A two-photon microscope imaging spectrometer has been proposed for use in scanning confocal two-photon microscopy. The proposed instrument would solve a spectrum-overlap that sometimes arises, as explained below.

Scanning confocal two-photon microscopy is a variant of fluorescence microscopy, in which fluorescent dyes are used as probes for selective monitoring of biological compounds and cells. In scanning two-photon confocal microscopy, a specimen is raster-scanned by a laser beam that is focused into the specimen through the objective lens of a microscope. Fluorescent light excited by two-photon absorption is collected by a photodetector. Light is collected from each pixel, then the output of the photodetector is digitized and stored. This process is repeated for all the pixels in the raster scan, thereby building up a digitized image that represents an "optical section" that is, in effect, a fluorescence cross section in the focal plane. Further repetition of the process on a succession of closely spaced focal planes yields three-dimensional image data.

If multiple dyes with overlapping fluorescence spectra are used, then one is faced with the problem of how to separate the individual fluorescence images, each of which reveals a different aspect of the structure and function of the specimen. The problem could be solved by processing image data acquired by the proposed two-photon microscope imaging spectrometer. The fluorescence-image data generated by this instrument would be resolved not only spatially but also spectrally; that is, a spectrum would be acquired for each pixel. If the spectrum of each pixel were a sum of overlapping, known fluorescence spectra, then by use of previously developed spectral-data-processing techniques, the intensity of each fluorescence spectrum (and thus the abundance of the corresponding dye in the pixel) could be computed.

In one version of the proposed instrument, a tunable filter (e.g., a liquid-crystal tunable filter) would be placed in front of the photodetector and would be tuned across the wavelength range of interest to acquire a spectrum for each pixel. One potential disadvantage of this version is that the spectrum-acquisition time could be long enough that one or more fluorescent dye(s) in the specimen could become photobleached during the concomitant long exposure to the laser beam used to excite the fluorescence. A second major problem is that the data acquisition time is too long for many biological problems.

A second version of the instrument would operate with a shorter exposure time per pixel, and thus less photobleaching. In this version, the fluorescent light from each pixel would be focused onto the entrance slit of a spectrometer. Inside the spectrometer, the light would be dispersed by wavelength along a linear array of photodetectors. To obtain sufficient sensitivity to acquire the spectrum for each pixel in a sufficiently short time, the array of photodetectors would likely have to consist of multiple photomultiplier tubes, a multielement photomultiplier tube, or an intensified charge-coupled-device (CCD) array.

This work was done by Gregory Bearman, Scott Fraser, and Rusty Landsford of Caltech for NASA's Jet Propulsion Laboratory. For further information, access the Technical Support Package (TSP) free on-line at www.nasatech.com/tsp under the Physical Sciences category.

In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to

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