Nis elements ar 64 bit version 3

For imaging Live-cell imaging was performed using a Nikon A1R confocal microscopy (Nikon Instruments) mounted onto a Nikon Eclipse Ti body equipped with CFI Plan Apochromat VC objectives (×60, 1.4 numerical aperture; Nikon) along with digital zooming of Nikon imaging software (NIS-elements AR 64-bit version 3.21, Laboratory Imaging). Chamlide TC system placed in a microscope stage was used to maintain environmental condition at 37 °C and 10% CO₂ (Live Cell Instrument, Inc.). For treatment of the chemical stimulus, HeLa cells were serum starved 3 h prior to imaging, and the medium was replaced with DPBS (Invitrogen) just before the imaging.

Prepared cells were plated on 96-well polymer coverslip bottom plates (μ-Plate 96-Well ibiTreat; ibidi). R-GECO1 fluorescence imaging with blue light illumination for OptoSTIM1 activation was performed using a Nikon A1R confocal microscope (Nikon Instruments), mounted onto an inverted Eclipse Ti body (Nikon), equipped with a CFI Plan Apochromat VC objective (× 60 /1.4-numerical aperture (NA)) and digital zooming Nikon imaging software (NIS elements AR 64-bit version 3.21; Laboratory Imaging). During image processing, cells were maintained at 10% CO₂ and 37 °C by incubating in a Chamlide TC system (Live Cell Instruments, Inc., Korea). Immediately before imaging, the medium was replaced with OPTI-MEM (Invitrogen). Blue light photo-excitation (power density, 500 μW mm⁻²) was delivered with a 488-nm laser at 5-second intervals for 1 minute, unless stated otherwise.

Images were analyzed with Nikon imaging software (NIS-elements AR 64-bit version 3.21; Laboratory Imaging), MetaMorph software (version 7.8.1.0; MDS Analytical Technologies) or ImageJ software (version 1.50b; U.S. National Institutes of Health; http://imagej.nih.gov/ij/). For quantification of cluster formation, clusters were defined as discrete puncta of fluorescence with criteria of fluorescence intensity (1,500–4,095 arbitrary units) and equivalent diameter (>0.2 μm). The percentage of clustered cells was calculated by dividing the number of cells that formed clusters upon blue light stimulation by the total population of transfected cells. The cluster ratio in cells was quantified using “Automated Measurement” and “ROI Statistics” tools in Nikon imaging software. The total cluster intensity (I_C) was divided by the total fluorescence intensity (I_W) of the whole cell. Cluster size distribution was determined by classifying different cluster areas using the “Annotated Measurement Results” in Nikon imaging software. Co-localization between two molecules with different fluorescent colors was quantified with Pearson’s correlation coefficient by using the “Co-localization” tool in Nikon imaging software. A “Kymograph” tool in MetaMorph software was used to draw kymographs. Statistical significance was evaluated using a two-tailed Student’s t-test.