Ff02 632 22 25

We built a device equipped with an optics enabling automated acquisition of florescence images of a sample. A schematic view of the optical design is shown in Fig. 1. We placed the sample in a cassette, and the sample was illuminated by the light emitting from an LED unit. The unit consists of two tunable LEDs (CBT-90-UV and CBT-90-B, Luminus), which have excitation filters of either 405 nm or 435 nm (FF01-406/15, FF02-438/24, Semrock), respectively. Fluorescence emitting from the sample was reflected by the dichroic mirror at wavelengths longer than 470 nm, collimated with an objective lens (MVPLAPO × 0.63, Olympus), and imaged on a monochrome CCD camera (Lt665R, Lumenera). An optical filter unit was placed at the position between the objective lens and the imaging lens (MVPLAPO × 1, Olympus). The optical filter unit consisted of fluorescence filters with the center wavelengths of 600 nm, 632 nm, 650 nm and 680 nm (FF01-600/14-25, FF02-632/22-25, FF01-650/13-25, and FF01-680/22-25, Semrock).Fig. 1

Schematic view of the device for automated detection of LN metastasis

Artificial cerebrospinal fluid (aCSF) of pH 7.4 and temperature of 37 °C was used to prepare RH1692 dye (Optical Imaging, Rehovot, Israel) solution (1 mg/ml). Bath application of the dye to the exposed cortex was performed for 60–90 min. The unbound dye was washed away by loading the cortex with aCSF for 30 min after the bath application. Then, 1.5–2% agarose was used to cover the cortex to minimize pulsation and movement artifacts. A 12 mm coverslip was put onto the agarose layer before agarose cooling. The coverslip was then fixed to the head plate with cyanoacrylate glue. A 100 W halogen lamp focused at 400 µm from the cortical surface was used for excitation. The excitation light was filtered with a filter centered at 632 nm (FF02-632/22-25, Semrock, NY, USA) and was reflected onto the cortex using a light guide. The signals coming back from the cortex were collected using a tandem lens macroscope with a long-pass emission filter at 675 nm (84–753, Edmund Optics, Barrington, NJ, USA). The macroscope was connected to a CCD (MV1-D1312-160-CL-12, PhotonFocus, Lachen, Switzerland) that recorded the signals at 150 Hz using a CELOX imaging system and VDAQ software (Optical Imaging, Rehovot, Israel). The data had a spatial resolution of 62.5 μm per pixel.