A proposed program of research would be oriented toward the development of fluorescent dyes for use in two-photon microscopy. Two-photon microscopy and its predecessor, one-photon microscopy, are variants of fluorescence microscopy, which has become a major technology for biological and physical sciences. The basic idea in fluorescence microscopy is to use fluorescent compounds as markers for various physical and biological processes so that by observation of fluorescence under microscopes, one can locate those processes with high resolution in space and time. Because the fluorescence emitted by a compound can be isolated by its characteristic excitation and emission wavelengths, the compound can be traced with high signal-to-noise ratio, even in a "messy" environment. In two-photon microscopy, excitation of a fluorescent dye involves the concurrent absorption of two photons of approximately twice the wavelength of the peak of the single-photon-absorption spectrum.

These Dye Molecules have been identified as potentially suitable for use in two-photon microscopy.

During the past few years, two-photon microscopy has evolved to the incipient development of a commercial two-photon microscope. Heretofore, two-photon microscopy has been performed with dyes optimized for one-photon microscopy; these dyes are unlikely to satisfy the requirements for future relatively inexpensive two-photon microscopes, which are expected to feature simplified optics and power-efficient, ultrafast lasers combined in such a way as to afford only about 1/25 of the sensitivity of previously constructed prototype two-photonmicroscopes.

The dyes to be developed in the proposed research would be optimized for two-photon microscopy. Preliminary research has revealed that at least three classes of dyes will be needed:

  1. Vital dyes could be used to label cells and follow them over time. These could include hydrophilic dyes that would be trapped in cytoplasm or hydrophobic dyes that would be carried in organelles or in cell membranes. Vital dyes must be optimized for minimal toxicity and slow bleaching.
  2. Hydrophilic marker dyes would be formulated for covalent linking of antibodies for use in immunocytochemical labeling of tissues. These dyes could be based partly on vital dyes. However, because these marker dyes would be used on fixed tissue, toxicity would not be of concern as in the case of vital dyes. Marker dyes must be very hydrophilic to minimize the "background" staining that would otherwise occur because the dye would interact with, and stick to, the tissue. Marker dyes must be optimized for brightness; bleaching is of less concern.
  3. Phototoxic agents would be used to sensitize cells or tissues for selective killing by laser light. A tumor or pathogen would be targeted, either by direct interaction with a dye or by formation of a targeting complex that could include, for example, antibodies to a tumor antigen. The dye-labeled tissue would be irradiated with light that the dye would absorb; photoactivated damage or toxic byproducts of dye bleaching would then kill the targeted cells. These dyes are required to be nontoxic until and unless illuminated.

The initial plan for the proposed research is straightforward, given that a few dyes (see figure) are already known to have properties that make them candidates for use in two-photon microscopy. These properties include absorption maxima in approximately the correct wavelength range and absorption cross sections about 50 times those of conventional dyes. The plan calls for the following coordinated efforts:

  1. Synthesize dyes that (a) have two-photon-absorption wavelengths tuned for specific applications; (b) exhibit hydrophilicity suitable for control of biodistribution and, possibly, toxicity; and (c) are functionalized to provide for routine attachment to antibodies, caging complexes, and other biologically relevant compounds.
  2. Determine lipo/hydrophilicity of each dye.
  3. Measure two-photon cross sections at wavelengths from 780 to 1,000 nm, determine one- and two-photon-fluorescence quantum efficiencies, and characterize bleaching rates and byproducts.
  4. 4. Test each dye in a biological setting to determine toxicity with and without illumination and to determine performance under a microscope.

This work was done by Scott E. Fraser, Seth R. Marder, and Joseph W. Perry of Caltech for NASA's Jet Propulsion Laboratory.

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

Intellectual Property group
Mail Stop 202-233
4800 Oak Grove Drive
Pasadena, CA 91109
(818) 354-2240

Refer to NPO-20150.

This Brief includes a Technical Support Package (TSP).

Unfortunately the TSP Flourescent Dyes for Two-Photon Microscopy (reference NPO-20150-) appears to be missing from our system.

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