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Comparing histologic evaluation of prostate tissue using nonlinear microscopy and paraffin H&E: a pilot study


Rapid histological assessment of large areas of prostate tissue is required for many intraoperative consultation scenarios such as margin evaluation. Nonlinear microscopy (NLM) enables imaging of large (whole mount) specimens without freezing or cryotoming. This study demonstrates rapid histological imaging of unsectioned prostate cancer surgical specimens using nonlinear microscopy and compares features of prostate pathology to standard paraffin embedded H&E histology. Fresh or formalin fixed specimens were stained in 2.5 min with fluorescent nuclear and stromal dyes. Nonlinear microscopy images of unsectioned tissues were generated by nonlinear (two-photon) excitation of the fluorophores, where fluorescence is only emitted from tissue at the microscope focus, avoiding the need for physical sectioning. The images were displayed in real time using a color scale similar to H&E, then tissues were processed for standard paraffin embedded H&E histology. Seventy nonlinear microscopy and corresponding paraffin H&E images of fresh and fixed prostate specimens (15 cancer, 55 benign) from 24 patients were read by genitourinary pathologists to assess if nonlinear microscopy could achieve an equivalent evaluation to paraffin embedded H&E histology. Differences between nonlinear microscopy images and paraffin H&E slides, including cytoplasmic color and stromal density, were observed, however nonlinear microscopy images could be interpreted with minimal training. Nonlinear microscopy enabled visualization of benign, atrophic and hyperplastic glands and stroma, ejaculatory ducts, vasculature and inflammatory changes. Nonlinear microscopy enabled identification of typical and variants of adenocarcinoma, as well as Gleason patterns. Perineural invasion and extraprostatic extension could also be assessed. Nonlinear microscopy images closely resemble paraffin H&E slides and enable rapid assessment of normal prostate architecture, benign conditions, and carcinoma in freshly excised and fixed specimens. Nonlinear microscopy can image large regions of tissue, equivalent to multiple frozen section tissue blocks, within minutes because cryotoming/microtoming are not required, making it a promising technique for intraoperative consultation.

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  1. 1.

    Schlomm T, Tennstedt P, Huxhold C, et al. Neurovascular Structure-adjacent Frozen-section Examination (NeuroSAFE) increases nerve-sparing frequency and reduces positive surgical margins in open and robot-assisted laparoscopic radical prostatectomy: experience after 11069 consecutive patients. Eur Urol. 2012;62:333–40.

  2. 2.

    Beyer B, Schlomm T, Tennstedt P, et al. A feasible and time-efficient adaptation of NeuroSAFE for da Vinci robot-assisted radical prostatectomy. Eur Urol. 2014;66:138–44.

  3. 3.

    Von Bodman C, Brock M, Roghmann F, et al. Intraoperative frozen section of the prostate decreases positive margin rate while ensuring nerve sparing procedure during radical prostatectomy. J Urol. 2013;190:515–20.

  4. 4.

    Wang M, Kimbrell HZ, Sholl AB, et al. High-resolution rapid diagnostic imaging of whole prostate biopsies using video-rate fluorescence structured illumination microscopy. Cancer Res. 2015;75:4032–41.

  5. 5.

    Wang M, Tulman DB, Sholl AB, et al. Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy. Sci Rep. 2016;6:27419.

  6. 6.

    Rais-Bahrami S, Levinson AW, Fried NM, et al. Optical coherence tomography of cavernous nerves: a step toward real-time intraoperative imaging during nerve-sparing radical prostatectomy. Urology. 2008;72:198–204.

  7. 7.

    Fried NM, Rais-Bahrami S, Lagoda GA, et al. Identification and imaging of the nerves responsible for erectile function in rat prostate, in vivo, using optical nerve stimulation and optical coherence tomography. IEEE J Sel Top Quantum Electron. 2007;13:1641–5.

  8. 8.

    Aron M, Kaouk JH, Hegarty NJ, et al. Second Prize: Preliminary Experience with the Niris TM Optical Coherence Tomography System during Laparoscopic and Robotic Prostatectomy. J Endourol. 2007;21:814–8.

  9. 9.

    D’Amico a V, Weinstein M, Li X, et al. Optical coherence tomography as a method for identifying benign and malignant microscopic structures in the prostate gland. Urology 2000;55:783–7.

  10. 10.

    Glaser AK, Reder NP, Chen Y, et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat Biomed Eng. 2017;1:0084.

  11. 11.

    Giacomelli MG, Yoshitake T, Cahill LC, et al. Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins. Biomed Opt Express. 2018;9:2457.

  12. 12.

    Denk W, Strickler J, Webb W. Two-photon laser scanning fluorescence microscopy. Science. 1990;248:73–6.

  13. 13.

    Tao YK, Shen D, Sheikine Y, et al. Assessment of breast pathologies using nonlinear microscopy. Proc Natl Acad Sci USA. 2014;111:15304–9.

  14. 14.

    Cahill LC, Giacomelli MG, Yoshitake T, et al. Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope. Lab Invest. 2018;98:150–60.

  15. 15.

    Giacomelli MG, Husvogt L, Vardeh H, et al. Virtual hematoxylin and eosin transillumination microscopy using epi-fluorescence imaging. PLoS ONE. 2016;11:e0159337.

  16. 16.

    Yadav R, Mukherjee S, Hermen M, et al. Multiphoton microscopy of prostate and periprostatic neural tissue: a promising imaging technique for improving nerve-sparing prostatectomy. J Endourol. 2009;23:861–7.

  17. 17.

    Durand M, Jain M, Aggarwal A, et al. Real-time in vivo periprostatic nerve tracking using multiphoton microscopy in a rat survival surgery model: a promising pre-clinical study for enhanced nerve-sparing surgery. BJU Int. 2015;116:478–86.

  18. 18.

    Tewari AK, Shevchuk MM, Sterling J, et al. Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications in prostate cancer surgery. BJU Int. 2011;108:1421–9.

  19. 19.

    Yossepowitch O, Briganti A, Eastham JA, et al. Positive surgical margins after radical prostatectomy: a systematic review and contemporary update. Eur Urol. 2014;65:303–13.

  20. 20.

    Gillitzer R, Thuroff C, Fandel T, et al. Intraoperative peripheral frozen sections do not significantly affect prognosis after nerve-sparing radical prostatectomy for prostate cancer. BJU Int. 2011;107:755–9.

  21. 21.

    Vasdev N, Agarwal S, Rai BP, et al. Intraoperative frozen section of the prostate reduces the risk of positive margin whilst ensuring nerve sparing in patients with intermediate and high-risk prostate cancer undergoing robotic radical prostatectomy: First Reported UK Series. Curr Urol. 2016;9:93–103.

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This study was supported in part by the National Institutes of Health R01-CA178636–05, R01-CA075289–20, F32-CA183400–02 and Air Force Office of Scientific Research AFOSR contracts FA9550–12–1–0551 and FA9550–15–1–0473.

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Conflict of interest

JGF, MGG, TY and LCC are inventors on patent application WO2017139649: Method and apparatus for imaging unsectioned tissue specimens. The remaining authors declare no conflict of interest.

Correspondence to Seymour Rosen.

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