Improved region of interest selection and colocalization analysis in three-dimensional fluorescence microscopy samples using virtual reality

Abstract

Although modern fluorescence microscopy produces detailed three-dimensional (3D) datasets, colocalization analysis and region of interest (ROI) selection is most commonly performed two-dimensionally (2D) using maximum intensity projections (MIP). However, these 2D projections exclude much of the available data. Furthermore, 2D ROI selections cannot adequately select complex 3D structures which may inadvertently lead to either the exclusion of relevant or the inclusion of irrelevant data points, consequently affecting the accuracy of the colocalization analysis. Using a virtual reality (VR) enabled system, we demonstrate that 3D visualization, sample interrogation and analysis can be achieved in a highly controlled and precise manner. We calculate several key colocalization metrics using both 2D and 3D derived super-resolved structured illumination-based data sets. Using a neuronal injury model, we investigate the change in colocalization between Tau and acetylated α-tubulin at control conditions, after 6 hours and again after 24 hours. We demonstrate that performing colocalization analysis in 3D enhances its sensitivity, leading to a greater number of statistically significant differences than could be established when using 2D methods. Moreover, by carefully delimiting the 3D structures under analysis using the 3D VR system, we were able to reveal a time dependent loss in colocalization between the Tau and microtubule network as an early event in neuronal injury. This behavior could not be reliably detected using a 2D based projection. We conclude that, using 3D colocalization analysis, biologically relevant samples can be interrogated and assessed with greater precision, thereby better exploiting the potential of fluorescence-based image analysis in biomedical research.

Encapsulation of Plasmid DNA by Nanoscale Metal–Organic Frameworks for Efficient Gene Transportation and Expression

“…Furthermore, through z ‐axis scanning at different sections by CLSM (Figure S15, Supporting Information), 3D reconstructed models based on the intracellular fluorescence intensity of blue, green, and red markers were applied to reveal the 3D distribution of the nanostructures inside the cells and to compare their lysosome escape ability after 12 h incubation via 3D colocalization analysis, more reasonable and precise manner than 2D projections . In Figure S15g1,g2 in the Supporting Information, the green fluorescence overlapping with red fluorescence surrounds and encapsulates the blue ones in three dimension, clearly showing that the pEGFP‐C1 could be positioned and released inside the entire structure of cells via the delivery of ZIF‐8 or ZIF‐8‐PEI 25 kD vectors.…”

Accuracy and Reproducibility of Linear and Angular Measurements in Virtual Reality: a Validation Study

“…The advanced visualization in the immersive view creates a high-fidelity virtual experience in which the user can interact with multi-dimensional objects and perceive relationships at their true scale in the same space. The VR nature helps users to identify critical features by applying scientific judgment, and instant manual measurement can be carried out on the identified area of interest [8,9]. The physical extent, alignment, separation, and other dimensional properties become much more perceptible as the user maneuvers through the virtual space in the immersive environment [6,10].…”

Colocalization Analysis of Cytoplasmic Actin Isoforms Distribution in Endothelial Cells

“…On the other hand, the choice of the correlation coefficient can seriously affect the results [ 18 , 19 ]. In a number of cases, the accuracy of ROI selection is extremely important (in particular, in some cases, it is essential to choose a three-dimensional region of interest, 3D ROI) [ 20 ]. In addition, although for flat endothelial cells with thin lamellae, this choice of ROI does not seem to be so fundamental, it should be borne in mind that in endothelial cells growing in a dense confluent monolayer, thickness of the cytoplasm near the cell nuclei can significantly exceed the thickness of the lamellae.…”