The microscopy setup consisted of an epifluorescence microscope (Ti-E, Nikon), an objective lens (40× CFI Plan Apo Lambda, 0.95 NA, Nikon), a relay optics box for dual-color imaging (GA03; G-Angstrom, Japan), and an electron multiplier-type CCD camera (EM-CCD, iXon DV887 or DU897; Andor Technology PLC, UK). A slit was placed at the imaging surface of the microscope in the relay optics. A dichroic mirror (FF458-Di02, Semrock) located just outside the slit split the optical pathway after the imaging surface into two pathways for cyan (CFP) and yellow (YFP1G) fluorescence. The two pathways converged on the acceptance surface of the EM-CCD camera side by side. Band-pass filters were set for each pathway (467–499 nm for CFP and 510–560 nm for YFP). Multiphoton fluorescence recovery after photobleaching (MP-FRAP) experiments were conducted as described elsewhere [17 (link)]. Image analyses were carried out in the ImageJ software (NIH, USA).
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Dual-color FRAP Microscopy Setup
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Other organizations : Quantitative BioSciences, Tokyo Medical University, Waseda Bioscience Research Institute in Singapore
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Variable analysis
independent variables
- Multiphoton fluorescence recovery after photobleaching (MP-FRAP) experiments
- Fluorescence recovery
- Epifluorescence microscope (Ti-E, Nikon)
- Objective lens (40× CFI Plan Apo Lambda, 0.95 NA, Nikon)
- Relay optics box for dual-color imaging (GA03; G-Angstrom, Japan)
- Electron multiplier-type CCD camera (EM-CCD, iXon DV887 or DU897; Andor Technology PLC, UK)
- Slit placed at the imaging surface of the microscope in the relay optics
- Dichroic mirror (FF458-Di02, Semrock) located just outside the slit
- Band-pass filters set for each pathway (467–499 nm for CFP and 510–560 nm for YFP)
- Multiphoton fluorescence recovery after photobleaching (MP-FRAP) experiments were conducted as described elsewhere [17].
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