TIRFM images were acquired by an Olympus IX-81 microscope equipped with a TIRF port, an Andor iXon+ DU-897D electron-multiplying charge-coupled device camera, and an Olympus 100× 1.49 NA objective lens. The acquisition was controlled by MetaMorph software (Molecular Devices). For the imaging options, the exposure time was 100 ms for a 512 × 512–pixel image unless specially indicated. Confocal images were acquired by a FLUOVIEW FV1000 confocal laser scanning microscope (Olympus) with a 60× 1.42 NA oil objective lens. All the images were acquired while keeping the cells in PBS unless specially indicated and were confirmed not to overexpose by the software. All the images were analyzed and processed with ImageJ (National Institutes of Health). The MFI and the total fluorescence intensity (total FI) as AU of BCRs, lipid biosensors, and signaling molecules were calculated based on the intensity analysis as described previously (Lakadamyali et al., 2004 (link); Liu et al., 2010b (link),c (link), 2012 (link)).
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Quantifying Membrane Protein Dynamics
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Corresponding Organization : Tsinghua University
Other organizations : Chengdu Institute of Biology, Institute of Biophysics, Chinese Academy of Sciences, University of Science and Technology of China, Zhejiang University
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Variable analysis
independent variables
- Microscope setup
- Imaging options
- Mean fluorescence intensity (MFI) of BCRs, lipid biosensors, and signaling molecules
- Total fluorescence intensity (total FI) of BCRs, lipid biosensors, and signaling molecules
- Cells were kept in PBS during imaging unless specially indicated
- Images were confirmed not to overexpose by the software
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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