Two-photon laser microscopy
First Claim
1. A laser scanning microscope comprising:
- stage means for receiving target material to be imaged, the target material including fluorescent means responsive to excitation by photons in a short wavelength spectral range to produce characteristic fluorescence;
lens means positioned to direct light toward said stage and having an object plane in target material at said stage means;
a source of subpicosecond monochromatic coherent light pulses of high instantaneous energy intensity comprised of photons in a long wavelength spectral range to which target material at said stage does not respond by single photon excitation to produce its characteristic fluorescence, said pulses having a high repetition rate;
detector means;
means directing said coherent light pulses along an optical path including said lens means to impinge on target material at said stage means, said lens means focusing said light pulses at said object plane so that said long wavelength light pulses provide sufficient instantaneous intensity to produce in target material at said object plane simultaneous absorption of two incident photons to thereby excite characteristic fluorescence in target material at said stage means, said fluorescence providing output light which travels on said optical path to detector means.
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Abstract
A laser scanning microscope produces molecular excitation in a target material by simultaneous absorption of two photons to thereby provide intrinsic three-dimensional resolution. Fluorophores having single photon absorption in the short (ultraviolet or visible) wavelength range are excited by a stream of strongly focused subpicosecond pulses of laser light of relatively long (red or infrared) wavelength range. The fluorophores absorb at about one half the laser wavelength to produce fluorescent images of living cells and other microscopic objects. The fluorescent emission from the fluorophores increases quadratically with the excitation intensity so that by strongly focusing the laser light, fluorescence as well as photobleaching are confined to the vicinity of the focal plane. This feature provides depth of field resolution comparable to that produced by confocal laser scanning microscopes, and in addition reduces photobleaching. Scanning of the laser beam, by a laser scanning microscope, allows construction of images by collecting two- photon excited fluorescence from each point in the scanned object while still satisfying the requirement for very high excitation intensity obtained by focusing the laser beam and by pulse time compressing the beam. The focused pulses also provide three-dimensional spatially resolved photochemistry which is particularly useful in photolytic release of caged effector molecules.
This invention was made with Government support under Grant Nos. P41RR04224 awarded by the National Institute of Health; NSF-BBS-8714069 awarded by the National Science Foundation, and NSF-DMB-8609084 awarded by the National Science Foundation. The Government has certain rights in the invention.
501 Citations
21 Claims
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1. A laser scanning microscope comprising:
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stage means for receiving target material to be imaged, the target material including fluorescent means responsive to excitation by photons in a short wavelength spectral range to produce characteristic fluorescence; lens means positioned to direct light toward said stage and having an object plane in target material at said stage means; a source of subpicosecond monochromatic coherent light pulses of high instantaneous energy intensity comprised of photons in a long wavelength spectral range to which target material at said stage does not respond by single photon excitation to produce its characteristic fluorescence, said pulses having a high repetition rate; detector means; means directing said coherent light pulses along an optical path including said lens means to impinge on target material at said stage means, said lens means focusing said light pulses at said object plane so that said long wavelength light pulses provide sufficient instantaneous intensity to produce in target material at said object plane simultaneous absorption of two incident photons to thereby excite characteristic fluorescence in target material at said stage means, said fluorescence providing output light which travels on said optical path to detector means. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A laser scanning microscope, comprising
stage means for receiving a target material having an absorption energy level peak responsive to single photon excitation by light of a predetermined wavelength; -
lens means positioned to direct light to said stage means and having an object plane in target material at said stage means; a laser source of subpicosecond a laser light pulses, said laser light having a wavelength about twice said predetermined wavelength; mirror means directing said laser light pulses along an optical path including said lens means to cause said pulses to impinge on target material at said object plane, said lens means focusing said laser light pulses on a focal point in the target material, the intensity of said pulses producing in the region of said focal point a two-photon excitation energy level equivalent to the single-photon excitation energy level which corresponds to said single photon absorption peak. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method of fluorescence microscopy by a two-photon excitation technique, comprising:
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providing a sample containing fluorescent molecules which radiate photons of a first characteristic energy; illuminating said sample with a beam of rapidly repeating, intense, subpicosecond pulses of laser light comprising photons of a second characteristic energy, wherein said second characteristic energy is about one-half said first characteristic energy; focusing said illumination to a focal point having a submicron diameter with said sample to produce an illumination intensity sufficiently high at said focal point to produce molecular excitation and fluorescence of said sample by simultaneous absorption of two incident photons; scanning the submicron diameter focal point of said beam in a raster pattern through said sample; and detecting the fluorescence produced by said sample. - View Dependent Claims (18, 19, 20)
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21. A method for producing localized photolytic release of caged biologically active compounds by a two-photon excitation technique, comprising:
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providing a sample containing caged biologically active molecules which are excitable by photons of a first characteristic energy; illuminating said sample with a beam of rapidly repeating, intense, subpicosecond pulses of laser light comprising photons of a second characteristic energy wherein said second characteristic energy is about one-half said first characteristic energy; focusing said illumination to a focal point within said sample to produce an illumination intensity sufficiently high only at said focal point to produce molecular excitation and consequent release of caged biologically active compounds by simultaneous absorption of two incident photons of said second characteristic energy.
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Specification