Ff01 650 13 25

Cells were placed in a 37 °C humid incubator by controlling the temperature and CO₂ flow using H201-couple with temperature and CO₂ units. Live chromatin imaging was performed using a DMI8 inverted automated microscope (Leica Microsystems) featuring a confocal spinning disk unit (CSU-X1-M1N, Yokogawa). An integrated laser engine (ILE 400, Andor) was used for excitation with a selected wavelength of 647 nm and 140 mW as excitation power. A 100× oil immersion objective (Leica HCX-PL-APO) with a 1.4 NA was chosen for a high-resolution imaging. Fluorescence emission of the SiR-Hoechst was filtered by a single-band bandpass filter (FF01-650/13-25, Semrock, Inc.). Image series of 150 frames (5 fps), with exposure time of 150 ms per frame, were acquired using Metamorph software (Molecular Devices) and detected using sCMOS cameras (ORCA-Flash4.0 V2) and 1 × 1 binning, with sample pixel size of 65 nm. All series were recorded at 37 °C.

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