In vivo microscopy: will the microscope move from our desk into the patient?

For more than a century, the essential elements of surgical pathology have remained constant: tissue excision; production of glass slides; and static histopathologic evaluation with an upright microscope. Although there have been tremendous advances in our field, the diagnostic prowess of a pathologist still rests in the faithful microscope sitting on our desk. With the emergence of high-resolution imaging techniques, such as multiphoton microscopy (MPM) and optical coherence tomography (OCT), we now have a way to visualize tissue in vivo, in patients, and in real-time without tissue excision. These techniques provide a means to conduct in vivo microscopy (IVM). Multiphoton microscopy is a nonlinear optical imaging technique that generates en face microscopic resolution images by exciting fluorescence emission from intrinsic fluorophores within tissues or exogenously applied fluorophores. Optical sectioning is used to conduct three-dimensional imaging up to several hundred microns deep within tissue. Optical coherence tomography is an optical imaging modality that uses near infrared light to generate cross-sectional images based on endogenous tissue contrast. Optical coherence tomography provides near-microscopic resolution images (~10 lm, comparable to low-power bright-field microscopy) at penetration depths up to 2 to 3 mm, but OCT resolution is approaching near-cellular (1–2 lm) resolution with recent technologic advancements. Optical coherence tomography technology provides rapid volumetric imaging, allowing for large tissue volume imaging within seconds. Both imaging techniques have been applied to many organ systems for disease detection, such as the gastrointestinal tract, coronary arteries, lungs, and urinary tract. In their article titled ‘‘Multiphoton Microscopy: A Potential Intraoperative Tool for the Detection of Carcinoma In Situ in Human Bladder,’’ Jain et al assess the diagnostic potential of MPM to identify and differentiate benign from malignant flat bladder lesions. The authors recognize a significant clinical problem in the diagnosis of bladder carcinoma in situ (CIS). White light cystoscopy, used to guide bladder biopsy after positive urine cytology findings, misses approximately 50% of CIS lesions. This is thought to be due in part to the flat architecture of CIS lesions and lack of cellular resolution with white light cystoscopy. The authors propose that performing MPM during cystoscopy would provide cellular resolution imaging and could guide biopsy to improve detection of urothelial CIS. In a blinded assessment, the authors demonstrate that urologic pathologists assessing MPM of ex vivo bladder tissues achieved a 99% accuracy rate in determining whether the samples were benign or malignant. Multiphoton microscopy assessments were based on architectural and cytologic features that parallel features used in traditional microscopy, including nuclear to cytoplasmic ratio, pleomorphism, and polarity/ organization of urothelial layers. The achieved sensitivity and specificity were high (97% and 100%, respectively), with strong interobserver agreement (j: 0.93). These results are very promising and the authors demonstrate that MPM has significant potential to improve CIS detection as a biopsy guidance tool during cystoscopy.