Fig 1.
Overview of the image analysis workflow.
The data set is exported as a Multi-Tiff file and analyzed using an algorithm developed in MATLAB. Image processing: The processing stage consists of green/red image separation, spatial and temporal noise filtering, background normalization and contrast enhancement. Image analysis: A migration map is generated automatically by classifying the signal variation of individual pixels over the time series. The user selects the location of the ROIs (a single mouse click for each), the regions are automatically created from these seed points and a graph of mean fluorescence of each ROI over time is automatically produced.
Fig 2.
A zoomed confocal image of a HeLa cell transiently expressing photo-converted Dendra2 (A) before and (B) after application of a median filter; (C) before and (D) after subtraction of the pre-conversion red image (background); (E) before and (F) after application of the Contrast Stretching Transform.
Fig 3.
A statistical model of the image sequence.
Construction of (A) the mean image M and (B) the standard deviation image S of a time series of a single HeLa cell transiently expressing photo-converted Dendra2.
Fig 4.
(A) Dendra2; (B) Dendra2UBC9; (C) & (D) Dendra2-Fibrillarin. The sub-cellular migration of a selection of Dendra2 fusion proteins from the PC region is described in their mean (left) and corresponding classification images (right). The key is as follows: White: accumulation; Light grey: diffusion towards; Dark grey: injection; Darkest grey: background; Black: unclassified. The initial PC region is highlighted with a cross.
Fig 5.
Comparison of automated selection methods for demarcating Regions of Interest (ROI) applied to the nucleoli.
(A) Nucleolar ROI selection using a seeded region growing algorithm; (B) Nucleolar ROI selection using a fixed area around a selected point.
Fig 6.
Automated analysis of Dendra2 sub-cellular trafficking in HeLa cells.
(A) User defined cellular ROIs using the seeded region growing algorithm. P1 is the PC point; P2 is a distant nucleoplasm region; P3 is a nucleolus and P4 is a region of cytoplasm. (B) Quantification of photo-converted Dendra2 fluorescence in user defined ROIs over time.
Fig 7.
Comparison of Dendra2UBC9 protein trafficking obtained for both AndorIQ2 and MATtrack software.
(A) The selected ROIs; (B) Dendra2UBC9 trafficking in HeLa cells analyzed using AndorIQ2; (C) The same sample analyzed using MATtrack; (D) Analysis using MATtrack with photobleaching compensation removed for direct comparison with AndorIQ2.