Orca r2

TP12 worms were immobilized with 0.1 µm polystyrene beads on a 6% NGM agarose pad. 10 µm glass beads (Duke Scientific) for indenting the worms were spread onto a coverslip, which was inverted to cover the agarose pad holding the worms. To image the worms, we used a high-magnification camera (Orca-R2, Hamamatsu) on an inverted microscope (Leica) with an EGFP filter set and a high-numerical aperture 63x oil immersion lens, to yield a shallow depth of field ≈0.1 µm for optical sectioning. When glass beads were trapped between the cuticle of the animal and the coverslip, we were able to capture fluorescence images of COL-19::GFP in the cuticle at >10 different focal planes. At each focal plane, we measured the radius of the bead and the radius of the cuticle deformation (by identifying where the cuticle was in focus). We then calculated the depth of the plane based on the radius of the bead at the focal plane. Experimental data shown are a combination of all focal planes for two adult animals.

The chips were placed on the stage of a Nikon ECLIPSE Ti inverted fluorescence microscope, which was equipped with a CFI plan fluor 10×/0.30 N.A. objective lens (Nikon, Tokyo, Japan), charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics, Hamamatsu City, Japan), mercury lamp (Intensilight; Nikon), XYZ automated stage (Ti-S-ER motorized stage with encoders; Nikon), and filter cubes for the fluorescence channels (DAPI and GFP HYQ; Nikon). For image acquisition, the exposure times were set to 100 ms for (DAPI) Hoechst 33258, 5 ms for (GFP HYQ) Calcein AM, 2 s for (TRITC) Annexin V, and 100 ms for the (TRITC) AdipoRed assay.

PER2::LUC is a luciferase fusion protein that reports its expression bioluminescently. Time-dependent changes in PER2::LUC expression were measured in real-time via a cooled-CCD camera (imaging) or a photomultiplier (luminometry). Bioluminescence imaging was performed in a previously described custom-made, light-sealed inverted microscope system [26 (link)], which has a simplified optical path optimized for low-light imaging, with a 4× objective lens and a 0.35× relay lens (Olympus). The cultured sample was maintained at 37 °C in a stage-top incubator (Tokai HIT, Shizuoka, Japan), and time-lapse imaged with an Orca R2 cooled-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) with external water-cooling (20 °C; TGV-10, Taipei, Taiwan). Images were taken under 1 h exposure at 4×4 binning, at a sampling interval of 1 h. For luminometry, an equal number of control and test samples were measured on the same 8-dish wheel in Kronos Dio (ATTO, Tokyo, Japan); light from each dish was measured under 1 min exposure at sampling interval of 10 min, with the nominal temperature of 37 °C and actual temperature maintained between 37.6–37.7 °C, measured with iButton datalogger in the dish (DS1921L, Maxim Integrated, San Jose, CA, USA). Bioluminescence traces were detrended using the HP filter as described previously [23 (link)].

Cells were sparsely seeded in wells of a culture plate 24 h prior to filming. Time-lapse imaging was conducted using an Olympus IX81 microscope attached to a Hamamatsu Photonics Orca-R2 cooled CCD camera (Hamamatsu Photonics., Welwyn Garden City, UK). Phase contrast images were captured at varying positions using a motorised stage (LUDL Electronic Products, Hawthorne, NY, USA) every 5 min for 16 h. Individual cells were manually tracked throughout consecutive frames using an ImageJ software plugin (ImageJ1, National Institutes of Health (NIH), Bethesda, MD, USA). Using these x-y coordinates, migrational velocity and persistence were determined using Mathematica 6.0 (Wolfram Research Ltd., Witney, UK) custom-written notebooks kindly provided from Professor Graham Dunn and Daniel Soong, King’s College London.

A Leica DMI4000B (Leica Microsystems) inverted fluorescence microscope equipped with a Hamamatsu Orca R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics) was used to acquire all immunofluorescent images. All devices were controlled by MetaMorph Imaging System software (Universal Imaging). Images were evaluated and processed using Metamorph Imaging System software or ImageJ software. The pixel intensity plots (line scan analyses) were performed by the microscopist using ImageJ whereby the line tool was used to first draw a 1.5-μm line perpendicularly across the invagination. Following this, the “plot profile” tool was used to obtain the pixel intensity value (from 0 to 255) corresponding to the protein of interest, actin, and when indicated, CellTracker Blue. Lines were excluded or shifted if intense signal from cellular structures (such as stress fibers), random artifacts, or other nearby invaginations interfered with the profile of interest. Line scan analyses were replicated at least 3 times (and up to 6 times) for each protein examined.