Tissue imaging using two-photon video rate microscopy

Non-invasive optical diagnosis of cellular and extracellular structure and biochemistry in thick tissue is becoming a reality with the maturation of the two-photon imaging. Today, the slow imaging speed of typical two-photon microscopes is a major hurdle in realizing their clinical potential. We have developed a high-speed two-photon microscope optimized for acquiring 3-D tissue images in real time. The scanning speed improvement of this system is obtained by the use of an air bearing polygonal mirror. The maximum achievable scanning rate is 40 microseconds per line, which is about 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real-time by an intensified CCD camera. Using this instrument, we have monitored the movements of protozoas and mapped the collagen/elastin fiber structures in excised human skin.

[1]  P. Corcuff,et al.  In vivo vision of the human skin with the tandem scanning microscope. , 1993, Dermatology.

[2]  D. Choy,et al.  Fluorescence spectra from cancerous and normal human breast and lung tissues , 1987, Annual Meeting Optical Society of America.

[3]  J. Squier,et al.  Dispersion pre‐compensation of 15 femtosecond optical pulses for high‐numerical‐aperture objectives , 1998, Journal of microscopy.

[4]  B. Chance,et al.  Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry. , 1959, The Journal of biological chemistry.

[5]  B. Chance,et al.  Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. , 1979, The Journal of biological chemistry.

[6]  S. Hell,et al.  Multifocal multiphoton microscopy. , 1998, Optics letters.

[7]  B. Athey,et al.  Real‐time two‐photon confocal microscopy using a femtosecond, amplified Ti:sapphire system , 1996, Journal of microscopy.

[8]  E. Gratton,et al.  Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin. - eScholarship , 1997 .

[9]  Stanley B. Brown,et al.  Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect , 1998, Photochemistry and photobiology.

[10]  R. Webb,et al.  In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast. , 1995, The Journal of investigative dermatology.

[11]  B R Masters,et al.  Three‐dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope , 1997, Journal of microscopy.

[12]  D W Tank,et al.  Direct Measurement of Coupling Between Dendritic Spines and Shafts , 1996, Science.

[13]  W A Mohler,et al.  Stereo-4-D reconstruction and animation from living fluorescent specimens. , 1998, BioTechniques.

[14]  Enrico Gratton,et al.  Two-photon fluorescence and confocal reflected light imaging of thick tissue structures , 1998, Photonics West - Biomedical Optics.

[15]  D. Piston,et al.  Quantitative Subcellular Imaging of Glucose Metabolism within Intact Pancreatic Islets (*) , 1996, The Journal of Biological Chemistry.

[16]  A. Katz,et al.  Two-photon excitation of fluorescence from chicken tissue. , 1997, Applied optics.

[17]  P. So,et al.  Two-Photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures. , 1998, Optics express.