Multiphoton microscopy of prostate and periprostatic neural tissue: a promising imaging technique for improving nerve-sparing prostatectomy

Abstract

Background and purpose: Various imaging modalities are under investigation for real-time tissue imaging of periprostatic nerves with the idea of improving the results of nerve-sparing radical prostatectomy. We explored multiphoton microscopy (MPM) for real-time tissue imaging of the prostate and periprostatic neural tissue in a male Sprague-Dawley rat model. The unique advantage of this technique is the acquisition of high-resolution images without necessitating any extrinsic labeling agent and with minimal phototoxic effect on tissue.

Materials and methods: The prostate and cavernous nerves were surgically excised from male Sprague-Dawley rats. The imaging was carried out using intrinsic fluorescence and scattering properties of the tissues without any exogenous dye or contrast agent. A custom-built MPM, consisting of an Olympus BX61WI upright frame and a modified MRC 1024 scanhead, was used. A femtosecond pulsed titanium/sapphire laser at 780-nm wavelength was used to excite the tissue; laser power under the objective was modulated via a Pockels cell. Second harmonic generation (SHG) signals were collected at 390 (+/-35 nm), and broadband autofluorescence was collected at 380 to 530 nm. The images obtained from SHG and from tissue fluorescence were then merged and color coded during postprocessing for better appreciation of details. The corresponding tissues were subjected to hematoxylin and eosin staining for histologic confirmation of the structures.

Results: High-resolution images of the prostate capsule, underlying acini, and individual cells outlining the glands were obtained at varying magnifications. MPM images of adipose tissue and the neural tissues were also obtained. Histologic confirmation and correlation of the prostate gland, fat, cavernous nerve, and major pelvic ganglion validated the findings of MPM.

Conclusion: Real-time imaging and microscopic resolution of prostate and periprostatic neural tissue using MPM is feasible without the need for any extrinsic labeling agents. Integration of this imaging modality with operative technique has the potential to improve the precision of nerve-sparing prostatectomy.

FIG. 1.

Multiphoton microscopy of rat prostate at low magnification (4×). A single optical section from the middle of the tissue is shown. The colored image is the processed composite of second harmonic generation (SHG) signal and autofluorescence (AF) from the prostatic tissue. Seen are the outlines of autofluorescent acini composed of prostatic cells (green) and SHG signal from the fibrous stroma around the acini (red). Scale bar: 500 μm.

FIG. 2.

Multiphoton microscopy of prostate using a 20× objective showing individual acini surrounded by the fibromuscular stoma. The colored image is the processed composite of second harmonic generation (SHG) signal (left) and autofluorescence (AF) (middle) from the prostatic tissue.

FIG. 3.

High magnification (20×) multiphoton microscopy image of rat femoral nerve. Seen are the second harmonic generation signal from the fibrocollagenous sheath (red) and autofluorescence (green) from the nerve, presumably coming from the axoplasm and the cytoplasm of Schwann cells. Note how the sheath wraps around the nerve bundle. Scale bar: 100 μm.

FIG. 4.

Low magnification (4×) multiphoton microscopy image of rat cavernous nerve. A single optical section from the middle of the tissue is shown. Second harmonic generation signal is from the fibrocollagenous sheath (red) and autofluorescence (green) is from the nerve. Scale bar: 500 μm.

FIG. 5.

High magnification (20×) multiphoton microscopy image of another rat prostate specimen, showing several optical sections through the specimen (roughly 10 μm apart). In the superficial optical sections, we see a second harmonic generation (SHG) signal from the fibrous capsule. Deeper sections show prostatic cells (green) and SHG signal from the intervening fibrous stroma (red). Scale bar: 100 μm.

FIG. 6.

Multiphoton microscopy (MPM) images and the corresponding hematoxylin and eosin (H&E) stained histologic images for validation of the findings. (A) Prostate lobe at low magnification (4×); (B) prostate acini at high magnification (20×); (C) cavernous nerve at high magnification (20×)—the arrow shows the perineural sheath; (D); major pelvic ganglion (MPG); (E) fat at high magnification (20×).

FIG. 6.

Multiphoton microscopy (MPM) images and the corresponding hematoxylin and eosin (H&E) stained histologic images for validation of the findings. (A) Prostate lobe at low magnification (4×); (B) prostate acini at high magnification (20×); (C) cavernous nerve at high magnification (20×)—the arrow shows the perineural sheath; (D); major pelvic ganglion (MPG); (E) fat at high magnification (20×).