Colocalization AnalyzerCoLocalization analysis for 3D fluorescence microscopy is available in Huygens Essential and Huygens Professional since version 3.0, allowing you to obtain quantitative information about the amount of spatial overlap between structures in different data channels, for 3D images and 3D-time series.
As this overlap can be defined in many ways, the colocalization analyzer gives you nine different colocalization coefficients most commonly used in literature that are part of the following major groups:
- The coefficients due to Manders M1,2 and K1,2
- intersection coefficients
For more information about these coefficients, see Colocalization Theory and Colocalization Basics.
Within the softwareThe colocalization analyzer is divided into three tabs. The main function, calculating the coefficients for all time frames, is visible in the first tab:
- choose your coefficients,
- select your channels,
- select a specific time frame or calculate the coefficients for all time frames at once,
- set the background using the 2D-histogram or the histogram per colorchannel, using the togglebutton,
- look at the 2D histogram more closely using the magnifier, by clicking on the histogram,
- save the 2D histogram with or without the background sliders and intensity-bars,
- apply RBNCC,
- use three different types of background estimators to automatically set the background,
- and of course the final calculation of the colocalization coefficients, by pressing Compute.
In the second tab, you can visualize the colocalization with the surface renderer. The surface can only be calculated if a colocalization map is calculated which you can choose in the first tab.
- set the threshold of the surface renderer to see which voxels contribute at least that much to the colocalization coefficient,
- twist and tilt the scene for a better perspective,
- turn the MIP renderer on or off per channel or both at the same time to see the whole data in comparison with the colocalized region,
- and finally by clicking on an object in the scene, analyze a colocalized region.
The object analyzer can be found in the third tab:
- look at the statistics of the object you clicked on in the Surface-Renderer-tab,
- and quickly analyze all objects.
This object analysis tab allows you to quickly determine the properties of the different colocalization regions in your data. This is realized by visualizing the colocalization map as iso-colocalization surfaces.
In the colocalization analyzer these surfaces are computed at the same time as the coefficients.
The surface objects show regions in which the degree of colocalization exceeds a certain value. By clicking on the objects local colocalization parameters are computed and reported. To relate the iso-colocalization objects to the original data the surface objects can be blended with a Red-Green MIP projection of the colocalizing channels, or with a colored MIP of another reference channel. The color range in which these objects will be displayed can be modified using a Hue Selector.
Object level analysisNotice that the Object Analyzer in the Huygens Software also provides colocalization measurements at the object level. The Colocalization Analyzer works more at the level of the whole image, so that local statistics of the colocalizing regions can be easily retrieved.
Both analyzers work, in a sense, in complementary ways.
The Object Analyzer allows you to define objects and see how much they overlap, in volume or intensity. Objects defined like this can overlap with other objects, or not.
The Colocalization Analyzer explores the whole image to search for colocalizing regions based on the usual colocalization coefficients. These regions are then segmented and treated as objects to analyze. These objects are therefore always volumes of intersection.
How to use itSee Colocalization Tutorial.
For a tutorial video on how to use the Colocalization Analyzer see here.