McClean: Quickie ImageJ Quantification
This is a quick and 'sloppy' way to segment cells and get an idea of their intensity. This is NOT really appropriate for carefully quantifying your data, but can at least give you an initial idea very quickly.
- FIJI ImageJ
Instructions for "quickie" segmentation of cells and estimation of intensity data:
- Import your sequence of interest
- File->Import->Image Sequence
- In "Sequence Options" -> "or enter pattern" type "c1" (or c2, or c3, whichever wavelength you want, you can also use regular expressions in this box)
- Threshold the images
- Hit "Apply"
- "Black Background" should be checked, hit "OK"
- Clean up the threshold image (the mask)
- Process->Binary->Fill Holes (to fill in any holes in cells, ie vacuoles)
- Process->Binary->Watershed (to break cells apart)
- Get "particles" (or cells in this case)
- Analyze->Analyze Particles
- Make sure "Add to Manager" is checked
- Hit "OK"
- Repeat step 1
- Select all the ROI's in the ROI Manager
- use shift, click the top ROI, then scroll to the bottom and click the last one
- Make sure measurements you want are checked
- Analyze->Set Measurements
- Hit "Measure" in the ROI manager
- Save the Results manu that pops up after you clicked "Measure"
- Close all windows (including the ROI manager and results) and start again
Dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.
- Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!)
- Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate.
- Positioning your agar plate on the stage is like flying a helicopter, not a jet. Landing, come over high, then descend. Taking off, go straight up, then depart. The needle stands higher than the platform!
- Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate.
- Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate.
- Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface).
- Pick tetrads.
- Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it.
- Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
- Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
- To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
- Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
- Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.
- Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.
- Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
- You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.
- Remove the YPD plate from the stage, taking care not to break the microneedle.
- Incubate the plate for 2-3 days at room temperature.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.