SIM imaging

Cells were fixed for 15 min in 4% paraformaldehyde (Ted Pella) in 100 mM sucrose and then washed two times in phosphate-buffered saline, pH 7.4. An aliquot of cells was placed on a glass slide and covered with a number 1.5 coverslip. SIM images were acquired with an Applied Precision OMX Blaze (GE Healthcare). A 60× 1.42 NA Plan Apo oil objective was used, and emission was collected onto two PCO Edge sCMOS cameras (Kelheim, Germany) with each camera dedicated to one specific channel. For CFP/mTurqouise2/YFP experiments, a 440/514/561 dichroic was used with 460–485 nm and 530–552 nm emission filters for CFP/mTurqouise2 and YFP, respectively. The 440 nm line and 514 nm line were used for excitation of CFP/mTurqouise2 and YFP, respectively. For GFP/mCherry experiments, a 405/488/561/640 dichroic was used with 504–552 nm and 590–628 nm emission filters for GFP and mCherry, respectively, using a 488 nm laser line (GFP) and 561 nm laser line (mCherry). To limit spectral cross-talk, all SIM data was acquired in alternating excitation mode. SIM reconstruction was performed with the Applied Precision software Softworx with a Wiener filter of 0.001. Color alignment from different cameras in the radial plane was performed using the color alignment slide from GE Healthcare (Pittsburg, PA). In the axial direction, color alignment was performed using 100 nm tetraspeck beads (Life Technologies, Kelheim, Germany). For image preparation, the SIM reconstructed images were scaled 2 × 2 with bilinear interpolation then smoothed with a Gaussian blur of pixel radius 0.8. In many cases, for illustration purposes, a max projection in z over the relevant slices was done.

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