Using Image Stitching to Analyze Adherent Cell Retention following Rigorous Automated Cell Washing

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/.

¹ S. Preibisch, S. Saalfeld, P. Tomancak (2009) "Globally optimal stitching of tiled 3D microscopic image acquisitions", Bioinformatics,25(11):1463-1465. Webpage PDF



By: BioTek Instruments, Peter J. Brescia, Jr., MSc, MBA, Applications Scientist