Perfect focus system

Approximately 5*10⁵ mildly sonicated SCD-grown cells were immobilized on the glass surface of a 35 mm glass-bottom dish coated with Concanavalin A (100 μg/m; ~5000 cells per 1 mm² glass surface). Immobilized cells were stained for 1 hour with CDCFDA (10 μg/ml in 3 mL of SCD medium), washed twice with fresh SCD_Cd medium and covered with 3 mL of the same medium containing 6 μg/ml PI.
Time-lapse experiments were performed by using a Nikon A1R inverted microscope equipped with a 100× oil immersion objective (NA 1) and a Andor camera (NEO 5.5 sCMOS). Temperature (30 °C) and humidity were controlled throughout the measurement by a Okolab incubating system. DCFDA (535 nm) and PI (620 nm) emission signals were acquired every 15 minutes for about 10 hours (exposure time 200 ms). Focusing was maintained throughout the measurement by Nikon perfect focus system. At longer incubation times cells tend to detach from the plate floating out of the field. Given that quantitative single cell analysis was limited by cell motion and duplication during the time lapse experiment, a population analysis was performed by using the NIS Elements imaging software (v. 4.5). Mean CDCFDA and PI fluorescence was extrapolated from each image after background subtraction and plotted vs time. Fiji software (http://fiji.sc) was used for image post-processing.

Wide-field fluorescence microscopy was performed on an inverted microscope (Nikon Ti-E, Nikon, Japan) using a mercury lamp (Nikon, Japan) using standard DAPI, FITC and Cy5 filter sets (Nikon, Japan). Images were acquired with an electron-multiplied CCD camera (IXON DU-897E; Andor Technologies, Ireland). The EM-CCD camera settings were as follows; 900ms acquisition time, electron multiplier gain 10 and the readout mode set to 1MHz at 16-bit. Tile scans of 4 x 4 fields of view (~0.5mm x 0.5 mm) were acquired for each fluorescence channel using a 60×, NA = 1.49 oil immersion objective. The focal plane was maintained by a Nikon Perfect Focus System (Nikon, Japan) and a motorised stage ensured good overlap between image channels. The microscope platform was automatically controlled using the NIS Elements software (Nikon, Japan) during acquisitions.
All image analysis to determine the single cell fluorescence intensity in each channel was automated using algorithms written for FiJi.[23 ] To compensate for the epifluorescence illumination profile of the microscope, tile scans of fluorophores in a mixed solution were acquired and used to flatten images.

Live-cell imaging was performed using a motorized Nikon Ti inverted microscope (Nikon, Tokyo, Japan) equipped with a Plan Apo × 60/1.4 oil immersion objective lens, a 491-nm laser (FRAP-3D laser launch; Photometrics) and CSU-22 scanning head (Yokogawa Electric Corp., Tokyo, Japan), Evolve EM-CCD camera (Photometrics) and the Nikon Perfect Focus System. Cells were maintained at 37 °C in a 5% CO₂ humidified environment using an on-stage incubator (Chamlide, Live Cell Instrument, Seoul, South Korea). Time-lapse imaging was controlled using MetaMorph software (100 ms exposure, EM-gain 100, 15% laser power, Molecular Devices, LLC, Sunnyvale, CA). The image analysis was completed using Fiji software (ImageJ, National Institutes of Health (NIH), Bethesda, MD).

The microscope was a Nikon Eclipse Ti driven by the Elements software package. The microscope features an Evolve electron-multiplying charge-coupled device (Photometrics), an Intensilight epifluorescence source (Nikon), a CFI Apo 100× (numerical aperture 1.49) objective (Nikon) and a total internal reflection fluorescence launcher with three laser lines: 488 (10 mW), 561 (50 mW), and 638 nm (20 mW). This microscope also includes the Nikon Perfect Focus System, an interferometry-based focus lock that allowed the capture of multipoint and time-lapse images without loss of focus. In all the reported experiments, we used the following Chroma filter cubes: TRITC, Cy5, and RICM.