Immunofluorescence microscopy was performed as previously described61 (link),62 (link) on a Zeiss LSM710 microscope (Carl Zeiss, Thornwood, NY). Live-cell imaging was conducted with a NIKON Eclipse Ti Microscope System equipped with an environmental chamber (temperature controlled at 37°C and CO2 at 5%), high-speed EM charge-coupled device cameras (iXon DU897 from Andor and Evolve 512 from Photometrics), and NIS-Elements Ar Microscope Imaging Software. TIRF Images were acquired with an Apo TIRF 100X objective (N.A. 1.49) and iXon DU897. Spinning-disk confocal z-stacks were taken with a Plan Apo VC 60x H objective (N.A. 1.40) and Evolve 512. Dual-color imaging was achieved by fast switching excitation lasers so that images from green and red channels were aligned automatically.
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Advanced Microscopy Techniques for Live-Cell Imaging
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Other organizations : National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development
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Variable analysis
independent variables
- Immunofluorescence microscopy
- Live-cell imaging
- Microscopic observations
- Temperature (37°C)
- CO2 (5%)
- Objective lenses (Apo TIRF 100X, Plan Apo VC 60x H)
- Cameras (iXon DU897, Evolve 512)
- Microscope systems (Zeiss LSM710, NIKON Eclipse Ti)
- Imaging software (NIS-Elements Ar)
- Previously described protocols in references 61 and 62
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