US5034613A — Two-photon laser microscopy

Claim 1

Independent Claim 192 words

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.

Claim 2

Dependent Claim 192 words

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.

Claim 3

Dependent Claim 44 words

The microscope of claim 1, wherein said light source produces subpicosecond pulses of sufficient instantaneous intensity and repetition rate that target material at said stage means will absorb energy simultaneously from at least two incident long wavelength photons substantially only in said object plane.

Claim 4

Dependent Claim 65 words

The microscope of claim 1, wherein said lens means is located with respect to said stage means to focus said long wavelength light from said source to a submicron diameter at said object plane to produce a sufficiently high intensity at said object plane to produce fluorescence in target material at said stage means and insufficient intensity outside said focal plane to produce such fluorescence.

Claim 5

Dependent Claim 48 words

The microscope of claim 1, wherein said lens means is arranged to focus said long wavelength light into a conical configuration to produce converging and diverging light on opposite sides of said object plane, whereby said long wavelength light is concentrated at a focal point on said plane.

Claim 6

Dependent Claim 84 words

The microscope of claim 1, further including target material carried by said stage means, and wherein said long wavelength light from said source is in the red wavelength spectral range, and wherein said lens means focuses said light at a focal point in said target material at said stage means, said target material being a fluorophore having a single photon absorption peak in the ultraviolet wavelength spectral range and being capable of absorbing two photons in the red wavelength spectral range to produce fluorescence.

Claim 7

Dependent Claim 42 words

The microscope of claim 5, wherein said lens means is located to focus said long wavelength light at a focal point in said target material to produce a light intensity which excites fluorescence in a limited ellipsoidal volume around said focal point.

Claim 8

Dependent Claim 148 words

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.

Claim 9

Dependent Claim 148 words

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.

Claim 10

Dependent Claim 46 words

The microscope of claim 7, wherein said long wavelength light is in the red wavelength spectral range, and wherein said lens means focuses said light at a focal point in target material at said stage means, said target material including a fluorophore having said absorption peak.

Claim 11

Dependent Claim 47 words

The microscope of claim 7, wherein said long wavelength impinging light pulses provide photons of light energy in the red wavelength spectral range to target material at said stage, and wherein the combined energy of two photons of said impinging light is required to produce fluorescence therein.

Claim 12

Dependent Claim 25 words

The microscope of claim 9, wherein said lens means is adjustable to select focal points at different depths within target material at said stage means.

Claim 13

Dependent Claim 23 words

The microscope of claim 10, further including scanning means to move said focal point with respect to target material at said stage means.

Claim 14

Dependent Claim 27 words

The microscope of claim 11, further including detector means responsive to light in said optical path for detecting fluorescence produced by target material at said stage means.

Claim 15

Dependent Claim 18 words

The microscope of claim 12, wherein said detector means is a photosensitive array which responds to said fluorescence.

Claim 16

Dependent Claim 37 words

The microscope of claim 7, further including target material at said object stage, wherein said target material is a biological cell responsive to said two-photon excitation energy level produced at said focal point by said light pulses.

Claim 17

Dependent Claim 32 words

The microscope of claim 14, wherein said target material at said stage means includes means responsive to said light pulses at said focal point to produce localized release of biologically active chemicals.

Claim 18

Dependent Claim 21 words

The microscope of claim 7, further including target material at said object stage, wherein said target material is a photon-activatable reagent.

Claim 19

Independent Claim 124 words

A method of fluorescence microscopy by a two-photon excitation technique, comprising: 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.

Claim 20

Independent Claim 124 words

A method of fluorescence microscopy by a two-photon excitation technique, comprising: 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.

Claim 21

Dependent Claim 36 words

The method of claim 17, wherein said step of illuminating includes directing a beam of light having an illumination intensity sufficient to produce molecular excitation substantially only at said focal point to thereby suppress background fluorescence.

Claim 22

Dependent Claim 45 words

The method of claim 17, wherein said step of illuminating includes directing a beam of light having an illumination intensity sufficient to produce molecular excitation substantially only at said focal point to thereby suppress photobleaching of said sample material at locations outside the focal plane.

Claim 23

Dependent Claim 21 words

The method of claim 17, wherein the step of providing a sample includes providing a sample of a living biological specimen.

Claim 24

Independent Claim 116 words

A method for producing localized photolytic release of caged biologically active compounds by a two-photon excitation technique, comprising: 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.

Claim 25

Independent Claim 116 words

A method for producing localized photolytic release of caged biologically active compounds by a two-photon excitation technique, comprising: 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.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1994-07-22	1995-01-21	1995-07-23	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1998-07-22	1999-01-21	1999-07-23	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2002-07-22	2003-01-21	2003-07-23	$7700.00	Window Closed / Lapsed

Two-photon laser microscopy

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