While microplate well imaging provides the ability to
determine single cell responses to stimuli, there is a limitation to viewing
the full cell population response. The
greater the magnification, the more the field of view is limited. It is possible to automate image capture resulting
in a series of images that can then be used to reconstruct the entire desired
field of view by stitching images, such that the complete well dimensions can
be assessed. This is true of applications in cell migration or cell
counting. In the case of the latter,
cell staining accompanied by a wash step to remove excess stain can be
problematic if the wash cycle is disruptive to the monolayer of cells adhered
to the microplate well surface. Recently, the need to determine the retention of
cells plated at several cell densities after being subjected to a rigorous
automated cell washing protocol was examined.
NIH3T3 cells with a GFP background were plated at densities of 2.5 and 5
K cells per well were plated and allowed to adhere overnight. The following day
the entirety of each well of the plates were imaged, both pre- and post-washing
and staining, using a 2X3 montage and a 2.5X objective which included a slight
overlap in the captured images. The captured images were then stitched together
using the ImageJ plugin, Stitching,
using the Grid/Collection stitching method1. Below is an example of 6 pre-processing
images captured using a Cytation 3 Cell Imaging Multi-Mode Microplate Reader
stitched using the above method.
The magnified region shown in the lower image is representative of the intersection of two upper-most images that have been stitched together. Several wells for each seeding density were examined in this way, both pre- and post-washing, to determine optimized automated washer/dispenser setting to assure no disruption of the adherent cell monolayer. Below is a comparison using automated cell counting at several cell densities both pre- and post-staining.
Keep an eye out for more detailed information regarding the methods used in future publications on the BioTek Technical Resources webpage: http://www.biotek.com/resources/.
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