Patent ID: 8686363

Claim:
A method for multi-color, subdiffraction imaging of a three-dimensional space, comprising: providing a sample that includes a three-dimensional space having one or more features of interest that are smaller than an optical diffraction limit; labeling the three-dimensional space with two or more different fluorophores having spectrally overlapped excitation and emission wavelengths; positioning the sample in a hyperspectral stimulated emission depletion (â€œHSTEDâ€) microscope system that is configured to generate an excitation light beam and a depletion light beam that is substantially coincident with the excitation light beam; illuminating an optical section of the three-dimensional space with the excitation light beam and the depletion light beam to excite fluorescence emission from the two or more different fluorophores in the optical section and, substantially simultaneously, depleting emission from substantially all but a selected subset of the two or more different fluorophores in the optical section; collecting polychromatic fluorescence emission data from the spectrally distinct emission of each of the two or more different fluorophores, wherein the collecting step further includes, collecting fluorescence emission data from a multitude of optical sections of the three-dimensional space, and assembling the fluorescence emission data from the multitude of optical sections to produce a three-dimensional, high-resolution image of the three dimensional space; and processing the polychromatic fluorescence emission data to identify and localize the fluorescent emission from each of the two or more different fluorophores.