Colocalization in Confocal Microscopy
1Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
2Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
HEp-2 cells (Hemmerich, Jena, Germany)
(aCBP/p300, aRNA-polymerase II, DAPI)
Zebrafish (Bastmeyer, Konstanz, Germany)
(Two different neuronal markers)
HEK cells (Böhmer, Jena, Germany)
(free GFP, YFP_SHP1 fusion)
Colocalization of fluorescently labeled molecules (immunostainings,
fluorescent proteins, etc.) is often used as the first indicator
of functional interaction
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
3Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Coincidence of two or
more fluorescent signals
inside the same
detection volume
Intensities (and
position) of the signals
inside the detection
volume may vary
Quantitative Colocalization Analysis
Prerequisites
1. Acquisition of crosstalk-free images
2. Identical size and position of
detection volumes for different color
channels
Colocalization in Confocal Microscopy
4Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Have look inside...
Innovative detection architecture
- single and META-detectors
Real confocal architecture
- multiple pinhole conceptequal sections
with multiple pinhole
concept
unequal sections
with single pinhole
Colocalization in Confocal Microscopy
5Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
The thickness of the confocal sections varies with:
(1) objective (num. aperture)
(2) size of the confocal pinhole
(3) wavelength of the light
Dyes Pinhole Optical slice
DAPI, Hoechst 1 Airy unit 0.7 µmeGFP, FITC, Alexa488 1 Airy unit 0.9 µmDsRed (RFP), Cy3, Rhod 1 Airy unit 1.0 µmCy5 1 Airy unit 1.1 µm
Dyes Pinhole Optical slice
DAPI, Hoechst 137 µm 1 µmeGFP, FITC, Alexa488 135 µm 1 µmDsRed (RFP), Cy3, Rhod 132 µm 1 µmCy5 128 µm 1 µm
Multiple pinhole concept
Single click for
optimal pinhole
settings of all
channels!
Colocalization in Confocal Microscopy
6Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
HEp-2 cells (Hemmerich, Jena, Germany)
(aCBP/p300, aRNA-polymerase II, DAPI)
Zebrafish (Bastmeyer, Konstanz, Germany)
(Two different neuronal markers)
HEK cells (Böhmer, Jena, Germany)
(free GFP, YFP_SHP1 fusion)
Colocalization is often indicated by the color overlay (green + red = yellow)
Several factors (exposure time, brightness, contrast etc.) render these
overlays problematic
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Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
7Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Image Window
Quantitative Colocalisation Data
Scattergram Tool Box
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
8Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Scattergram – Pixel-wise Correlation of Image Channels
Colocalization in Confocal Microscopy
9Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
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)
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
10Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Interactive link between scattergram and image display
- Select ROIs in image display and view corresponding pixels
in scattergram
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
11Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Scatterregion 3:
Colocalizing pixels
Scatterregion 1:
Pixels in channel 1 only
Scatterregion 2:
Pixels in channel 2 only
Scatterregion 4:
Sub-threshold pixels
(background intensities)
Scattergram and thresholding with crosshair
- Use the crosshair tool for setting intensity thresholds in scattergram
Crosshair
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
12Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
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
Colocalization in Confocal Microscopy
13Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Cross-Hair Function – Clear Separation of Colocalization
Colocalization in Confocal Microscopy
14Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Scattergram
Data table
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 r2)
- Export data
- Save and reload threshold settings (scatter ROIs and crosshair)
for use with multiple images
Save and
reload of
threshold
settings
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
15Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Colocalization in Confocal Microscopy
16Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Channel 2
Channel 1
Colocalising pixels = 4
Total number pixels in Ch1 = 6 = 0,66
Colocalization in Confocal Microscopy
17Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Channel 2
Channel 1
Intensities of colocalising pixels = 90
Total intensities of pixels in Ch1 = 105 = 0,85
25 25
20 20
5 10
Colocalization in Confocal Microscopy
18Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
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.
Colocalization in Confocal Microscopy
19Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
R=-1 R=-0 R=1
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.
If intensity of A is high then
intensity of B is low.
Example: Expression of protein A
suppresses expression of protein B
Colocalization in Confocal Microscopy
20Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Reference Structures for Definition of Colocalization
Colocalization in Confocal Microscopy
21Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Reference Structures for Definition of Colocalization
Colocalization in Confocal Microscopy
22Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
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
Colocalization in Confocal Microscopy
23Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Analysis of Expression-Level Ratios
Each of the scatter ROIs represents a
particular cell with a distinct CFP/YFP
expression ratio.
CFP
YF
P
Colocalization in Confocal Microscopy
24Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Paste colocalizing pixels into new image set
Quantitative Colocalization Analysis
Colocalization in Confocal Microscopy
25Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
Cut Mask Function - 3D Images of Colocalized Structures
Colocalization in Confocal Microscopy
26Carl Zeiss - TASC - Dr. René Hessling 29/10/2015
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 r2)
Export of the analysis results
Quantitative Colocalization Analysis
A comprehensive tool
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