Automated analysis of images for molecular quantification in immunohistochemistry

Abstract

The quantification of the expression of different molecules is a key question in both basic and applied sciences. While protein quantification through molecular techniques leads to the loss of spatial information and resolution, immunohistochemistry is usually associated with time-consuming image analysis and human bias. In addition, the scarce automatic software analysis is often proprietary and expensive and relies on a fixed threshold binarization. Here we describe and share a set of macros ready for automated fluorescence analysis of large batches of fixed tissue samples using FIJI/ImageJ. The quantification of the molecules of interest are based on an automatic threshold analysis of immunofluorescence images to automatically identify the top brightest structures of each image. These macros measure several parameters commonly quantified in basic neuroscience research, such as neuropil density and fluorescence intensity of synaptic puncta, perisomatic innervation and col-localization of different molecules and analysis of the neurochemical phenotype of neuronal subpopulations. In addition, these same macro functions can be easily modified to improve similar analysis of fluorescent probes in human biopsies for diagnostic purposes based on the expression patterns of several molecules.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

“…Visualization and quantification of fluorescently labeled cells are important for studying human developmental biology . However, accurate analysis of fluorescent signals in images is often cofounded by autofluorescence and poor image segmentation .…”

Dark exposure affects plasticity‐related molecules and interneurons throughout the visual system during adulthood

“…On these planes, four small areas of the neuropil (300 μm 2 each) were selected for analysis, in order to avoid blood vessels and cell somata. Images were processed using different customized macros as previously described (Guirado, Carceller, Castillo‐Gómez, Castrén, & Nacher, ). In short, noise was reduced using a Gaussian blur filter and images were binarized, creating a mask containing the 2% brightest pixels; the outline of this mask was used in the original image to measure the number and fluorescence intensity of puncta.…”

“…Images were processed using different customized macros as previously described (Guirado, Carceller, Castillo-Gómez, Castrén, & Nacher, 2018).…”

<p>Role of the CXCR4/ALK5/Smad3 Signaling Pathway in Cancer-Induced Bone Pain</p>

“…The relative mean fluorescence intensity (MFI) for ALK5 immunofluorescence in the entire superficial dorsal horn, including 16 spinal cord sections from four rats (4 sections from each rat from each group) was measured using ImageJ software. 1,25 The fold change associated with the control group was set at 1.0 for quantification.…”