MOSAIC (MOdels, Simulations, and Algorithms for Interdisciplinary Computing) , the image-processing algorithms developed at the MOSAIC Group for fluorescence microscopy , are available as plugins for the popular free image processing software ImageJ.
microManager (also called μManager) is a ImageJ Plugin for control of automated microscopes. It lets you execute common microscope image acquisition strategies such as time-lapses, multi-channel imaging, z-stacks, and combinations thereof. μManager works with microscopes from all four major manufacturers (Leica, Nikon, Olympus and Zeiss), most scientific-grade cameras and many peripherals (stages, filter wheels, shutters, etc.) used in microscope imaging.
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Fluorescent images of brain sections and astrocyte cultures were captured using a confocal microscope (Olympus FluoView FV1200) at either 10 or 60 magnification. For immunohistochemistry, slices chosen for analyses were anatomically-matched between comparing groups, and included samples from rostral to caudal regions. All images were converted to grey-scale and normalized to background staining. Fluorescently-labeled cells were quantified in specifically-defined regions of the cortex using the ITCN plugin for ImageJ ( ), where fixed parameters of cell width and threshold are pre-set such that only cells reaching the minimum signal will be counted. For Cux1 and Ctip2, regions of interest (ROIs) were positioned over cortical regions with each ROI further subdivided into eight equal bins from the pia to the inner border of the cortex, to assess neuron distribution across the layers of the cortex (Fig. 2a) [33]. The distribution was expressed as a percentage of the numbers of labeled-cells in each bin divided by the total numbers within each ROI [33]. In addition, fluorescent signal intensities of myelin staining and GFAP immunoreactivity were measured by mean grey values (ImageJ). Similarly with immunocytochemistry on astrocytes, the cell perimeter was first outlined and mean grey values of GFAP fluorescence, as well as surface area were determined as previously described. All image-capturing and threshold parameters were kept the same for each measurement between comparing groups.
FHFL and FL designed the study. FHFL prepared all cells and brain tissues, performed all immunofluorescent experiments, image acquisition, data analysis and manuscript writing. FHFL and TKYL performed Western blots and data analysis. PS helped with breeding and maintaining Fmr1 KO mouse colony. FL supervised all projects and contributed in writing the manuscript. All authors read and approved the final manuscript.
a Adhesion of GFP-expressing EcN to Caco-2:HT29-MTX co-cultures (3:1): 1, 2 ImageJ visualizations of inoculated GFP-expressing bacteria, 3 Inverted image, 4 Automated cell counting method in ImageJ (using the ITCN plug-in). b Inhibition of EcN adhesion to Caco-2/HT29-MTX co-cultures. The bacteria were incubated with the co-culture for 1 h in the presence of 5 mg/mL of FRU, SUC, FOS, or INU. Bars with different letters indicate the significant difference for each column (p
We used both the automated and manual cell counting methods to eliminate possible bias. The manual cell counting method, a plate count method involving serial dilution, is commonly used for cell counting. However, one of the major disadvantages of the manual cell counting method is that more than one colony can grow from one bacterium and bacteria can be lost during serial dilution and harvesting processes (i.e., when attached bacteria are scraped off culture plates), while the automated cell counting method requires no additional procedures such as serial dilution as well as staining method. Such differences in methods may explain the discrepancy of results. In addition, practically speaking, automated cell counting methods allow high-throughput screening which is not achievable with manual counting methods albeit it was not the case for the current study. To take an example, Navarro et al. utilized the automated image-based screening method for the identification of small molecules against targeted bacteria in vitro (Navarro et al. 2013). For those reasons, the automated method is widely accepted as a useful alternative of manual counting method.
Area selected for densitometry study. Representative image from WT mice showing Iba1+ staining after perforant pathway transection. The selected area (black squares) represented the area analyzed for densitometry. Scale bar = 50μm.
CD206 and Laminin expression after PPT. (A) Representative images from WT and GFAP-IL6Tg mice showing CD206 staining in the ML of the DG at 7 dpl. Black arrows indicate CD206+ cells. Scale bar = 20μm. (B) Representative images, from WT and GFAP-IL6Tg mice, of double IHC combining MHCII (red) and Laminin (green) at 7 dpl. White arrows indicate MHCII+ cells in the perivascular space. Scale bar = 10μm
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