Orca flash 4.0 lt scmos camera

Data shown in Figs. 1 and 5 were acquired on a Zyla 4.2 sCMOS camera (Andor) with a CSU-W1 spinning disk (Yokogawa) mounted on a Ti2 eclipse microscope (Nikon). The Apo Plan 100 × oil/NA1.4 and 60 × oil/NA1.4 objectives and a 405/488/561/638 nm laser (Omicron) were used in combination with an Okolab stage top incubation chamber (Okolab) in case of live-cell experiments. For SIM (3D structured illumination microscopy) images shown in Figs. 3 and 4, an N-SIM E (Nikon) was used, built on a Ti-Eclipse microscope (Nikon). Data were acquired using a z Piezo drive (Mad city labs), an Apochromat TIRF 100 × Oil/NA 1.49 objective, an Orca flash 4.0 LT sCMOS camera (Hamamatsu), a LU-N3-SIM 488/561/640 laser unit (Nikon) and a motorized N-SIM quad band filter combined with a single 525/50 emission filter using the laser line 488 at maximum output power. Z-stacks were acquired with a step size of 200 nm. Both microscopes were controlled by NIS-Elements software (Nikon). Slice reconstruction (NIS-Elements, Nikon) was performed using reconstruction parameters IMC 0.7, HNS 0.7, OBS 0.2.

Cell–cell adhesion flipping assays were performed as previously described^{44 (link),45 (link)}. Briefly, a suspension of fluorescently labeled cells in PBS⁺⁺ was sedimented for 10 min in a chamber slide containing unlabeled confluent monolayers of adherent cells. A picture was taken before flipping the dish. Slides were then filled and carefully dipped in a large vessel containing pre-warmed PBS⁺⁺. They were then rotated by 180° and maintained in an upside-down position for 15 min, allowing cells that did not adhere to the monolayer to detach. The chamber slide was then rotated back 180° and carefully removed from the large vessel. A second picture was taken after flipping, around the same position. Finally, the percentage of cells in suspension adhering to the monolayer of adherent cells was calculated. For this assay, wide-field images were rapidly acquired on a Zeiss Axio Observer 7 epifluorescence microscope equipped with a EC Plan-Neofluar 5× NA 0.16 dry objective and an ORCA-Flash 4.0 LT sCMOS camera (Hamamatsu).

Staining of worms with tetramethylrhodamine-phalloidin (catalog # P1951, MilliporeSigma, St. Louis, MO) was performed as described (28 (link), 89 (link)). Samples were observed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope (Nikon Instruments, Tokyo, Japan) with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Images were captured by a Hamamatsu ORCA Flash 4.0 LT sCMOS camera (Hamamatsu Photonics, Shizuoka, Japan) and processed by NIS-Elements AR V5.02.01 (Nikon Instruments) and Adobe Photoshop CS3.
To measure fluorescence intensity of AIPL-1::GFP, live worms were mounted and immobilized with 25% Pluronic F-127 (catalog # 2730-50G, Biovision, Milpitas, CA) in M9 buffer containing 0.5 mM levamisole and 0.1% tricaine methanesulfonate. Fluorescence images were captured with the same settings for all samples in NIS-Elements using a Nikon Eclipse TE2000 inverted microscope with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Fluorescence intensity of each worm was determined using ImageJ as average fluorescence intensity at 10 randomly selected points within the cytoplasm of the body wall muscle in the head region.

ΔΨm and mtROS were determined in neuron cultures by staining with 100 nM TMRM and 2.5 μM MitoSOX probes (Invitrogen), respectively [12 (link)]. In the TMRM staining, the non-potentiometric probe MitoTracker Green FM (100 nM, Invitrogen) was also included to normalize TMRM signal by the signal of mitochondrial mass. Images were acquired in an Axioskop2 Plus (Zeiss, Oberkochen, Germany) with Lambda 10–2 Optical Filter Changer (Sutter Instrument, Novato, California, USA) coupled to an ORCA-Flash4.0 LT sCMOS camera (Hamamatsu, Hamamatsu, Japan) from Optical and Confocal Microscopy Facility at CBMSO. Specificity of TMRM staining was assessed by the addition of 5 μM oligomycin or 5 μM FCCP to collapse ΔΨm.

Stained sections were examined and imaged using Olympus BX61 DSU fluorescence microscope with a Hamamatsu ORCA-FLASH4.0LT + SCMOS CAMERA. Images were taken in stacks. All images were taken and processed using the integrated software program Olympus cellSens Software Version 4. ImageJ (NIH) was used to perform integration and analysis of images.

To image mitochondrial ATP-Mg²⁺ levels, cells were plated onto 8-well Lab-Tek chamber slides (Thermo Fisher, 154534) and transfected 48 h prior to the experiments. We used a plasmid encoding a ratiometric mitochondrial targeted ATP-Mg²⁺ mitGO-Ateam2 probe [43 (link)]. Transfections were performed using Lipofectamine 3000 following the manufacturer’s instructions. Experiments were performed in HEPES Buffered Saline Solution (137 mM NaCl, 1.25 mM MgSO₄, 10 mM HEPES, 3 mM KCl, 2 mM NaHCO₃, 2 mM CaCl₂) supplemented with 5 mM glucose and 2 mM glutamate. Additions of the stimulus were made as a bolus in the same medium. Cells were excited for 100 ms at 485 ± 27 nm, and the emitted fluorescence was alternately collected through an FF495-Di03 dichroic at 520 ± 35 nm (GFP) and 567 ± 15 nm (OFP). Images were collected every 2.5 or 15 s using a filter wheel (Lambda 10-2, Sutter Instruments; Chroma) and recorded with an ORCA-Flash4.0 LT sCMOS camera (Hamamatsu) mounted on an Axiovert200 inverted microscope (Zeiss) equipped with a 63X/1.4 Plan-Apochromatic oil objective. The emission ratio GFP/OFP reflects mitochondrial levels of ATP-Mg²⁺.
ROIs were selected on single-cell fluorescence recordings and analyzed using MetaMorph (Universal Imaging) and ImageJ (NIH). For quantification, the time of the decrease in the GFP/OFP fluorescence ratio by 20% after stimulation was determined.