Multiphoton Microscopy Development

多光子显微镜已成为亚微米活体成像的首选方法

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.