Dictyostelium cells transformed with mCherry-myoII were plated on glass dishes. FCS experiments were performed on interphase cells at ambient temperature (∼20–23°C; normal growth temperatures for Dictyostelium cells) using a Zeiss AxioObserver with 780-Quasar confocal module and FCS, with a C-Apochromat 40× (NA 1.2) water objective. 100 nM Rhodamine 6G was used for pinhole alignment and structural parameter calculation. A structural parameter of six was used, and the confocal volume was measured to be 0.45 fL. Data were fit to a single-component 3D diffusion model, including a triplet-state component with an upper limit of 8 µs (Srivastava and Robinson, 2015 (link); Kothari et al., 2017 (link)).
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Fluorescence Correlation Spectroscopy of mCherry-myoII in Dictyostelium
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Corresponding Organization : Johns Hopkins University
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Variable analysis
independent variables
- MCherry-myoII transformation in Dictyostelium cells
- Diffusion dynamics of mCherry-myoII in interphase Dictyostelium cells
- Ambient temperature (∼20–23°C)
- Normal growth temperatures for Dictyostelium cells
- Zeiss AxioObserver with 780-Quasar confocal module and FCS
- C-Apochromat 40× (NA 1.2) water objective
- 100 nM Rhodamine 6G for pinhole alignment and structural parameter calculation
- Structural parameter of six
- Confocal volume of 0.45 fL
- Single-component 3D diffusion model with triplet-state component (upper limit of 8 µs)
- 100 nM Rhodamine 6G used as a positive control for pinhole alignment and structural parameter calculation
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