Tie inverted wide field microscope

Cells were spotted onto an agar pad (A + with 1% agar) and placed onto a microscope slide. Cells were imaged on a customized Nikon TiE inverted wide-field microscope with a Near-IR-based Perfect Focus system. Images were acquired with an ORCA Flash4.0 V2 + Digital sCMOS camera (Hamamatsu) using a Nikon CF160 Plan Apochromat Lambda 100 × oil immersion objective (1.45 N.A.). Chlorophyll fluorescence of thylakoid membranes was imaged using a 640 nm LED light source (SpectraX) for excitation and a standard Cy5 emission filter. GFP localization was imaged using a 470 nm LED light source (SpectraX) for excitation and a standard GFP emission filter.

Overnight cultures of Cglu were back-diluted to an OD₆₀₀=0.025, grown to an OD₆₀₀ between 0.15 and 0.5 depending on fitness of mutant, and normalized to 1 mL of OD₆₀₀=0.3. WT cells ectopically expressing P_TAC-GFP were grown with Kan and 0.1 mM IPTG. Cells were pelleted for 3 min at 5000×g, resuspended in 100 µL BHIS with 100 µM 6-TMR-Tre (Tocris), and incubated in the dark for 30 min. Cells were then re-pelleted, washed 2× with BHIS, and resuspended in BHIS. Wild-type and mutant cells were mixed together in a 1:5 ratio and spotted onto BHIS 1% agarose pads. Images were acquired on a Nikon Ti-E inverted widefield microscope with a motorized stage, a perfect focus system, and a 1.45 NA Plan Apo ×100 Ph3 DM objective lens with Cargille Type 37 immersion oil. Fluorescence imaging was performed with Lumencore SpectraX LED Illumination with images in the ET-GFP and ET-mChrery channels using Chroma 49002 and 49008 filter sets, respectively. Acquired images were taken with an Andor Zyla 4.2 Plus sCMOS camera (65 nm pixel size) with Nikon Elements acquisition software (v5.10). Images were rendered for publication with Fiji and contrast was normalized between the samples.

All samples were imaged on a Nikon Ti-E inverted widefield microscope equipped with a fully motorized stage and perfect focus system. Images were acquired using a 1.45 NA Plan Apo ×100 Ph3 DM objective lens with Cargille Type 37 immersion oil. Fluorescence was excited using a Lumencore SpectraX LED light engine and filtered using ET-GFP (Chroma, 49002) and ET-mCherry (Chroma, 49008) filter sets. Images were recorded on an Andor Zyla 4.2 Plus sCMOS camera (65 nm pixel size) using Nikon Elements (v5.10) acquisition software. For subsequent deconvolution procedures, three 200 nm spaced Z-planes were acquired for both fluorescence channels using 100% LED output power and 50 ms exposure. Temperature was maintained at 30 °C using a custom-made environmental enclosure. After a 20 min acclimatization period, cells were imaged at a 2.5 min acquisition frame rate for a total observation time of 1–4 h.