Jul 25, 2022
Tube Radius Calculation
- 1Hurley Lab
Protocol Citation: Liv Jensen 2022. Tube Radius Calculation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9y3rqg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61846
Keywords: Tube Radius Calculation, Fluorescence ratio calibration, ASAPCRN
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Abstract
This protocol details Tube Radius Calculation.
Attachments
416-898.pdf
42KB
Materials
Materials:
- Image stack of multichannel confocal fluorescence images
- Pressure data over time for GUV aspiration pipette
- Force data over time for bead held in optical trap
- FIJI
- Image segmentation and quantification script (Python)
Fluorescence ratio calibration:
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:
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 Ktub as a parameter in the quantification function to normalize fluorescence ratio to tube radius.