Use of multiphoton excitation through optical fibers for fluorescence spectroscopy in conjunction with optical biopsy needles and endoscopes

#The present invention relates to the use of multiphoton excitation in conjunction with optical biopsy needles and endoscopes.

#Many crucial biological functions are mediated or accomplished by biomolecules and tissue structures that are intrinsically fluorescent. As a result, there is an opportunity to diagnose and study important biological events by measuring and localizing the spectra and tissue fluorescence emission. To investigate in vivo internal processes and structures in large organisms, such as human beings and agricultural animals, endoscopic procedures which penetrates body cavities or even solid tissue may be required.

#Endoscopy video imaging in body cavities ordinarily utilizes back-scattered white light applied through the endoscope to form a low-resolution color image of the internal surfaces of these cavities. Physicians often use the changes in shapes and changes in local apparent color (which are often due to changes in blood distribution) to recognize disease states, such as malignant tumors or inflammation. Unfortunately, these clues are frequently not sufficient, especially for detection of the early onset of disease. Diagnostic improvements have been made by quantitative measurements of the light scattering and of tissue fluorescence emission.

#FIG. 1 is a perspective drawing showing the spatial distribution of ultraviolet light emitted from a single mode optical fiber as well as the corresponding autofluorescence in accordance with the prior art. Here, the drop in intensity of radiation is reciprocal to the square of the distance from the tip of the optical fiber.

#FIG. 2 is a perspective drawing showing the spatial distribution of ultraviolet light of two photon excitation fluorescence emitted from a single mode optical fiber in accordance with the present invention. Here, the pattern of illumination is nearly the same as for one-photon excitation. However, because the rate of two photon excitation is proportional to the square of the pulsed illumination intensity, the fluorescence intensity decreases from the tip of the fiber approximately as the reciprocal fourth power of the distance from the tip of the fiber providing a highly localized effective focal volume for excitation of the fluorescence. For higher order fluorescence excitation, the power law concentration of the emission is even stronger, reciprocal six power for three-photon excitation for example, although the sharpness of the effective focal volume could be reduced if a larger diameter optical fiber is used.

#FIG. 3 shows a two photon excitation profile at the fiber output in the form of a Gaussian Intensity Profile. This figure is a representation of the spatial distribution of the two-photon excited fluorescence emission from a uniform fluorophore shown for a plane along the axis of the optical fiber beginning at the exit from the fiber. The normalized maximum drops by half in about 10-15 micrometers along the axis and has a half maximum half-width of about 2 μm at the exit of this typical single mode fiber.

#One aspect of the present invention relates to a method of detecting disease within a particular tissue of a plant or animal. This method involves activating the particular plant or animal tissue by application of radiation through at least one optical fiber under conditions effective to promote a simultaneous multiphoton excitation of the particular plant or animal tissue and to emit an intrinsic fluorescence characteristic. The intrinsic fluorescence characteristic is compared to fluorescence emitted by exciting healthy tissue of the particular plant or animal under the same conditions used to carry out the activating step. The particular tissue of a plant or animal where the intrinsic fluorescence characteristic differs from the fluorescence, emitted by exciting healthy tissue of the particular plant or animal under the same conditions used to carry out such activity, is then identified as potentially diseased.

#Another aspect of the present invention involves a method of producing an image of an internal surface of a particular tissue within a plant or animal. In this method, the particular plant or animal tissue is activated with radiation applied through an optical fiber under conditions effective to promote a simultaneous multiphoton excitation of the internal surface of the particular tissue within the plant or animal and produce an autofluorescence. The autofluorescence is collected to produce an image of the tissue under the internal surface by recording a set of adjacent volumes.

#The present invention also relates to a method of detecting and localizing fluorescence within a subject by applying radiation to an internal region of the subject through at least one optical fiber. Each fiber terminates in a tip proximate to the internal region. Radiation is applied under conditions effective to cause simultaneous multiphoton absorption of fluorophore molecules within the internal region and, as a result, fluorescent excitation is proximate to the tip of the at least one optical fiber.