Cell Imaging Techniques

Abstract

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Development and validation of an alternative parameter for quantification of signals emitted by fluorescently labelled bacteria in microscopic images

“…Fluorescence signals of stained bacterial cells are usually measured using mean grey value of objects (MGV) (Poulsen et al, 1995;Poulsen et al, 1993), mean grey value of objects corrected with the background (MGVcwB) (Strack et al, 2013) or the signal to background ratio (SBR) (Fuchizawa et al, 2008;Waters and Swedlow, 2007). Since these parameters are known to depend considerably on the conditions at which the experiments are performed (Webb and Brown, 2013) and on the background fluorescence signal (Waters, 2009;Waters and Swedlow, 2007), another parameter, the coefficient of variation (CV), was investigated for its robustness and suitability in the analysis of fluorescent signals obtained from bacterial cells in microscopic images.…”

“…The parameters MGVcwB and SBR are also dependent on the fluorescence signal of the image background. The increased fluorescent signal of the image background may be caused by non-specific binding of the fluorescent label to molecules in the matrix, incorrect protocol implementation, or improper equipment handling (Bouvier and del Giorgio, 2003;Webb and Brown, 2013). These fluctuations in MGVcwB and SBR measurements will directly affect the comparability of the fluorescent signals of bacterial cells between, or within experimental studies (Daims and Wagner, 2007;Pang et al, 2012;Waters, 2009;Waters and Swedlow, 2007;Zhou et al, 2007).…”

Development of Quantitative Tools for the Characterization and Analysis of the Blood Brain Barrier in Normal and MPS IIIB Mice

“…Epifluorescence microscopy is the standard form of imaging for most immunofluorescence image analysis 39,40 . In this method, a light source such as a mercury arc lamp is used to excite the fluorophores in the tissue sample.…”

“…Light that is emitted from the sample's excited fluorophores is then directed upward toward the objective lens and detector. The emitted light passes back through the dichroic mirror and a barrier filter which blocks the shorter wavelengths of the excitation light from reaching the detector [39][40][41] . The result of this method of microscopy is a 2D image of a single focal plane containing signal only from the fluorophores excited in the tissue sample.…”

“…While most of the light is redirected to the focal point of the detector out of focus light from the surrounding excited tissue area still reaches the detector and creates a haze across the image. In higher objectives and with thicker tissues the amount of out of focus light increases making it difficult to obtain a focused image 39,40,43 . Additionally, epifluorescence microscopes cannot focus through multiple planes limiting the field of view of the images acquired to a single focus plane as well as increasing the amount of out of focus light in the image 39,43,44 .…”