Prater KE, Prater RD, Biggerstaff JP
3-D confocal laser-scanning microscopy is an ideal way to visualize a sample with a curved surface. An application of this method involves the quantification of activated blood leukocytes and platelets adhering to the surface of medical grade catheters. Human blood was perfused across treated and untreated catheters using a microperfusion system and fixed with paraformaldehyde. Portions of each catheter were immunostained and 5 random fields were imaged using a Leica SP2 microscope (20× objective; 2× optical zoom). Image stacks were loaded into image analysis software and transformed into a max intensity projection. From this 2-D projection, area occupied by fluorescence was calculated by implementing a binary threshold layer above isotypic control. Area fluorescence was divided by the mean area of a single cell, generating a cell count per unit area. Results are presented as adherent cells/mm2 ± SEM. The treated surface showed reduced adherent cell counts (monocytes: 0.67 ± 1.03, polymorphs: 1.33 ± 3.99, lymphocytes: 0.62 ± 1.86, platelets: 1.33 ± 3.99) compared to the untreated surface (monocytes: 302.43 ± 215.55, polymorphs: 1647.56 ± 615.54, lymphocytes: 38.46 ± 29.59, platelets: 4076.23 ± 2867.3). This method can be used to capture and quantify large amounts of data that may be obscured or otherwise unavailable when imaging curved surfaces, such as in biocompatibility assessment of medical devices.
Prater KE, Prater RD, Biggerstaff JP. 2012. 3-dimensional imaging and quantification of cell adherence on curved surfaces of medical grade devices using confocal laser-scanning microscopy. FASEB Journal 26.