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. Sertoli's-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.