colocalization in confocal microscopy · 2016-01-11 · colocalization in confocal microscopy carl...

Colocalization in Confocal Microscopy

1Carl Zeiss - TASC - Dr. René Hessling 29/10/2015

Quantitative Colocalization Analysis



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




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



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



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!



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

? ??




Image Window

Quantitative Colocalisation Data

Scattergram Tool Box




Scattergram – Pixel-wise Correlation of Image Channels



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)




Interactive link between scattergram and image display

- Select ROIs in image display and view corresponding pixels

in scattergram




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 Analysis – Set Background Threshold

Positioning of the Cross at mean

intensity value of image ROI plus two

time the standard deviation for each

cannel



Cross-Hair Function – Clear Separation of Colocalization



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




Channel 2

Channel 1

Colocalising pixels = 4

Total number pixels in Ch1 = 6 = 0,66



Channel 2

Channel 1

Intensities of colocalising pixels = 90

Total intensities of pixels in Ch1 = 105 = 0,85

25 25

20 20

5 10



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.



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



Reference Structures for Definition of Colocalization



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



Analysis of Expression-Level Ratios

Each of the scatter ROIs represents a

particular cell with a distinct CFP/YFP

expression ratio.

CFP

YF

P



Paste colocalizing pixels into new image set




Cut Mask Function - 3D Images of Colocalized Structures



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


A comprehensive tool

colocalization in confocal microscopy · 2016-01-11 · colocalization in confocal microscopy carl...

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