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 (or more) 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. More 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 also prototyped a planar version of TED, a monolithic lightguide, and began preliminary testing. Using hollow first-surface reflection designs, monolithic TED is currently being used to recover lost light in epi-CARS microscopy, to enhance the sort of data we recovered for water in arteries and to remove certain artifacts of epi collection geometry. We had, in previous years, tested dendrimeric oxygen probe moleculesthat phosphoresced. We found this slower than optimal, and probe targeting was tenuous. 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 thesefirst probes while reworking others for greater range and reliability as DNA-based transfections. Cellular tests of probe plasmids were calibrated with known O2 buffers. We have targeted Mb-mCherry, for example, to mitochondria, where we can directly image oxygen levels near their biggest sinks. We have also tested for anoxia within nuclei. Testing of intracellur oxygen levels in differing metabolic conditions are underway. 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. For example, we are able to count and learn binding rates for various proteins and their receptors on cells, using RICS (raster FCS), and how transcription factors bundle into large complexes.
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