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