Dm il epifluorescence microscope

Animals were mounted in 3 μl of 15 mM tetramisole (Sigma-Aldrich, L9756) in M9 buffer suspension containing 45-μm polystyrene microspheres (Polysciences, 07314-5), and sealed between 2 coverslips (Corning, 2845-18 and 2975-246) with vaseline. Alternatively, for time-lapse recordings (Movies S1, S2 and S3), a recently described procedure was used.⁷⁵ (link) Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany), a Yokogawa CSU X1 scan head (Yokogawa Electric Corporation, Tokyo, Japan), and a Hamamatsu ImagEM EM-CCD (Hamamatsu Photonics, Hamamatsu, Japan). All acquisitions were performed at 21°C to 23°C using a Leica HC PL APO 40x/1.3 oil or a Leica HC PL APO 63x/1.4–0.6 oil objective.
Animals stained with DAPI were imaged using a Leica DM IL epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica HC PL FL 10×/0.3 objective and a Motic Moticam 1SP camera (Motic Deutschland GmbH, Wetzlar, Germany) with Motic acquisition software.
3D reconstructions were performed using imod (http://bio3d.colorado.edu/imod/) for cell outlines and Endrov (http://www.endrov.net/) for autophagosome movement.

Fluorescence microscopy images were obtained from an inverted Leica DMIL epifluorescence microscope (Leica Microsystems Pty Ltd, USA), fitted with transmitted light LED back illumination and an EL6000 Fluoro system (mercury lamp). FITC fluorescence was detected with a GFP filter cube (excitation filter: BP470/40, dichroic mirror: 500, emission filter: 525/50). Images were observed under a 10 × NA0.22 air objective, and captured using ProgRes Capture Pro 2.7 software by an integrated ProgRes CFscan CCD camera (JENOPTIK laser, Optik, Systeme GmnH, Germany). The exposure time for recording the image was 5 s, and the gain factor was 1.0.
ImageJ 1.44p (National Institute of Health, USA) was used for the quantitative analysis of the fluorescence microscopy images obtained under the same instrumental setting (lamp intensity, magnification and exposure time). Mean grey values in 20 circular areas (diameter 80 μm) from both spot and background regions of the patterning images were measured to compare protein adsorptions between these two regions.