Approximately 1 × 105 U373 cells were plated 16 h before imaging on 25-mm (diameter) no. 1.5 glass coverslips. The imaging medium was phenol red–free DMEM supplemented with 10% FCS and 20 mM HEPES (pH 7.4). For imaging (Kural et al., 2012 (link)), the coverslips were placed on a temperature-controlled 5% CO2 humidified chamber (20/20 Technology, Wilmington, NC) mounted on the piezo-electric driven stage of a Mariana imaging system (Intelligent Imaging Innovations, 3I, Denver, CO) based on an Axiovert 200M inverted microscope (Carl Zeiss, Thornwood, NY), a CSU-X1 spinning-disk confocal unit (Yokogawa Electric Corporation, Tokyo, Japan), a spherical aberration-correction device (SAC; Infinity Photo-Optical, Boulder, CO), and a 63× objective lens (Plan-Apochromat, NA 1.4, Carl Zeiss). The SAC was placed between the oil-based objective lens and the camera to resolve the spherical aberration introduced by the refractive index mismatch between living cells and the glass optics. Three-dimensional time series were obtained using Slidebook 5 (Intelligent Imaging Innovations).
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High-Resolution Imaging of Live Cells
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Other organizations : Boston Children's Hospital, Harvard University, The Ohio State University, Janelia Research Campus, Howard Hughes Medical Institute
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Variable analysis
independent variables
- None explicitly mentioned
- Three-dimensional time series
- Approximately 1 × 10^5 U373 cells were plated 16 h before imaging
- Imaging medium was phenol red–free DMEM supplemented with 10% FCS and 20 mM HEPES (pH 7.4)
- Coverslips were placed on a temperature-controlled 5% CO2 humidified chamber
- Imaging was performed on a Mariana imaging system based on an Axiovert 200M inverted microscope, a CSU-X1 spinning-disk confocal unit, a spherical aberration-correction device, and a 63× objective lens
- No positive or negative controls were explicitly mentioned in the provided information.
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