Colocalization in Confocal Microscopy. Quantitative Colocalization Analysis

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1 Quantitative Colocalization Analysis 1

2 Quantitative Colocalization Analysis Colocalization of fluorescently labeled molecules (immunostainings, fluorescent proteins, etc.) is often used as the first indicator of functional interaction HEp-2 cells (Hemmerich, Jena, Germany) (acbp/p300, arna-polymerase II, DAPI) HEK cells (Böhmer, Jena, Germany) (free GFP, YFP_SHP1 fusion) Zebrafish (Bastmeyer, Konstanz, Germany) (Two different neuronal markers) 2

3 Quantitative Colocalization Analysis Coincidence of two or more fluorescent signals inside the same detection volume Intensities (and position) of the signals inside the detection volume may vary Prerequisites 1. Acquisition of crosstalk-free images 2. Identical size and position of detection volumes for different color channels 3

4 Have look inside... Innovative detection architecture - single and META-detectors Real confocal architecture - multiple pinhole concept equal sections with multiple pinhole concept unequal sections with single pinhole 4

5 Multiple pinhole concept The thickness of the confocal sections varies with: (1) objective (num. aperture) (2) size of the confocal pinhole (3) wavelength of the light Single click for optimal pinhole settings of all channels! Dyes Pinhole Optical slice DAPI, Hoechst 1 Airy unit 0.7 µm egfp, FITC, Alexa488 1 Airy unit 0.9 µm DsRed (RFP), Cy3, Rhod 1 Airy unit 1.0 µm Cy5 1 Airy unit 1.1 µm Dyes Pinhole Optical slice DAPI, Hoechst 137 µm 1 µm egfp, FITC, Alexa µm 1 µm DsRed (RFP), Cy3, Rhod 132 µm 1 µm Cy5 128 µm 1 µm 5

6 Quantitative Colocalization Analysis Colocalization is often indicated by the color overlay (green + red = yellow) Several factors (exposure time, brightness, contrast etc.) render these overlays problematic??? HEp-2 cells (Hemmerich, Jena, Germany) (acbp/p300, arna-polymerase II, DAPI) HEK cells (Böhmer, Jena, Germany) (free GFP, YFP_SHP1 fusion) Zebrafish (Bastmeyer, Konstanz, Germany) (Two different neuronal markers) 6

7 Quantitative Colocalization Analysis Scattergram Image Window Tool Box Quantitative Colocalisation Data 7

8 Scattergram Pixel-wise Correlation of Image Channels 8

9 Quantitative Colocalization Analysis Interactive link between scattergram and image display - Select ROIs in scattergram and view corresponding pixels in image display Free color assignment for pixels in ROI (RGB or overlay) 9

10 Quantitative Colocalization Analysis Interactive link between scattergram and image display - Select ROIs in image display and view corresponding pixels in scattergram 10

11 Quantitative Colocalization Analysis Scattergram and thresholding with crosshair - Use the crosshair tool for setting intensity thresholds in scattergram Crosshair Scatterregion 2: Pixels in channel 2 only Scatterregion 3: Colocalizing pixels Scatterregion 4: Sub-threshold pixels (background intensities) Scatterregion 1: Pixels in channel 1 only 11

12 Quantitative Analysis Set Background Threshold Positioning of the Cross at mean intensity value of image ROI plus two time the standard deviation for each cannel 12

13 Cross-Hair Function Clear Separation of Colocalization 13

14 Quantitative Colocalization Analysis Interactive link between scattergram, image display and data table - View quantitative parameters for up to 99 individual ROIs No. of pixels Area / relative area Mean intensities / SD Colocalization coefficients Weighted colocalization coefficients Overlap coefficient after Manders Correlation coefficients (r and r 2 ) - Export data - Save and reload threshold settings (scatter ROIs and crosshair) for use with multiple images Scattergram Save and reload of threshold settings Data table 14

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16 Channel 2 Colocalising pixels = 4 Total number pixels in Ch1 = 6 = 0,66 Channel 1 16

17 Channel Intensities of colocalising pixels = 90 Total intensities of pixels in Ch1 = 105 = 0,85 Channel 1 17

18 The nominator brings in a significant value only when the intensities of Ch1 and Ch2 belong to a pixel of one of the colocalizing objects. Thus, the nominator is propotional to the number of colocalizing objects. Likewise, the denominator is proportional to the number of colocalizing and non-colocalizing objects in both components of an image. Advantage: the overlap coefficient is not sensitive to differences in signal intensities that could result from different fluorochrome concentration, photo-bleaching or detector settings. 18

19 R=-1 R=-0 R=1 If intensity of A is high then intensity of B is low. Example: Expression of protein A suppresses expression of protein B No correlation! If intensity of A is high then intensity of B is also high. Example: Protein A and protein B are expressed in tandem. 19

20 Reference Structures for Definition of Colocalization 20

21 Reference Structures for Definition of Colocalization 21

22 Analysis of Expression-Level Ratios COS-7 Cells, double transfected with YFP(free) and CFP (ER-localization frequency). Specimen; Anette and Dr. Frank Böhmer, University Jena, Germany) Distinct horizontally stretched pixel clouds in the scattergram 22

23 Analysis of Expression-Level Ratios YFP Each of the scatter ROIs represents a particular cell with a distinct CFP/YFP expression ratio. CFP 23

24 Quantitative Colocalization Analysis Paste colocalizing pixels into new image set 24

25 Cut Mask Function - 3D Images of Colocalized Structures 25

26 Quantitative Colocalization Analysis A comprehensive tool Display and Analysis of Colocalization Experiments: Interactively linked image display, scattergram and data table Interactive or automatic determination of thresholds Overlay of image channels and results of colocalization analysis Quantitative colocalization analysis with up to 99 ROIs No. of pixels Area / relative area Mean intensities / SD Colocalization coefficients Weighted colocalization coefficients Overlap coefficient after Manders Correlation coefficients (r and r 2 ) Export of the analysis results 26