Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy

Abstract

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+ and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms the dynamic range that could be detected by the software packages was 1 x 10 6 -1 x 10 9 cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our 'Region of Interest' method, with the software's 'Standard Area Method' of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By preincubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250ug/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells.

MYC Can Induce DNA Breaks In vivo and In vitro Independent of Reactive Oxygen Species

“…Fluorescent images of f100 cells from multiple fields per sample were viewed under a 20Â objective using a FITC filter in a Confocal Microscope (Nikon, PCM2000, with a Spot Camera) and quantitated using Metamorph software (Universal Imaging Corp., Downingtown, PA; refs. 32,33).…”

Integrin β1 Regulates Phagosome Maturation in Macrophages through Rac Expression

“…The percentage of colocalization between these two colored markers (weighted for integrated brightness) within each cell was quantitatively analyzed by Metamorph offline software (27,28). Whereas the percentage of E. coli colocalized with Lysotracker Red reached 42% in WT macrophages at 1 h, colocalization was Ͻ20% in integrin ␤ 1 -deficient macrophages at this time point (Fig.…”

Different binarization processes validated against manual counts of fluorescent bacterial cells

“…In an effort to standardize results, digital images of fluorescent labelled organisms or structures can be processed by (semi-)automated image analysis software packages (Grivet et al, 1999;O'Mahony et al, 2005;Selinummi et al, 2005;Thiel and Blaut, 2005). In this automatization process a grayscale image is converted into a binary (two possible pixel values, normally black and white) image.…”

“…However, the application of an automated thresholding algorithm to discriminate the background (white; value = 0) from the objects of interest (black; value =1)) is challenging. Therefore, the use of a fixed threshold is accepted in current software approaches (O'Mahony et al, 2005). Some authors report the use of an empirically obtained fixed threshold based on their own reference dataset (Schönholzer et al, 1999;Thiel and Blaut, 2005).…”