Abstract:
A scanning confocal microscopy system, especially useful for endoscopy with a flexible probe which is connected to the end of an optical fiber( 9 ). The probe has a grating( 12 ) and a lens( 14 ) which delivers a beam of multi-spectral light having spectral components which extend in one dimension across a region of an object and which is moved to scan in another dimension. The reflected confocal spectrum is measured to provide an image of the region.

Description:
This application is a 371 of PCT/US99/04356 filed Feb. 26, 1999 which claims the priority benefit of U.S. Provisional Application No. 60/076,041, filed Feb. 26, 1998. 
    
    
     DESCRIPTION 
     The present invention relates to systems (method and apparatus) for confocal microscopy for the examination or imaging of sections of a specimen of biological tissue, and particularly to such systems using multi-spectral illumination and processing of multi-spectral light. 
     Currently, the use of fast scanning confocal microscopy is limited to accessible surfaces of the skin and the eye. The reason for this is that the only reliable methods for optical scanning must be performed in free space. In addition, the size of these optical scanners prohibit their use in small probes such as endoscopes or catheters. It is a feature of the invention to miniaturize the fast scanning mechanism and increase the number of medical applications of confocal microscopy to include all surfaces of the body, gynecologic applications, probe-based applications, and internal organ systems. 
     Multi-spectral light was proposed for use in confocal microscopy, but only for imaging vertically-spaced regions of a body under examination. See B. Picard, U.S. Pat. No. 4,965,441, issued Oct. 25, 1990. An interferometer using a grating to obtain multi-spectral light which is resolved in the interferometer to obtain a spectroscopic image is disclosed in A. Knuttal, U.S. Pat. No. 5,565,986, issued Oct. 15, 1996. A lens having a color separation grating which obtains a multi-spectral light is disclosed in U.S. Pat. No. 5,600,486, issued Feb. 4, 1997. Such multi-spectral proposals are not effective for high resolution imaging using a compact, flexible probe. A confocal microscope system according to this invention can be miniaturized and incorporated into a compact probe. In addition, by allowing light delivery through a single optical fiber, the probe may also be easily incorporated into catheters or endoscopes. Thus, a confocal microscope in accordance with the invention allows imaging of all accessible surfaces of the body and increases the biomedical applications of confocal microscopy by an order of magnitude. 
     Briefly described, a confocal microscopy system embodying the invention illuminates a region of interest in a body into which said probe may be inserted with a confocal spectrum extending along one dimension. Optics in said probe or physical movement of said probe enabled by attachment thereto of a flexible light conductive member (which may be an optical fiber), enables scanning of said spectrum along one or two additional dimensions thereby providing for two or three dimensional imaging of the region. The reflected confocal spectrum may be detected or decoded spectroscopically, preferably with a heterodyne detection mechanism which may be implemented interferometrically. 
    
    
     The invention will be more apparent from the following drawings wherein 
     FIG. 1 is a schematic diagram of a spectrally encoded confocal probe in accordance with the invention where specific wavelengths are shown for illustrative purposes, their exact values depending on the optical parameters of the system. 
     FIG. 2 is a plot of spectrally encoded light obtained by confocal detection using direct spectral detection in accordance with this invention, where different wavelengths are detected by turning the spectrometer grating. 
     FIG. 3 is a schematic diagram showing a system embodying the invention using a spectrometer for measurement of the spectrum, I(λ), which corresponds to reflectance from different transverse locations, x, on the specimen. 
     FIG. 4 is a schematic diagram of a system embodying the invention having spectrally encoded confocal detection using interference spectroscopy. 
     FIG. 5A-D are schematic diagrams showing: (a) image formation; (b) translation of the optical fiber in the y direction; (c) rotation of the optical fiber in the forward firing mode; and (d) rotation of the optical fiber in the side firing mode. 
     FIG. 6 is a schematic diagram showing cross-sectional image formation by scanning the optical fiber or the objective lens along the z axis using a system embodying the invention. 
     FIG. 7 is another schematic diagram of a system embodying the invention wherein optical zoom is achieved by moving the focus of an intermediate lens in and out of the image plan of the objective. 
    
    
     Referring now to the figures, multi-spectral encoding for confocal microscopy uses a broad bandwidth source  10  as the input to the microscope. In the probe  8  of the microscope, the source spectrum provided via an optical fiber  9  is dispersed by a grating  12  and focused by an objective lens  14  onto the sample  16 . A lens  9   a  is preferably disposed between the optical fiber  9  and the grating  12  to collimate the light from the optical fiber, as shown in FIG. 1, however, lens  9   a  may be removed. The spot for each wavelength is focused at a separate position, x, on the sample (FIG.  1 ). The reflectance as a function of transverse location is determined by measuring the reflected confocal spectrum from the sample  16  returned from probe  8 . 
     The number of wavelengths or points that may be resolved is determined by:                  λ     δ                 λ       =   mN     ,           (   1   )                                
     where λ is the center wavelength, δλ is the bandwidth of the spectrum, N is the number of lines in the grating  12  illuminated by the polychromatic input beam  10 , and m is the diffraction order. If the total bandwidth of the source is Δλ, the number of resolvable points, n is defined by:              n   =       Δ                 λ       δ                 λ               (   2   )                                
     For an input source with a center wavelength of 800 nm, a bandwidth of 25 nm, an input spot diameter of 5 mm, a diffraction grating of 1800 lines/mm and a diffraction order of 1, n=281 points may be resolved by the spectrally encoded confocal system (FIG.  2 ). The parameters used in this example may be found in common, inexpensive optical components. The number of points may be increased by simply increasing the input spot diameter or the bandwidth of the source. Increasing the spot diameter increases the resultant probe diameter. Increasing the bandwidth of the source could be accomplished by using a broader bandwidth superluminescent diode, a rare earth doped fiber superfluorescent source, or a solid state modelocked laser. 
     Consider next the multi-spectral process. First, consider direct spectral measurement. The reflectance from the sample  16  as a function of transverse location is determined by measuring the reflected confocal spectrum from the sample arm  18 . The spectrum may be measured efficiently by incorporating the probe  8  in the sample arm of a Michelson interferometer  20  (FIG. 3) and detecting the light transmitted through a high resolution spectrometer  21  at the output port  19  of the interferometer. Thus, each wavelength measured corresponds to a separate position, x, on the sample (FIG.  3 ). The advantage to this method over traditional real time confocal microscopy is that the fast axis scanning (˜15 kHz) may be performed external to the probe  8  by the spectrometer  21  with approximately 0.1 nm spectral resolution for the parameters given above, well within reach of high quality spectrometers. 
     High sensitivity may be achieved through the use of the heterodyne detection. If the reference arm  22  is modulated, such as by modulator  23  with mirror  24  (FIG.  3 ), the interference of light from the sample arm  18  and the reference arm  22  will also be modulated. High signal-to-noise ratios may be then achieved by lock-in detection on the reference arm modulation frequency of detector  26 . 
     Another method for measuring the spectrum is interference or Fourier transform spectroscopy. This may be accomplished by inserting a linearly translating mirror  28  in the reference arm  22  and measuring the cross-correlation output  30  from the interference spectrometer due to the interference of the reflected light from the sample and reference arms  18  and  22 , respectively (FIG.  4 ). The advantages to this type of spectroscopic detection include the ability to achieve higher spectral resolutions than direct detection methods, efficient use of the returned light, inherent modulation of the reference arm  22  by the Doppler shift of the moving mirror  28 , and the capability to extract both reflectance and phase data from the sample  16 . The ability to extract phase data from the sample may allow detection of refractive index as a function of transverse position, x, which is useful to reveal the molecular composition of the sample as well as provide an additional source of image contrast other than the reflectivity of the sample specimen  16 . Finally, interferometric detection has the potential to allow elimination of high order multiple scattering from the confocal signal by coherence gating. 
     Consider finally image formation. The multi-spectral encoding of the transverse location, x, allows the performance of a one-dimensional raster scan. To obtain an image, a scan of another axis must be performed, which is usually slower. Methods of accomplishing this slow scanning of the y axis include moving the optical fiber  9  in the y direction (FIG.  5 B), or rotating the entire probe  8  around the optical fiber axis either in a forward scanning configuration (FIG. 5C) or a side-firing configuration (FIG.  5 D). Cross-sectional images may be created by scanning the optical fiber  9  or the objective lens  14  along the z axis (FIG.  6 ). Finally, a zoom mode may be created by scanning the optical fiber  9  (or another lens  32  between grating  12  and objective lens  14 ), in and out of the image plane of the objective lens (FIG.  7 ). Both linear motion along the y or z axis and rotation are easily accomplished in a compact probe by use of piezoelectric transducers. As shown in FIG. 5A, signals may be received by a computer  34  from spectroscopic detector  32  by a spectrometer (such as described in connection with FIG. 3) or Fourier transform (such as described connection with FIG. 4) representing an image of the a microscopic section of the sample, and the image displayed on a display coupled to the computer. 
     From the foregoing description, it will be apparent that the invention provides a confocal microscopy system which (a) is compact, optical fiber-based, capable of enabling confocal microscopy through a flexible catheter or endoscope; (b) is fast-scanning which takes place external to the probe; (c) allows phase information to be retrieved; and (d) provides a number of resolvable points proportional to the bandwidth of the source and the beam diameter on the grating. Variations and modifications in the herein described confocal microscopy system in accordance with the invention will undoubtedly suggest themselves to those skilled in the art. Accordingly, the foregoing description should be taken as illustrative and not in a limiting sense.