizing and nitric oxide producing reagents induce met-myoglobin this year. 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. The same principle is being applied to mobility of mitochondria. We have focused this year on disseminating the method to multiple collaborations, esp. myocytes and cancer cells, and refining global analyses of the images for more precise measurements.