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.
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