Taylor C. elegans Lab: Image Quantification Analysis Protocol
Image Quantification Analysis Protocol
- This protocol is to establish the light densities of images collected by fluorescence microscopy and their significance. This is most often done as part of the plate controls in a RNAi experiment (L4440 empty vector against GFP).
For general analysis:
- Image files in .tif format
- ImageJ software: http://rsbweb.nih.gov/ij/download.html
- spreadsheet software
- online software: http://graphpad.com/quickcals/ttest1.cfm
- sigmaplot may be helpful for generating graphs
- Open first image in ImageJ from computer
- In ImageJ goto Edit -> Specify -> Width: 250 Height: 600
- Edit -> Specify -> Rotate -> enter # of degrees to rotate until phylangeal bulb is aligned and centered with box
- Analyze -> Measure, data will collect in a separate window
- Use Edit -> Open Next to open the next file
- Repeat with rest of images
- Move data to spreadsheet
- Enter data according to instructions into the graphpad t test calculator
- Make graph
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- The goal in imaging is for all the images to be placed the same
- If the worm's til is interfering in the box, use the color selector tool to select the background then use that color to pint over unwanted lit areas with the paint brush tool (double click to change stroke size). Click and hold to draw.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Elena Vayndorf
or instead, discuss this protocol.