Abstract
Introduction:
In this study we have used Multiphoton Microscopy (MPM) for ex vivo tissue imaging in order to assess its potential as a future intraoperative imaging modality.
Methods:
We used two types of specimens for imaging: (1) intraoperative margins and biopsies; (2) tissue sections obtained from the excised prostate. 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 Bio-Rad 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 380–530 nm. The images obtained from SHG and from tissue fluorescence were then merged and color coded during post processing for better appreciation of details. The corresponding tissues were subjected to hematoxylin and eosin staining for histological confirmation of the structures.
Results:
High-resolution images of the periprostatic tissue, nerves, prostate capsule, underlying acini, and individual acinar cells were obtained at varying magnifications. Histological confirmation and correlation of the periprostatic tissue, prostate gland, fat, blood vessels and nerves validated the findings of MPM.
Conclusions:
We have utilized a novel approach for real time tissue imaging which seems to provide microscopy level resolution in fresh tissue, without the need for any extrinsic labeling agents.