Orca flash 4.0 v3 cmos camera

Cells for fluorescence microscopy were cultured, with or without CueO overproduction, as for the co‐immunoprecipitation experiments, and prepared in tunnel slides as previously described (Alcock et al., 2013 (link)). Where indicated cells were incubated with 50 μM CCCP for 10 min prior to imaging.
Fluorescence images were acquired using a Nanoimager (Oxford Nanoimaging) equipped with a 532 nm 1 W DPSS laser, a 100x oil‐immersion objective (Olympus, numerical aperture 1.4), and an ORCA‐Flash4.0 V3 CMOS camera (Hamamatsu). Images were collected in HiLo mode (49% laser angle) at 10% laser power. For figure composition, image stacks were imported into Fiji (Schindelin et al., 2012 (link)), averaged over 60 ms and scaled to display 1400 arbitrary units (a.u.) as the maximum (white) and 550 a.u. as the minimum (black).
Fluorescence imaging data are representative of experiments carried out a minimum of three times with independent biological replicas.
Cells for light microscopy were cultured in LB to mid‐log phase, diluted, spotted onto glass slides and imaged on a phase contrast microscope with a 40x objective.

The microgels were characterized by transmission electron microscopy (TEM) (SI Appendix, Figs. S1 and S2), CLSM (SI Appendix, Fig. S3), DLS and electrophoretic mobility measurements (SI Appendix, Fig. S4). The measurements were performed at 20 and 28 ^°C. The results are summarized in SI Appendix, Table S1. The mean aspect ratio (b/a) of the nonspherical microgels MG2 and MG3 was determined at 28 ^°C from statistical analysis over 100 microgels adsorbed at the cover glass in water, respectively. The three microgels are slightly positively charged. The microgels were also observed at superresolution using a 3D-structured illumination microscope (N-SIM, Nikon Healthcare) mounted on a Nikon Ti2 research microscope body equipped with an SR HP apochromat TIRF 100x/NA 1.49 oil-immersion objective lens and a Hamamatsu ORCA-Flash4.0 V3 CMOS camera. SIM images of the Alexa488-stained microgels were acquired at 20 ^°C with 700-ms exposure time and no binning and reconstructed using the N-SIM module in the NIS-Elements AR software package. Illumination modulation contrast, high-resolution noise suppression, and out-of-focus blur suppression were set to 2.50, 1.0, and 0.35, respectively. Finally, the images presented in this manuscript were adjusted for brightness and contrast (SI Appendix, Fig. S3).

Wide-field image capturing was performed on a Nikon Eclipse Ti-2 microscope (Nikon, Melville, NY) using a monochrome, Hamamatsu ORCA-Flash 4.0 V3 CMOS camera (Hamamatsu, Bridgewater, NJ) and PlanApo objectives (10x, 20x, 40x; NA = 0.30, 0.75, and 1.30, respectively). Filter sets (Chroma Technology, Bellows Falls, VT) were designed with careful attention to the spectra of the various fluorophores to make certain no bleed through exists between channels. An extended depth of field (EDF) algorithm was used to generate two-dimensional images from acquired Z-stacks (Elements Software; Nikon). The Nikon Elements EDF module facilitates merging of captured Z-stacks into two-dimensional images, using only the focused regions for each optical plane. Separate channels were pseudocolored and lookup tables were adjusted slightly for each image to reduce background noise and best depict labeling observed through the microscope. Images were saved as lossless JPEG2000 files prior to subsequent quantitative analyses. Masking of minimal artifact observed outside section contours in a few instances was performed (Adobe Photoshop, San Jose, CA) to enhance overall figure quality.