Fluoview fv10 asw software

The OV100 small animal imaging system (Olympus Corp., Tokyo, Japan), was used. The OV100 contains an MT-20 light source (Olympus Biosystems, Planegg, Germany) and DP70 CCD camera (Olympus) for subcellular imaging in live mice. The optics of the OV100 have been specially developed for macroimaging as well as microimaging with high light-gathering capacity. The instrument incorporates a unique combination of high numerical aperture and long working distance. Four individually optimized objective lenses, parcentered and parfocal, provide a 105-fold magnification range for seamless imaging of the entire body down to the subcellular level without disturbing the animal [17 (link), 18 (link)].
Imaging was also performed with an FV 1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan) with a XLUMPLFL 20× (0.95 NA) water-immersion objective. GFP was excited at 488 nm. Images were produced with FV10-ASW Fluoview software (Olympus) and ImageJ and were not modified beyond the standard adjustment of intensity levels [19 (link)].

Cells plated on two-well chamber slides were chronically treated with 5 μM, 10μM, or 20μM cisplatin for 4 or 24 hours and then fixed with 3.5% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100 for 10 min. Fixed cells were blocked with 5% FBS and stained with anti-γH2AX and Alexa-conjugated anti-mouse secondary antibodies. Images were acquired by confocal microscope OLYMPUS IX81. The fluorescence intensity of γH2AX in a cell was quantified by an OLYMPUS FLUOVIEW: FV10-ASW software. At least 100 cells from each cell lines were quantified.

The DNA transfection followed a Promega FuGENE protocol by the manufacturer as described in Refs. 45 (link), 59 (link), and 60 (link). The HEK293 cells were transfected after reaching 30–50% confluence in a LabTeck 4-well cover glass chamber with the mixtures of bGCAP-GFP or hRD3-GFP coding plasmids with mOrange-RetGC1 coding plasmid, at ∼1/100 molar ratio (58 (link), 59 (link), 60 (link)), using 3 μl of Promega FuGENE reagent per 1 μg of DNA. Confocal images were taken after 24–30 h of incubation in 5% CO₂ at 37 °C, using an Olympus FV1000 Spectral instrument with the respective 543- and 488-nm excitation for the red and the green fluorochromes in sequential mode. The images were processed, and the PCC values in whole-cell images were determined using Olympus FluoView FV10-ASW software as previously described (43 (link), 44 (link), 45 (link), 58 (link), 59 (link), 60 (link)). No changes to the original images were made except for minor γ correction applied to whole image for more clear presentation in print. Quantitative analyses were performed using the original images without corrections.

Larvae were imaged as described previously^{12 (link)}. Larvae were maintained in embryo media containing 0.0003% 1-phenyl 2-thiourea (PTU, Sigma-Aldrich P7629) starting at 20 h postfertilization for confocal imaging. Larvae were analyzed at 6 days postfertilization (dpf) by embedding in 0.5% low melting point agarose containing embryo media with 0.02% (w/v) Tricaine (Sigma-Aldrich, E10521). The agarose was submerged in embryo media containing 0.0003% PTU and 0.02% (w/v) Tricaine. Imaging was performed using an Olympus FV1000 with a 40 × water objective in conjunction with Olympus FluoView FV10-ASW software (RRID:SCR_014215). The excitation/emission wavelengths used for mito-GCaMP3 were 488/510 nm. Images of total eye mitochondrial clusters were collected at a z-depth of 2 µm, and blinded quantification was performed using ImageJ + Fiji software (SCR_002285).