Love:Protocols:Counting

Cell Counting (in the metamorph era) [Cut]

Staining

Scan slide

   1.place slide
   clean a glass slide thoroughly with ethanol; let dry and then carefully place the grid on the slide (open wells facing down).
   leave some PBS after the last wash on top of the grid, so that the seal is uniform and air is not trapped
  
   place glass slide on microscope
   use fast transmission settings (TRANS fast - transmission light; 2ms exp time; live) to find a rough focus
   make sure grid is slightly angled, so that the top-left corner of any given block is higher in the image than the top-right corner
      -to move the grid around, take advantage of the thin liquid layer that should be left and tap lightly the grid to move it.

   launch 'scan slide' application.
   set binning to 1 and wavelengths to 1+#of wavelengths to scan for the setup process.
    Transmission is used as the additional wavelength for calibration and alignment.

   2.adjust alignment
   move to right end top position and adjust focus  
   use dapi for the actual focus as fluorescence and transmission plane may not be confocal
      - use live DAPI mode to adjust focus: set z= 0 in the software; adjust z height on microscope, then if necessary fine tune it in the software;
        set the z position in order to obtain the highest values in the image histogram
   keep z position on microscope and software constant from now on.
   move to right end bottom position and adjust the focus (always in DAPI live mode), this time using the corresponding alignment screw on the slide holder.
   check alignment of the right end by scrolling up and down
   move to left end, center of the slide, and adjust focus using the corresponding alignment screw

   3.check illumination settings
   move to a random position in the center of the grid
   adjust illumination settings for all channels; take snaps and try minimizing the number of repetitive exposures
   to reduce scan time: keep the exposure time to the minimum necessary in every channel
   to improve image recognition: use exposure time that gives the best spread in the image histogram
   usually there is a threshold exposure, above which the improvement in image quality is not worth the increase in overall time

   4.Calibration
  snap a transmission picture (to make sure calibration process will be performed using this wavelength)
   start step by step calibration procedure following software instruction.
   pick a corner of one of the blocks as feature, in order to easily individuate it. Don't use wells alone, as it may be confusing later on.

   5. Set scan area
   using transmission live mode, go to outermost corner of PDMS (not only the corner of the features, but further on)
   make sure that you are really on outermost corner by scrolling a little around the edges both in the horiz and vert directions
   set first corner position to current
   repeat for the bottom right corner, paying attention to exagerate(!) the extent of your area. metamorph tends to scan a smaller area than the one inputted.

   6.start scanning
   set binning to 2, reduce wavelengths to #of wavelengths to scan
   set description and directory for file save. every single scan attempt should be in a different directory.
   cycle once more through all illumination settings to check focus and exp times.
   press 'scan' button

once done (usually ~3hrs), backup all files before proceeding

Counting algorithm - step 1 - parse into blocks

   in MM offline, open scan to be parsed.
   then open journal for step 1, making sure all references to files are respected (sometimes copies on the local and network drives may conflict)
   run journal
   save temp file to directory where all parsed blocks should be saved
   align grid to DAPI Fast Scan preview image (keeping the magenta feature on the bottom right corner)
   click on 'run journal for every grid posn'
   nb: metamorph usually crashes on the last journal loop; make sure all 52 blocks have been saved in the directory, then end program and restart.

Counting algorithm - step 2 - align regions

   open directory where parsed blocks are saved
   open journal for step 2
   run journal  (make sure all journals are on a local drive, for best performance)
   adjusting angle and size, align 40x40 grid with the features in the DAPI image
   press 'run journal for each grid posn'
   repeat for every block
  

backup region files before proceeding

Counting algorithm - step 3 - counting and logging cells

   using randomly selected files for all channels, calibrate settings for 'multiwavelength cell scoring' application
   after repeating for several blocks, save settings
   open journal for step 3
   go to line of code where multiwavelength cell scoring is called;
   edit settings for command: load settings you just calibrated
   save journal
   run journal

Check

   by checking the log numbers, the original tiffs for the blocks and comparing with the segmentation images in the directory control if results make sense and seem reliable
   use journal for step 4 and input number of blocks for which count has to be repeated
   recall to save in separate logs

import data into MATLAB

   open journal for step 3