A1r point scanning confocal system

Images were acquired using a Nikon wide-field epifluorescence microscope, Nikon A1R confocal, and Yokogawa W1 spinning disk confocal. All microscopes were run using Nikon Elements software. The widefield microscope was an inverted Nikon Eclipse TiE system with a 120BOOST LED-based illumination system and equipped with a Photometrics HQ2 CoolSnap camera and motorized XY stage. The Nikon A1R point scanning confocal system was run on an inverted Nikon Eclipse TiE base with 405-, 488-, 568- and 647nm excitation laser lines and four detectors: two GaAsP and two Alkali PMTs with a motorized XY stage. The Yokogawa W1 spinning disk confocal has an inverted Nikon Eclipse TiE base and 100mW 405-, 490-, 561-, and 640nm lasers, equipped with an Andor iXon 888 Life EMCCD camera linked with a 10-position filter wheel and a motorized XY stage. The spinning disk system was enclosed in an environmental chamber with temperature and local [CO2] control.

FRAP experiments of GFP or RFP-tagged proteins, transiently expressed in RAW-Dectin1 cells, were conducted on an A1R point-scanning confocal system (Nikon Instruments, Japan). For FRAP, Candida-BFP hyphae-infected cells were imaged in HPMI at 37°C. Images were acquired using a 60x/1.4 NA oil objective (Nikon), 1.2-AU pinhole, resonant scanning mode, and 16x line averaging. For a complete 2 min FRAP acquisition at 1.9 fps, after 5 s of initial imaging, a region of interest 3 μm in diameter was bleached for 1.06 s using the 405 laser at 100% power, followed by imaging for fluorescence recovery. Images were exported and analyzed for fluorescence intensity using Volocity software.
After background subtraction, fluorescence intensity units were normalized (see Figure 6 legend) using Microsoft Excel software, and transformed to a 0–1 scale, to correct for differences in bleaching depth and allow for comparison of up to 30 individual FRAP curves per condition. Graphpad Prism software was used to fit the FRAP curves to a single exponential, plotted as fractional recovery over time.