Laser scanning confocal microscopy of cervical tissue

Optical technologies are being used increasingly to perform real-time assessment of tissue abnormalities. Many groups have demonstrated that quantitative optical spectroscopic approaches, including fluorescence spectroscopy1-4 and reflectance spectroscopy,5, 6 have the potential to deliver a highly sensitive, specific, and costeffective diagnosis in real time and without the removal of tissue. These spectroscopic methods provide a means to extract diagnostically relevant structural and histochemical information on the basis of the interaction of light with chromophores within the tissue. Ramanujam et al7-9 developed a diagnostic algorithm that could differentiate normal squamous tissue, normal columnar tissue, low-grade squamous intraepithelial lesions, and highgrade squamous intraepithelial lesions on the basis of laser-induced autofluorescence of cervical tissue, collected in vivo for 3 excitation wavelengths. The algorithm performed with a sensitivity of 82% and a specificity of 68% in a blinded 95-patient study conducted in a diagnostic setting.9 Cantor et al10 performed a decision analysis showing that a see-and-treat strategy combining fluorescence spectroscopy and colposcopy would be more effective and less expensive than the current standard of care of colposcopy. They10 estimated that using fluorescence spectroscopy could save >$625 million annually in the United States. Over the past several years, in an attempt to further improve sensitivity and specificity, new optical technologies have been introduced, which provide direct imaging of tissue structure on the basis of the backscattering characteristics of a tissue. One particularly promising new technology is confocal microscopy,11 which samples small volumes of tissue, producing images with micron resolution at depths up to several hundred microns within the tissue. High-resolution confocal imaging can be used to obtain near real-time reflected light images of human epithelial tissue in vivo with micron resolution.11 In vivo confocal imaging can provide information about subcellular morphologic and biochemical changes in epithelial cells, which may be useful in the recognition and monitoring of epithelial precancers in organ sites such as the uterine cervix. Much of the work demonstrating the potential of confocal microscopy to image cell morphologic features has been carried out in pigmented tissue in which melanin within cells provided the confocal signal and image contrast.11 More recent work has demonFrom the Biomedical Engineering Program, University of Texas, Austina; and the Department of Pathology,b Thoracic/Head and Neck Medical Oncology,c and the Department of Gynecologic Oncology,d University of Texas M.D. Anderson Cancer Center. Supported by National Institutes of Health Shannon Award CA73920 and by grant No. P50 DE11906 from the National Institute of Disease and Cancer Research. Tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. Received for publication May 19, 1999; revised August 2, 1999; accepted December 1, 1999. Reprint requests: Rebecca R. Richards-Kortum, PhD, Engineering Science Building, Rm 08, Biomedical Engineering Program, University of Texas, Austin, TX 78712. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/104844 doi:10.1067/mob.2000.104844 Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid