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

Abstract

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.

Fig. 1

a The prostate specimens were transected, cross sections were rapidly stained with acridine orange and sulforhodamine 101, rinsed and then placed on the nonlinear microscope stage. 10x and 20x objectives were used to visualize the tissue. The tissue was translated in the x, y, and z direction to examine the entire cross section of tissue (whole mount) up to 100 µm in depth (analogous to serial sectioning). The H&E color display was generated using the acridine orange and sulforhodamine 101 fluorescence signals. The user display showed a photograph (right side) of the prostate cross section and a high magnification nonlinear microscopy (NLM) image (left side) of a user selected region of interest (marked in red). b For real time intraoperative consultation, the user can translate the specimen in the x-y direction to rapidly view different regions and change the focus in the z direction to view different depths. The images can be recorded to create a digital record. c Nonlinear microscopy can also automatically image the entire specimen cross section by stitching multiple high magnification fields. This operating mode is too time consuming for intraoperative consultation, but is useful for digital archiving purposes (analogous to slide scanning) and for comparing nonlinear microscopy images to standard paraffin embedded H&E slides

Fig. 2

Examples of prostate tissue architecture visualized using nonlinear microscopy imaging of freshly excised, unfixed specimens that were not physically sectioned versus corresponding standard paraffin embedded H&E slides after standard histological processing. a Nonlinear microscopy and b paraffin H&E of benign ducts with basal (blue arrow) and secretory (orange arrow) layers (20x objective), c nonlinear microscopy, and d paraffin H&E of a neural bundle (10x objective, 0.75 µm pixel), e nonlinear microscopy of stroma (10x objective, 0.75 µm pixel) with f corresponding paraffin H&E, and g nonlinear microscopy and h paraffin H&E of an artery (10x objective, 1 µm pixel). All scale bars are 100 µm

Fig. 3

Examples of prostate tissue architecture visualized by nonlinear microscopy imaging of fixed tissue that was not processed or physically sectioned versus corresponding standard paraffin embedded H&E slides. An artery and vein (a and b) and ejaculatory ducts (c and d) are shown. Scale bars are 100 µm. (10x objective, 0.75 µm pixel)

Fig. 4

A prostate specimen from a patient who underwent Bacillus Calmette−Guerin therapy. The characteristic histological patterns of this therapy are evident in nonlinear microscopy images of the fixed, unsectioned specimen and corresponding paraffin embedded H&E slide, including granulomatous inflammation (red) with giant cells (yellow arrow) and atrophic glands (blue arrow). (10x objective, 0.75 µm pixel)

Fig. 5

Nonlinear microscopy images of a freshly excised, unfixed specimen with benign prostatic hyperplasia and corresponding paraffin H&E slide. Complex hyperplastic glands are abundant throughout the tissue (red and blue box). (10x objective, 0.75 µm pixel)

Fig. 6

A nonlinear microscopy image of a fixed, unsectioned prostate specimen and corresponding paraffin H&E slide from a patient with typical adenocarcinoma displaying Gleason pattern 4, with ductal features (blue, yellow) and 3 (red). The nuclei are enlarged with prominent nucleoli (orange box). (20x objective)

Fig. 7

A nonlinear microscopy image of a freshly excised unfixed specimen and corresponding paraffin H&E slide exhibiting foamy gland variant of prostate cancer with Gleason score 6 (3 + 3). The cytoplasm of the cancer cells is less ‘foamy’ in the nonlinear microscopy images than the paraffin H&E (red, blue). Intraluminal crystalloids (yellow arrows) are present in the images. (10x objective, 0.75 µm pixel)

Fig. 8

A white-light photograph and nonlinear microscopy image of multi-centimeter, freshly excised, unfixed prostate tissue. Extra-prostatic extension and perineural invasion is observed among the periprostatic fat (red and blue). (10x objective, 1 µm pixel)

Fig. 9

A white-light photograph and nonlinear microscopy image of multi-centimeter, freshly excised, unfixed prostate tissue with tumor touching the blue ink. The tumor can be seen touching the ink in the nonlinear microscopy image (red and blue box, green arrows). The paraffin H&E slide did not show tumor touching the ink because of discrepancy between the nonlinear microscopy imaging plane and paraffin embedded H&E section. (10x objective, 1 µm pixel)