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
#*Image->Adjust->Threshold #*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
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
- Spin down 250 μL of sporulated culture.
- Resuspend in 250 μL sterile water.
- Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
- Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.)
- Gently add 100 μL water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
- Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).
- Mark the plate at opposite edges to indicate the center of the plate (see following diagram).
- Resuspend the digested cells from your chosen timepoint. by gently tapping or pipetting (they will probably have settled while you were looking at the digestions under the microscope). Drip 20 μl down the imaginary center line you just indicated, beginning from one edge.
- Let the digested culture fully absorb into the plate.
- Dissect (see dissection section). If you dissect on a different day, you must begin with a fresh plate and set up a new dissection. The sporulation cultures are good for weeks, though the spore viability may start to decrease.
Appearance of Tetrads
Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.
How to distinguish a tetrad from two budded cells adjacent to each other?
- Generally, the spores of a tetrad are smaller than a budded cell.
- Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
- The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.
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.