US20070070497A1 - System and method for enhancing confocal reflectance images of tissue specimens - Google Patents

Abstract

A system using cross polarization effects and an enhancement agent having citric or other similar alpha hydroxy acid to enhance confocal microscope reflectance images and particularly images of the nuclei of BCCs (basal cell carcinomas) and SCCs (squamous cell carcinomas) in the confocal reflectance images of excised tumor slices obtained during Mohs surgery by illuminating the tissue being imaged (a tumor slice) using polarized light. The reflected illumination is passed to a polarization analyzer, which passes the polarization component which is crossed with respect to the polarization of the illuminating light. The light from the analyzer is passed through the confocal aperture and detected. The section of the tissue either at the surface or within the tissue is scanned and the reflectance image is produced with enhanced visualization of the cellular or nuclear structure thereof thereby enabling determination of the extent of the tumor (cancerous cells) in the section. A method is also provided using the system for diagnosing cancerous cells in skin tissue.

Description

• This application claims the benefit of priority to U.S. Provisional Patent Application No. 60/241,140, filed Oct. 17, 2000, which is herein incorporated by reference, and describes an improvement over International Application No. PCT/US00/07008, which was filed on Sep. 14, 2001 in the United States Patent and Trademark Office as U.S. patent application Ser. No. 09/936,535.
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 of10-30minutes 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.
• Acetic acid (vinegar) has been used as an image enhancement agent which enhances the back-scatter of light from certain cells. This is described, for example, in U.S. Pat. No. 5,733,739. When a tissue is illuminated with light of one polarization and a reflected image is detected via light of cross polarization the contrast of certain cells in the detected image is enhanced, when the tissue is treated by acetic acid, especially when the image is viewed via cross-polarized light in a confocal microscope. Such a system is described in the above referenced International Application No. PCT/US00/07008.
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 and using an image enhancement agent other than acetic acid.
• 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 containing citric acid or another alpha hydroxy acid.
• It is a further feature of the present invention to provide a method for diagnosing cancerous cells in skin tissue using confocal microscopy and a citric or alpha hydroxy acid image enhancing agent to treat the tissue.
• 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, which contains an effective amount of citric or similar alpha hydroxy acid, is used in a bath in which the tissue is immersed while being imaged. It will be understood that the agent may also be applied to tissue by coating, dipping or encapsulating in a gel of citric or other alpha hydroxy acid solution.
• 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 citric or other alpha hydroxy acid to whiten epithelial cells and believe to compact the 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 (TM) 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 theconfocal microscope10 ofFIG. 1, a linearly polarized (p-state)laser beam12 is passed through a half wave plate (HWP)13 on arotation stage14. 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 polarizingbeam splitter16 is scanned, as by apolygon mirror18 andgalvanometric mirror19 across the specimen orsample22 having asurface22a. As shown inFIGS. 2B and 2C,sample22 may be a BCC/SCC sample in a sample holder orcontainer22bcontained in anenhancement solution bath26 havingwater28 under atissue ring33 which places thesample22 under tension. As shown inFIG. 1, themicroscope10, via anobjective lens23, images thetissue sample23 through anopening33ain thetissue ring33. For example, theopening33amay include a window having a material transparent to the beam.
• The target surface is the surface of the sample22 (such as a tissue tumor specimen), which may be at thesurface22aor 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 arotation stage20.
• The detected light is cross-polarized that is in the s-state as shown by the bulls-eye indication12ainFIG. 1 and labeled “detection s-state” inFIG. 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 ananalyzer24 also mounted on arotation stage25. For example,analyzer24 may be a linear polarizer. The light from theanalyzer24 is passed through theconfocal aperture28a, such as a pinhole, and a photo-detector28, such as an avalanche photodiode (APD) inFIG. 1. While p polarized light from a linearly polarizedlaser11 is shown inFIG. 1, the linearly polarizedlaser11 and thehalf wave plate13 can be replaced with a laser providing an unpolarized laser beam and a linear polarizer, respectively. Further, the linear polarizer and theanalyzer24 can then be replaced with a polarized beam splitter. Also, instead of rotating thehalf wave plate13 and theanalyzer24, they can be kept fixed in cross polarization states and thesample22 can be rotated.
• As shown inFIG. 1, optical components are provided inconfocal microscope10 to direct the beam fromlaser11 along a path tosample22, and include, beam expander-spatial filter42 (which, for example, may be provided by twolens42aand42bandaperture42c), HWP13,mirror43, ND filter44 (which, for example, may be a neutral density filter, such as provided by a circular variable attenuator manufactured by Newport Research Corporation), throughbeam splitter16 topolygon mirror18. The beam is then deflected bypolygon mirror18 through a lens45 (which for example, may be a f/2 lens), a lens46 (which for example, may be a f/5.3 lens), and deflected bygalvanometric mirror19 through a lens47 (which for example, may be a f/3 lens),QWP21 andobjective lens23 to sample22. The optical components along the path of the reflected light returned from thesample22 todetector28 include,objective lens23,QWP21,lens47, and deflected bymirrors19 and18 vialenses46 and45 tobeam splitter16. Thebeam splitter16 directs the returned light throughlens48,analyzer24, andpinhole28atodetector28. Theraster line17aandraster plane17binFIG. 1 are illustrated by dashed lines to denote the angular scan of the beam along araster line17agenerated by the rotation ofpolygon mirror18, while the angular movement ofgalvanometric mirror19 scans that raster line to form araster plane17b. In this manner, a confocal image of a tissue section can be captured by thecontrol electronics38 throughdetector28. To provide a start ofscan beam12cto synchronize thecontrol electronics38 with the start of each raster line, thebeam splitter16 directs part of the beam incident thebeam splitter16 torotating polygon mirror18, viamirror48, to split diode50 (e.g., photo-diode) which is connected to thecontrol electronics38 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 ofrespective mirrors18 and19.
• The system which is shown inFIG. 1 operates as follows:
• 1. RemoveQWP21. Rotate theHWP13 so that its fast axis is at 90 degrees with the illumination p-state (seeFIG. 2A). Thus, there is no change (rotation) of the direction of the p-state. Rotate theanalyzer24 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 excisedtissue sample22 is placed in awater bath26 with a tissue-ring33 placed on top (seeFIG. 2B).
• 3. Thewater bath26 containing thesample22 is placed under theobjective lens23, such that the tissue-ring33 fits into the objective lens housing31 (seeFIG. 2C). Thewater bath26 is on anXY translation stage34 to move thesample22. TheXY stage34 is on a lab-jack35 with which can move theentire assembly36 upwards, such that thesample22 is gently pressed between the tissue-ring33 and thewater bath26 to keep thesample22 still during the imaging.Arrow37 denotes the direction of such light pressure.
• 4. Rotate theHWP13 in small angular increments of 10 degrees and, correspondingly, theanalyzer24 in angular increments of 20 degrees, on theirrespective stages14 and25, such that theanalyzer24 is always cross-polarized with respect to the illumination polarization state. The confocal images of thesample22 change from bright to dark to bright as theHWP13 andanalyzer24 is rotated.
• 5. Set theHWP13 andanalyzer24 such that thesample22 appears dark (i.e., minimum brightness). Survey thesample22 by moving it with theXY stage34, to check that the sample appears dark everywhere in the confocal images.
• 6. Lower thewater bath26 using the lab-jack35. Remove the water from within thetissue ring33, and add an enhancement agent, namely citric or other alpha hydroxy acid (e.g., to provide a 5% by volume—ph 2.5—solution in the water). Raise the lab-jack35 and place thesample22, as before, under theobjective lens23. It will be understood that the concentration of the solution to be effective may be in the range of from 3-20%.
• 7. Survey thesample22 by moving it with theXY stage34, and focusing on the surface and at varying depths of the sample with the objective lens23 (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 aframe grabber39,video monitor40, orvideotape recorder41 viacontrol electronics38.
• 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.) TheQWP21 is inserted and rotated so that its optic axis is at 45 degrees to both the illumination and detection linear polarization states (seeFIG. 2D).
• With the confocal reflectancelight microscope10 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 citric or other alpha hydroxy 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% citric or alpha hydroxy acid, as described earlier. Citric or alpha hydroxy acid causes whitening of epithelial tissue and believe to cause the compaction of the 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 withconfocal microscope10 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 ofanalyzer24. When illuminated with linearly polarized light and confocally imaged through theanalyzer24, the brightness of the citric or alpha hydroxy 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 enhancing images in confocal microscopy and method for diagnosing skin cancer cells is provided using citric or other alpha hydroxy acid. Variations and modifications in the herein described system and method 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.

Claims (15)

1-7. (canceled)

8. A method for confocal imaging tissue comprising the steps of:

generating an illumination beam;

scanning the beam to the tissue;

receiving returned light from the tissue representing of a section of the tissue;

cross polarizing the illumination beam and the returned light with respect to each other;

detecting the returned light to form an image of the section of the tissue, and the step of treating said sample with a solution of alpha hydroxy acid which enhances the brightness of one or more tissue structures in the image of the section of the tissue.

9. The method according toclaim 8 wherein said polarization controlling step further comprises the step of changing polarization state of at least one o the illumination beam and the returned light to effect characteristics of tissue structures in the image of the tissue section to enable determination of which of the tissue structures are cancerous.

10. The method according toclaim 9 wherein said tissue is skin tissue.

11. The method according toclaim 8 wherein the tissue is one of naturally exposed tissue and surgically excised tissue.

12. The method according toclaim 8 further comprising the step of applying said solution to said tissue.

13. The method according toclaim 12 wherein said solution is applied to said tissue by one of coating, washing, dipping, or rinsing with said image enhancing agent.

14. The method according toclaim 12 wherein said solution is provided by a gel encapsulating said tissue.

15. The method according toclaim 12 wherein said solution is applied by placing said tissue in a bath of said solution.

16. The method according toclaim 8 wherein said solution is of 3-20% concentration of said acid.

17. The method according toclaim 8 wherein said solution is of 5% concentration of said acid.

18. The method according toclaim 8 further comprising the step of analyzing cells in said images to diagnose which of said cells are cancerous.

19. A method for detecting cancerous basal cell and squamous cell in dermal tissue with confocal reflected light imaging, said method comprising the steps of:

washing the tissue to be imaged with a solution of citric or other alpha hydroxy acid which whitens epithelial cells and compacts cromatin 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.

20-36. (canceled)

37. The method according toclaim 8 wherein said alpha hydroxy acid represents citric acid.

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