Softworx deconvolution package

HeLa cells were fixed and imaged with a 100× oil immersion objective (1.4 numerical aperture) on a DeltaVision deconvolution microscope (Applied Precision, Issaquah, WA) and a CoolSNAP HQ² camera (Photometrics, Tucson, AZ). Single plane, widefield images were collected for DIC, FITC (GFP/Venus), and TRITC (mCherry). Images were collected at 512 × 512 pixels. Exposures were typically 0.2 to 0.5 seconds and were sufficient to achieve a minimum 5:1 signal-to-noise ratio. Individual raw files were deconvolved using Applied Precision Softworx deconvolution package. For each GFP-Rab11-FIP2 construct, 10 cells were imaged. These cells were chosen based on a low expression level of GFP-Rab11-FIP2. Images were then deidentified and scored blindly. Images were scored as to the colocalization of GFP-Rab11-FIP2 with Cherry-MYO5B tail as Colocalized, Partial, or Separate. These distributions were plotted as a bar graph. Distributions and Chi Square analyses were performed with PRISM software for each pair.

HeLa cells were fixed and imaged with a 100× oil immersion objective (1.4 numerical aperture) on a DeltaVision deconvolution microscope (Applied Precision, Issaquah, WA) and a CoolSNAP HQ² camera (Photometrics, Tucson, AZ). Single plane, widefield images were collected for DIC, FITC (GFP/Venus), and TRITC (mCherry). Images were collected at 512 × 512 pixels. Exposures were typically 0.2 to 0.5 seconds and were sufficient to achieve a minimum 5:1 signal-to-noise ratio. Individual raw files were deconvolved using Applied Precision Softworx deconvolution package. Colocalization was quantified as Pearson’s Correlation Coefficient using the colocalization feature in Softworx, which measures a 3D volume. There was a statistically significant difference between groups (Rab4, Rab5, Rab8a, Rab14, and Rab11-FIP5) as determined by one-way ANOVA (F(5,42) = 2.412, p = .05).