Methods for improved selectivity in photo-activation and detection of molecular diagnostic agents

1. Imaging apparatus adapted for in vivo imaging a particular volume of plant or animal tissue containing at least one photo-active molecular agent, the apparatus comprising:

a source of collimated light, said light having a frequency effective to penetrate substantially into the tissue, said light being adapted to promote simultaneous two-photon excitation (TPE) of the molecular agent contained within the tissue;

beam expanding apparatus for expanding said collimated light into an expanded beam of collimated light;

focusing apparatus for focusing the expanded beam of collimated light throughout a range of focal lengths extending from a surface of said tissue to a depth substantially beyond said surface, said light source and focusing apparatus cooperating to promote TPE of the molecular agent,

wherein a focal point or focal plane is adjustable over a distance of up to 100 mm with respect to said tissue;

a detector located proximate to the tissue and positioned to detect said light emitted by the molecular agent and which travels a path that does not retrace an optical path of the light incident on the tissue, said detector configured to produce a detected signal characteristic of the particular volume at which the light source has been focused;

a processor coupled to said detector; and

a modulation system associated with said light source, said processor being coupled to said modulation system and adapted to produce a demodulated energy signal which is characteristic of the particular photo-activated molecular agent.

2. The apparatus of claim 1 , wherein said light source produces a pulsed output having a pulse repetition frequency above about 75 megahertz and a sub-nanosecond pulse duration.

3. The apparatus of claim 2 wherein said light source produces near-infrared light.

4. The apparatus of claim 3 wherein said light source produces pulse energies of about 20 nanojoules.

5. The apparatus of claim 3 wherein said light source comprises a laser.

6. Apparatus adapted for in vivo medical diagnostic imaging comprising:

light source means for directing a confined light at and into tissue to be imaged, said light being selected in frequency and energy to penetrate below a tissue surface and to promote TPE substantially only at a confocal region at a distance of up to 100 mm below said tissue surface, said light source means including beam expanding means and focusing means cooperating together to shape said confocal region;

means for varying a position of a confocal region of the light within a range of depths in the tissue to be imaged;

detector means positioned to receive and detect isotropic radiation emitted by a photo-activated molecular agent within the tissue after said agent has been excited using two-photon excitation;

a modulator means for modulating said light with a type of modulation and producing a modulated light; and

a processor coupled to said detector, said processor being coupled to said modulation system and adapted to produce a demodulated energy signal which is characteristic of said photo-activated molecular agent.

7. The apparatus of claim 6 :

wherein the light source means includes means for producing a collimated light beam; and

wherein the light source means includes focusing means for focusing the collimated light beam to a confocal region located with tissue at a point below the tissue surface.

8. The apparatus of claim 7 wherein the means for producing a collimated light beam comprises a pulsed laser operating the near infra-red spectrum.

9. The apparatus of claim 6 wherein said imaging apparatus is a microscope.

10. An apparatus adapted for in vivo microscopic imaging to a distance of up to 100 mm within tissue comprising:

a light source, said light source producing a beam of light directed to or into a material to be imaged, wherein said light source includes beam expanding means and beam focusing means cooperating together to shape said beam of light into a confocal region, said light being selected in frequency and energy to penetrate into or below a surface of the material and to promote two-photon excitation substantially only in a confocal region to be imaged;

a modulator, said modulator cooperating with said light source to modulate said light with a type of modulation and produce a modulated light;

a detector positioned to receive and detect radiation emitted by a photo-activated molecular agent within the material after said agent has been excited using two-photon excitation; and

a demodulator, said demodulator coupled to said detector and producing a demodulated energy signal which is characteristic of the particular photo-activated molecular agent.

11. An apparatus according to claim 10 wherein said demodulator is a processor.

12. Apparatus adapted to in vivo medical diagnostic imaging comprising:

a light source, said light source producing light directed to or into deep tissue to be imaged, said light being selected in frequency and energy to penetrate into or below a surface of the tissue and to promote two-photon excitation substantially only in a region to be imaged;

a beam expanding apparatus adapted for expanding said light into an expanded beam of light;

a focusing apparatus adapted for focusing said expanded beam of light, said focusing apparatus being able to vary the position of the light within a range of depths in the region of tissue to be imaged, said range of depths extending up to 100 mm into said tissue to be imaged; and

a detector positioned to receive and detect radiation emitted by a photo-activated molecular agent within the material after said agent has been excited using two-photon excitation,

wherein said light source means includes means for producing a collimated light beam and focusing means for focusing the collimated light beam to a confocal region located with tissue at a point below the tissue surface.

13. An apparatus according to claim 12 wherein said light source produces near-infrared light.

14. An apparatus according to claim 13 wherein said light source is a laser.

15. An apparatus according to claim 12 further comprising a modulator, said modulator cooperating with said light to modulate said light with a type of modulation and produce a modulated light.

16. An apparatus according to claim 15 further comprising a demodulator, said demodulator coupled to said detector and producing a demodulated energy signal which is characteristic of the particular photo-activated molecular agent.

17. An apparatus according to claim 16 , wherein said demodulator is a processor.

18. An apparatus according to claim 12 wherein said light source produces a pulsed output having a pulse repetition frequency above about 75 megahertz and a subnanosecond pulse duration.

19. An apparatus according to claim 12 wherein said light source produces pulse energies of about 20 nanojoules.