A Robust Co-Localisation Measurement Utilising Z-Stack Image Intensity Similarities for Biological Studies

Abstract

BackgroundCo-localisation is a widely used measurement in immunohistochemical analysis to determine if fluorescently labelled biological entities, such as cells, proteins or molecules share a same location. However the measurement of co-localisation is challenging due to the complex nature of such fluorescent images, especially when multiple focal planes are captured. The current state-of-art co-localisation measurements of 3-dimensional (3D) image stacks are biased by noise and cross-overs from non-consecutive planes.MethodIn this study, we have developed Co-localisation Intensity Coefficients (CICs) and Co-localisation Binary Coefficients (CBCs), which uses rich z-stack data from neighbouring focal planes to identify similarities between image intensities of two and potentially more fluorescently-labelled biological entities. This was developed using z-stack images from murine organotypic slice cultures from central nervous system tissue, and two sets of pseudo-data. A large amount of non-specific cross-over situations are excluded using this method. This proposed method is also proven to be robust in recognising co-localisations even when images are polluted with a range of noises.ResultsThe proposed CBCs and CICs produce robust co-localisation measurements which are easy to interpret, resilient to noise and capable of removing a large amount of false positivity, such as non-specific cross-overs. Performance of this method of measurement is significantly more accurate than existing measurements, as determined statistically using pseudo datasets of known values. This method provides an important and reliable tool for fluorescent 3D neurobiological studies, and will benefit other biological studies which measure fluorescence co-localisation in 3D.

Statistical analysis of molecule colocalization in bioimaging

“…Thus, we confirmed that pixel randomization leads to false positive colocalization compared to spot randomization . Finally, we underline that spot randomization is based on synthetic images, and requires to define and estimate image model parameters (noise, intensity, number of spots; ).…”

Influence of the chirality of carbon nanodots on their interaction with proteins and cells

“…Afterwards, the cells were washed with 200 μL PBS once and 300 μL of fresh pre-warmed RPMI medium without phenol red was added prior to imaging by confocal microscopy using z-stack analysis (Supplementary Figs. 67 and 68 ) 80 . A laser diode emitting at 405 nm and a bandpass emission filter BP 420–480 nm were used for the excitation and emission collection of CNDs.…”

“…Both scenarios however can be distinguished using confocal microscopy. To confirm that the majority of fluorescence signal from CNDs associated with cells originates from internalized CNDs z-stacks were recorded 80 with CLSM (LSM 510, Zeiss, Germany) with a Pla N- Apochromat ×63/1.40 Oil DIC M27 objective. Herein, Calcein - AM (Molecular Probes) was applied to label living cells (i.e.…”

Streptococcus pneumoniae Invades Endothelial Host Cells via Multiple Pathways and Is Killed in a Lysosome Dependent Manner

“…Of each cell with clear intracellular S. pneumoniae, individual pictures, and Z-stack series (green, red, and grey phase contrast) were taken with a Leica DM IRE2 inverted confocal microscope setup, and subsequently analyzed with ImageJ and its co-localization plug-in function (http://rsb.info.nih.gov/ij/). For the representative image a XY plane merger was created as well as 2 orthogonal views of the YZ and XZ plane [13] were also generated with ImageJ for illustrative purposes.…”