Abstract:
A confocal scanning microscope system ( 10 ) using cross polarization effects and an enhancement agent (acetic acid) to enhance confocal microscope reflectance images of the nuclei of BCCs (basal cell carcinomas) and SCCs (squamous call carcinomas) in the confocal reflectance images of excised tumor slices. The confocal scanning microscope system having a laser ( 11 ) for generating an illumination beam ( 12 ), a polygon mirror ( 18 ) for scanning the beam to a tissue sample ( 22 ) and for receiving a return beam from the tissue sample and detector ( 28 ) for detecting the returned beam to form an image. The system further includes a half-waveplate ( 13 ) having a rotatable stage ( 14 ) and a quarter-wave plate ( 21 ) having a rotatable stage ( 20 ) disposed in the optical path of the illumination beam and at least a linear polarizer ( 24 ) having a rotatable stage ( 25 ) disposed in the optical pat of the returned beam from the tissue sample.

Description:
This application is a Continuation of application Ser. No. 09/936,535, filed Sep. 14, 2001, now U.S. Pat. No. 6,720,547, a National Application under 35 U.S.C. §371 of International Pat. application Ser. No. PCT/US00/07008, filed Mar. 17, 2000, which claims the benefit of priority to U.S. Provisional Application Nos. 60/125,033, filed Mar. 18, 1999, and 60/146,819, filed Aug. 2, 1999, which are herein incorporated by reference. 

   FIELD OF THE INVENTION 
   The present invention relates to confocal microscopy and particularly to a system (method and apparatus) for enhancing images of tissue at the surface or internally of a tissue sample so as to enable rapid and accurate screening of tissue for the determination of the nuclear and cellular structure thereof. The present invention also relates to a method for diagnosing cancerous cells in skin tissue using confocal microscopy. The invention is especially suitable in providing enhanced images of the nuclei of BCC/SCC (basal cell carcinoma or squamous cell carcinoma) in confocal reflectance images of tumor slices obtained during Mohs micrographic surgery. Tissue may be either naturally exposed, or surgically excised tissue. 
   BACKGROUND OF THE INVENTION 
   Mohs micrographic surgery for BCCs and SCCs involves precise excision of the cancer with minimal damage to the surrounding normal skin. Conventionally, precise excision is guided by histopathologic examination for cancer margins in the excised tissue slices during Mohs surgery. Typically, 2–4 slices are excised, and there is a waiting time of 10–30 minutes for the surgeon and patient while each slice is being processed. 
   Confocal reflectance microscopes can noninvasively image nuclear and cellular detail in living skin to provide images of tissue sections, such a microscope is described in U.S. Pat. No. 5,880,880. The contrast in the images is believed to be due to the detected variations in the singly back-scattered light caused by variations in refractive indices of tissue microstructure. Within the epidermal (basal and squamous) cells, the cytoplasm appears bright and the nuclei as dark ovals. The underlying dermis consists of collagen bundles and that, too, appears bright with dark spaces in-between. Thus, when neoplastic epidermal cells invade the dermis as in BCCs and SCCs, confocal detection of the cancers is very difficult because the cells and nuclei lack contrast relative to the surrounding normal dermis. 
   SUMMARY OF THE INVENTION 
   It is the feature of the present invention to provide an improved system and method for confocal microscopy by cross polarizing the light illuminating a tissue sample and the light returned from the tissue sample representing a section of the tissue. 
   It is another feature of the present invention to use such cross polarizing of the light illuminating a tissue sample and the light returned from the tissue sample in combination with imaging the sample when immersed in an image enhancement agent. 
   It is a further feature of the present invention to provide a method for diagnosing cancerous cells in skin tissue using confocal microscopy 
   Briefly described, a system for providing enhanced images in confocal microscopy is provided by utilizing cross polarized light in the illumination of tissue and in the detection of light from which the images are formed, respectively, and where an image enhancing agent, such as acetic acid or vinegar solution, is used in a bath in which the specimen is immersed while being imaged. 
   It has been found in accordance with the invention that a confocal laser scanning microscope using cross polarized components of light in illumination and in the detection of the reflected light from tissue specimens immersed in such an enhancement agent solution images of the cellular structure are enhanced, enabling cells and voids in the structure and the cell condition to be readily observed. By virtue of the use of such cross polarized light in imaging of tumor slices obtained in the course of Mohs surgery, epidermis sections which may have holes in the collagen are imaged more accurately so that holes are unlikely to be confused with cells or cell structure. 
   A method is also provided for detecting cancerous basal cell and squamous cell in dermal tissue with confocal reflected light imaging having the steps of: washing the tissue to be imaged with a solution of acetic acid to whiten epithelial cells and compact chromatin of the tissue; imaging the tissue with a confocal microscope to provide confocal images of basal and squamous cells in which the confocal microscope directs light into the tissue and collects reflected light representing confocal images of the tissue; changing the polarization state of the light used by the confocal microscope to increase the contrast of the nuclei of basal and squamous cells in the confocal images; and analyzing the nuclei of the basal and squamous cells in the confocal images to diagnose which of such cells are cancerous. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The foregoing features and advantages of the invention will become more apparent from a reading of the following description in connection with the accompanying drawings wherein, 
       FIG. 1  is a schematic diagram of a Vivascope (™) confocal microscope which is available from Lucid Inc. of Rochester, N.Y. and is described in the above referenced U.S. Pat. No. 5,880,880; and 
       FIGS. 2A , B, C, and D are schematic depictions of various parts of the confocal microscope system and the cross-polarized illumination which is used therein. 
   

   DETAILED DESCRIPTION OF INVENTION 
   Referring to the drawings, in the confocal microscope  10  of  FIG. 1 , a linearly polarized (p-state) laser beam  12  is passed through a half wave plate (HWP)  13  on a rotation stage  14 . A confocal microscope especially suitable in practicing the invention is described in U.S. Pat. No. 5,880,880, issued Mar. 9, 1999, which is herein incorporated by reference. Other confocal microscopes may also be used. The illumination through the non-polarizing or partially polarizing beam splitter  16  is scanned, as by a polygon mirror  18  and galvanometric mirror  19  across the specimen or sample  22  having a surface  22   a . As shown in  FIGS. 2B and 2C , sample  22  may be a BCC/SCC sample in a sample holder or container  22   b  contained in an enhancement solution bath  26  having water  28  under a tissue ring  33  which places the sample  22  under tension. As shown in  FIG. 1 , the microscope  10 , via an objective lens  23 , images the tissue sample  23  through an opening  33   a  in the tissue ring  33 . For example, the opening  33   a  may include a window having a material transparent to the beam. 
   The target surface is the surface of the sample  22  (such as a tissue tumor specimen), which may be at the surface  22   a  or within the body of the sample, utilizing the techniques described in the above referenced U.S. Patent. The polarization of the incident light and the reflected light also can be modified using a quarter wavelength plate (QWP)  21  which is also removably mounted on a rotation stage  20 . 
   The detected light is cross-polarized that is in the s-state as shown by the bulls-eye indication  12   a  in  FIG. 1  and labeled “detection s-state” in  FIG. 2D . It is crossed or perpendicular or orthogonal to the p-state. Although preferably cross-polarized light is in s and p states, because the beam splitter may be non-polarizing or partially polarizing, other states are possible. The detected illumination of desired polarization is obtained with an analyzer  24  also mounted on a rotation stage  25 . For example, analyzer  24  may be a linear polarizer. The light from the analyzer  24  is passed through the confocal aperture  28   a , such as a pinhole, and a photo-detector  28 , such as an avalanche photodiode (APD) in  FIG. 1 . While p polarized light from a linearly polarized laser  11  is shown in  FIG. 1 , the linearly polarized laser  11  and the half wave plate  13  can be replaced with a laser providing an unpolarized laser beam and a linear polarizer, respectively. Further, the linear polarizer and the analyzer  24  can then be replaced with a polarized beam splitter. Also, instead of rotating the half wave plate  13  and the analyzer  24 , they can be kept fixed in cross polarization states and the sample  22  can be rotated. 
   As shown in  FIG. 1 , optical components are provided in confocal microscope  10  to direct the beam from laser  11  along a path to sample  22 , and include, beam expander-spatial filter  42  (which, for example, may be provided by two lens  42   a  and  42   b  and aperture  42   c ), HWP  13 , mirror  43 , ND filter  44  (which, for example, may be a neutral density filter, such as provided by a circular variable attenuator manufactured by Newport Research Corporation), through beam splitter  16  to polygon mirror  18 . The beam is then deflected by polygon mirror  18  through a lens  45  (which for example, may be a f/2 lens), a lens  46  (which for example, may be a f/5.3 lens), and deflected by galvanometric mirror  19  through a lens  47  (which for example, may be a f/3 lens), QWP  21  and objective lens  23  to sample  22 . The optical components along the path of the reflected light returned from the sample  22  to detector  28  include, objective lens  23 , QWP  21 , lens  47 , and deflected by mirrors  19  and  18  via lenses  46  and  45  to beam splitter  16 . The beam splitter  16  directs the returned light through lens  48 , analyzer  24 , and pinhole  28   a  to detector  28 . The raster line  17   a  and raster plane  17   b  in  FIG. 1  are illustrated by dashed lines to denote the angular scan of the beam along a raster line  17   a  generated by the rotation of polygon mirror  18 , while the angular movement of galvanometric mirror  19  scans that raster line to form a raster plane  17   b . In this manner, a confocal image of a tissue section can be captured by the control electronics  38  through detector  28 . To provide a start of scan beam  12   c  to synchronize the control electronics  38  with the start of each raster line, the beam splitter  16  directs part of the beam incident the beam splitter  16  to rotating polygon mirror  18 , via mirror  48 , to split diode  50  (e.g., photo-diode) which is connected to the control electronics  38  to provide a start of scan pulse at the beginning of each raster line. Motors, not shown, can provide the desired rotation and angular movement of respective mirrors  18  and  19 . 
   The system which is shown in  FIG. 1  operates as follows: 
   1. Remove QWP  21 . Rotate the HWP  13  so that its fast axis is at 90 degrees with the illumination p-state (see  FIG. 2A ). Thus, there is no change (rotation) of the direction of the p-state. Rotate the analyzer  24  so that it acts as a crossed polarizer and transmits the detection s-state (which is orthogonal to the illumination p-state). 
   2. The surgically excised tissue sample  22  is placed in a water bath  26  with a tissue-ring  33  placed on top (see  FIG. 2B ). 
   3. The water bath  26  containing the sample  22  is placed under the objective lens  23 , such that the tissue-ring  33  fits into the objective lens housing  31  (see  FIG. 2C ). The water bath  26  is on an XY translation stage  34  to move the sample  22 . The XY stage  34  is on a lab-jack  35  with which can move the entire assembly  36  upwards, such that the sample  22  is gently pressed between the tissue-ring  33  and the water bath  26  to keep the sample  22  still during the imaging. Arrow  37  denotes the direction of such light pressure. 
   4. Rotate the HWP  13  in small angular increments of 10 degrees and, correspondingly, the analyzer  24  in angular increments of 20 degrees, on their respective stages  14  and  25 , such that the analyzer  24  is always cross-polarized with respect to the illumination polarization state. The confocal images of the sample  22  change from bright to dark to bright as the HWP  13  and analyzer  24  is rotated. 
   5. Set the HWP  13  and analyzer  24  such that the sample  22  appears dark (i.e., minimum brightness). Survey the sample  22  by moving it with the XY stage  34 , to check that the sample appears dark everywhere in the confocal images. 
   6. Lower the water bath  26  using the lab-jack  35 . Remove the water from within the tissue ring  33 , and add an enhancement agent, namely acetic acid (e.g., to provide a 5% by volume—ph 2.5—solution in the water). Raise the lab-jack  35  and place the sample  22 , as before, under the objective lens  23 . 
   7. Survey the sample  22  by moving it with the XY stage  34 , and focusing on the surface and at varying depths of the sample with the objective lens  23  (which may be mounted on a Z-translation stage to move the objective lens towards and away from the sample). Confocal images are either videotaped or grabbed in this “crossed polarization” mode at a frame grabber  39 , video monitor  40 , or videotape recorder  41  via control electronics  38 . 
   8. Whenever or wherever necessary, confocal images are obtained in “brightfield” mode, to either determine lateral or depth location, or identify structures (examples: hair follicles, sweat ducts, epithelial margins) within the sample. (This is analogous to using reflectance imaging in conjunction with fluorescence imaging.) The QWP  21  is inserted and rotated so that its optic axis is at 45 degrees to both the illumination and detection linear polarization states (see  FIG. 2D ). 
   With the confocal reflectance light microscope  10  described herein, BCCs, SCCs in human skin are described herein without the processing (fixing, sectioning, staining) that is required for conventional histopathology of Mohs surgery. Rapid confocal detection is provided after strongly enhancing the contrast of nuclei in the cancer cells relative to the surrounding normal tissue using acetic acid and crossed polarization. 
   To improve the detection of BCCs and SCCs in confocal images in tissue, such as dermal tissue, which may be either naturally exposed, or surgically excised, the contrast of the nuclei of such cells is increased by the following method. The area of the tissue to be imaged is washed with 5% acetic acid, as described earlier. Acetic acid causes whitening of epithelial tissue and compaction of chromatin. The chromatin-compaction is believed to increase its refractive index, which then increases light back-scatter from the nuclei and makes them appear bright. Next, the tissue area is imaged with confocal microscope  10  in which the polarization state of the light directed to the tissue and collected by the confocal microscope is controlled by rotating the linear polarizer of analyzer  24 . When illuminated with linearly polarized light and confocally imaged through the analyzer  24 , the brightness of the acetic acid-stained nuclei does not vary much, whereas the brightness of the collagen varies from maximum to minimum. The back-scattered light from the inter-nuclear structure is significantly depolarized (probably due to multiple scattering), whereas that from the dermis preserves the illumination polarization (due to single back-scatter). With the light in a crossed polarized state, bright nuclei in the BCCs and SCCs are shown in the confocal images produced by the microscope in strong contrast against a dark background of surrounding normal dermis. BCCs and SCCs can be distinguished from normal tissue by the cellular organization, cell size, cell shape, nuclear morphology, and cellular differentiation. One example of cellular organization is anaplasia. One example of cell size and shape and nuclear morphology is dysplasia. One example of cellular differentiation is pleomorphism. 
   Thus, the bright clusters of nuclei in the cancer cells are detectable at low resolution, as in conventional histopathology. Mosaics of low-resolution confocal images can be assembled to produce confocal maps of the BCCs or SCCs within the entire excised tissue. Detection of the cancers is made within minutes; thus, the total savings in time for a Mohs surgery can be hours. 
   Others cancers and tissue abnormalities may also be detected by using this approach any time a cellular tissue needs to be distinguished from acellular background. For example, dermal melanocytes, mucosal tissue in stromal tissue, breast epithelium in a stromal matrix. 
   From the foregoing description, it will be apparent that an improved system for enhanced imaging in confocal microscopy and method for diagnosing skin cancer cells have been described. Variations and modifications in the herein described system, method, and in the enhancement agent used therein will undoubtedly become apparent to those skilled in the art. Accordingly, the foregoing description should be taken as illustrative and not in a limiting sense.