Tube Radius Calculation
Liv Jensen
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Abstract
This protocol details Tube Radius Calculation.
Attachments
Steps
Fluorescence ratio calibration:
Measure the radius of the GUV and of the aspiration pipette from imaging data.
Calculate radius from:
In FIJI, create new image stacks from two rectangular selections: one containing a section of the membrane tube, and one containing the approximately horizontal section of the guv.
Save these new stacks as .tif files and paste paths into segmentation and quantification script.
Use the output of the segmentation and quantification script to calculate the ratio of fluorescence intensity of the membrane tube and the GUV surface (Itub / IGUV).
Plot tube radius as calculated in step 2 vs. the fluorescence ratio calculated in step 5. Perform a linear fit of the data, recording the slope, Ktub, for use in subsequent experiments.
Calculating tube radius with fluorescence images and Ktub:
In FIJI, create new image stacks from two rectangular selections: one containing a section of the membrane tube, and one containing the approximately horizontal section of the guv.
Save these new stacks as .tif files and paste paths into segmentation and quantification script.
Input Ktubas a parameter in the quantification function to normalize fluorescence ratio to tube radius.
