Mvplapo 1

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

In brief, the adult wings were collected 48 h after eclosion. The wings were emerged in 75% ethanol and washed on a shaker for 5–6 times with 6–8 min for each time for removing debris and dust on their surface. The cleaned wings were placed on a glass slide and embedded in glycerol. They were observed using a Multifocus Imaging System of Fluorescence Microscope (MV PLAPO 1×, Olympus, Tokyo, Japan) equipped with a CCD camera (DFC450 C, Leica, Wetzlar, Germany) for imaging.