Joshua Sterling

Multiphoton Microscopy in the Evaluation of Human Bladder Biopsies

CONTEXT Multiphoton microscopy (MPM) is a nonlinear imaging approach, providing cellular and subcellular details from fresh (unprocessed) tissue by exciting intrinsic tissue emissions. With miniaturization and substantially decreased cost on the horizon, MPM is an emerging imaging technique with many potential clinical applications. OBJECTIVES To assess the imaging ability and diagnostic accuracy of MPM for human bladder biopsies. DESIGN Seventy-seven fresh bladder biopsies were imaged by MPM and subsequently submitted for routine surgical pathology diagnosis. Twelve cases were excluded because of extensive cautery artifact that prohibited definitive diagnosis. Comparison was made between MPM imaging and gold standard sections for each specimen stained with hematoxylin-eosin. RESULTS In 57 of 65 cases (88%), accurate MPM diagnoses (benign or neoplastic) were given based on the architecture and/or the cytologic grade. The sensitivity and specificity of MPM in our study were 90.4% and 76.9%, respectively. A positive (neoplastic) diagnosis on MPM had a high predictive value (94%), and negative (benign) diagnoses were sustained on histopathology in two-thirds of cases. Architecture (papillary versus flat) was correctly determined in 56 of 65 cases (86%), and cytologic grade (benign/low grade versus high grade) was assigned correctly in 38 of 56 cases (68%). CONCLUSIONS The MPM images alone provided sufficient detail to classify most lesions as either benign or neoplastic using the same basic diagnostic criteria as histopathology (architecture and cytologic grade). Future developments in MPM technology may provide urologists and pathologists with additional screening and diagnostic tools for early detection of bladder cancer. Additional applications of such emerging technologies warrant exploration.

Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications in prostate cancer surgery

Study Type – Diagnostic (exploratory cohort)

Pilot Study of the Correlation of Multiphoton Tomography of Ex Vivo Human Testis with Histology

PURPOSE Although microdissection testicular sperm extraction has become first line therapy for sperm retrieval in men with nonobstructive azoospermia, there are challenges to the procedure, including difficulty differentiating between seminiferous tubules with normal and abnormal spermatogenesis. Multiphoton microscopy illuminates tissue with a near infrared laser to elicit autofluorescence, which enables real-time imaging of unprocessed tissue without labels. We hypothesized that we could accurately characterize seminiferous tubular histology in humans using multiphoton microscopy. MATERIALS AND METHODS Seven men with normal or abnormal spermatogenesis underwent testicular biopsies, which were imaged by multiphoton microscopy. We assessed these images in blinded fashion. The diagnosis rendered with multiphoton microscopy was then correlated with that of hematoxylin and eosin stained tissue. We evaluated the ability of multiphoton microscopy to differentiate normal from abnormal seminiferous tubules by examining autofluorescence characteristics and diameters, as imaged by multiphoton microscopy. Assessment was repeated with stained slides and results were compared. RESULTS The overall concordance rate between multiphoton microscopy and stained slides was 86%. The seminiferous tubules of patients with nonobstructive azoospermia were smaller than those of controls when measured by multiphoton microscopy and staining (p <0.05). The proportion of normal tubules and the diameters obtained with multiphoton microscopy were not different from those obtained with hematoxylin and eosin (p >0.05). CONCLUSION Multiphoton microscopy can be used to differentiate normal from abnormal spermatogenesis. Its characterization of seminiferous tubular architecture is similar to that provided by hematoxylin and eosin staining. Further investigation of the clinical applications of multiphoton microscopy may improve surgical sperm retrieval outcomes for patients with nonobstructive azoospermia.

Identification of Spermatogenesis With Multiphoton Microscopy: An Evaluation in a Rodent Model

PURPOSE Microdissection testicular sperm extraction has replaced conventional testis biopsies for men with nonobstructive azoospermia and it has become first line treatment. The current problem is that the decision to retrieve tubules is based only on appearance and there is no guarantee that the tubules removed contain sperm. Multiphoton microscopy enables label-free immediate visualization of many biological processes in living tissue at subcellular resolution. MATERIALS AND METHODS We used multiphoton microscopy to study the different developmental stages of spermatogenesis using neonatal, pubertal and adult rat testes. We used a testis hypothermia plus ischemia model to study different testicular histopathologies with multiphoton microscopy. To assess the risk of photo damage DNA fragmentation in testis biopsies imaged at different intensities was assessed by TUNEL assay. RESULTS Multiphoton microscopy identified the stage of spermatogenesis in a seminiferous tubule in fresh tissue without using exogenous labels. We noted significant differences in fluorescence and spectroscopic characteristics between tubules with and without sperm. Sertolis-cell only tubules had abundant autofluorescence in the 420 to 490 and 550 to 650 nm wavelength ranges while tubules containing sperm had autofluorescence only in the 420 to 490 nm range. On DNA fragmentation assay sperm from tubules imaged by multiphoton microscopy had minimal DNA fragmentation at the laser intensities needed to distinguish tubules with and without sperm. CONCLUSIONS Multiphoton microscopy has the potential to facilitate real-time visualization of spermatogenesis in humans and aid in clinical applications, such as testicular sperm extraction for men with infertility.

Full field optical coherence tomography can identify spermatogenesis in a rodent sertoli-cell only model

Background: Microdissection testicular sperm extraction (micro-TESE) has replaced conventional testis biopsies as a method of choice for obtaining sperm for in vitro fertilization for men with nonobstructive azoospermia. A technical challenge of micro-TESE is that the low magnification inspection of the tubules with a surgical microscope is insufficient to definitively identify sperm-containing tubules, necessitating tissue removal and cytologic assessment. Full field optical coherence tomography (FFOCT) uses white light interference microscopy to generate quick high-resolution tomographic images of fresh (unprocessed and unstained) tissue. Furthermore, by using a nonlaser safe light source (150 W halogen lamp) for tissue illumination, it ensures that the sperm extracted for in vitro fertilization are not photo-damaged or mutagenized. Materials and Methods: A focal Sertoli-cell only rodent model was created with busulfan injection in adult rats. Ex vivo testicular tissues from both normal and busulfan-treated rats were imaged with a commercial modified FFOCT system, Light-CT TM , and the images were correlated with gold standard hematoxylin and eosin staining. Results: Light-CT TM identified spermatogenesis within the seminiferous tubules in freshly excised testicular tissue, without the use of exogenous contrast or fixation. Normal adult rats exhibited tubules with uniform size and shape (diameter 328 ±11 μm). The busulfan-treated animals showed marked heterogeneity in tubular size and shape (diameter 178 ± 35 μm) and only 10% contained sperm within the lumen. Conclusion : FFOCT has the potential to facilitate real-time visualization of spermatogenesis in humans, and aid in micro-TESE for men with infertility.

Multiphoton Imaging and Laser Ablation of Rodent Spermatic Cord Nerves: Potential Treatment for Patients With Chronic Orchialgia

PURPOSE Microsurgical denervation of the spermatic cord has been done to treat chronic orchialgia. However, identifying the site of spermatic cord nerves is not feasible with an operating microscope or robotic stereoscope. We used multiphoton microscopy, a novel laser imaging technology, to identify and selectively ablate spermatic cord nerves in the rat. MATERIALS AND METHODS The spermatic cords of adult male Sprague-Dawley® rats were initially imaged in vivo under a low power multiphoton microscopy laser. After assessing the number, diameter and site (vasal vs perivasal) of the nerves a higher power laser using the same objective was used to ablate the nerves. The precision of nerve ablation and the preservation of surrounding structures were determined by histological analysis. We assessed the heterogeneity of the number of nerves with the Wilcoxon signed rank test. RESULTS The average number of nerves per spermatic cord was 10, which was similar bilaterally (p = 0.13). The vas and perivasal structures had a similar number of nerves (p = 0.4). The median diameter of all nerves was 32 μm. Confirmation of nerve ablation, and preservation of the vas deferens and vasculature were anatomically validated by histological analysis. CONCLUSIONS Multiphoton microscopy can identify and ablate nerves selectively in vivo in the rat. It can potentially be used for spermatic cord denervation to treat chronic orchialgia. Such imaging may increase the efficacy of nerve ablation and can avoid the potential risks of testicular atrophy and hydrocele associated with spermatic cord microsurgical denervation.

Microsurgical denervation of rat spermatic cord: safety and efficacy data

To describe a microsurgical technique for denervation of the spermatic cord and use of multiphoton microscopy (MPM) laser to identify and ablate residual nerves after microsurgical denervation. To evaluate structural and functional changes in the rat testis and vas deferens after denervation.

Noninvasive Detection of Inflammatory Changes in White Adipose Tissue by Label-Free Raman Spectroscopy

White adipose tissue inflammation (WATi) has been linked to the pathogenesis of obesity-related diseases, including type 2 diabetes, cardiovascular disease, and cancer. In addition to the obese, a substantial number of normal and overweight individuals harbor WATi, putting them at increased risk for disease. We report the first technique that has the potential to detect WATi noninvasively. Here, we used Raman spectroscopy to detect WATi with excellent accuracy in both murine and human tissues. This is a potentially significant advance over current histopathological techniques for the detection of WATi, which rely on tissue excision and, therefore, are not practical for assessing disease risk in the absence of other identifying factors. Importantly, we show that noninvasive Raman spectroscopy can diagnose WATi in mice. Taken together, these results demonstrate the potential of Raman spectroscopy to provide objective risk assessment for future cardiometabolic complications in both normal weight and overweight/obese individuals.

Real-time multiphoton microscopy of human periprostatic tissue architecture for improving identification of vital tissue structures during nerve-sparing radical prostatectomy

Introduction: There is a paucity of data invesigating the relationship between histopathological ariables of oncologic importance and prostate volme, and we aimed to investigate this. Patients and methods: 2207 consecutive atients who underwent robotic-assisted radical rostatectomy (RARP) were studied. Preoperative emographic and both preand post-operative istopathological parameters were compared mong the small prostate (<40 cc), intermediate ize (40—70 cc), and large prostate (>70 cc) groups. Results: Patients with smaller prostates were ounger, had slightly lower BMIs, and lower PSAs han those with larger prostates (p < 0.001). They lso had worse histopathological criteria (Gleaon, core positivity, and maximum percent cancer) n preoperative biopsy and had worse radical pecimen Gleason sums (p < 0.001), percent caner (p < 0.001), and pathological stage (p = 0.016). 1.5% of men in the small prostate group suffered positive surgical margin (PSM) compared to 8.3% nd 5.6% in the intermediate and large prostate roups, respectively (p = 0.008). Basilar, posterolatral, and multifocal PSMs were commoner in the mall prostate group. Conclusions: Younger men have smaller prostates nd worse preoperative histopathological parameers despite lower PSAs. Men with small prostates ndergoing RARP have worse final Gleason sums, umour volume, extraprostatic extension (EPE), nd PSM rates than those with larger prostates.

Identification of Spermatogenesis in a Rat Sertoli-Cell Only Model Using Raman Spectroscopy: A Feasibility Study

PURPOSE We determined whether Raman spectroscopy could identify spermatogenesis in a Sertoli-cell only rat model. MATERIALS AND METHODS A partial Sertoli-cell only model was created using a testicular hypothermia-ischemia technique. Bilateral testis biopsy was performed in 4 rats. Raman spectra were acquired with a probe in 1 mm3 samples of testicular tissue. India ink was used to mark the site of spectral acquisition. Comparative histopathology was applied to verify whether Raman spectra were obtained from Sertoli-cell only tubules or seminiferous tubules with spermatogenesis. Principal component analysis and logistic regression were used to develop a mathematical model to evaluate the predictive accuracy of identifying tubules with spermatogenesis vs Sertoli-cell only tubules. RESULTS Raman peak intensity changes were noted at 1,000 and 1,690 cm(-1) for tubules with spermatogenesis and Sertoli-cell only tubules, respectively. When principal components were used to predict whether seminferous tubules were Sertoli-cell only tubules or showed spermatogenesis, sensitivity and specificity were 96% and 100%, respectively. The ROC AUC to predict tubules with spermatogenesis with Raman spectroscopy was 0.98. CONCLUSIONS Raman spectroscopy is capable of identifying seminiferous tubules with spermatogenesis in a Sertoli-cell only ex vivo rat model. Future ex vivo studies of human testicular tissue are necessary to confirm whether these findings can be translated to the clinical setting.