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Color shift

In a Fluorescence Microscope , different WaveLengths (colors) can be recorded simultaneously in a Multi Channel 3D image . When in the microscope setup the different wavelenghts follow different optical paths, it frequently happens that each recorded channel is shifted spatially in a different direction. This produces a misalignment of the intensity distributions that dramatically affects further analysis, like CoLocalization .

This effect is independent of what is usually understood by Chromatic Aberration , which is something that happens on the lenses of the objective. But both concepts are related at some point: the resulting effect is that different colors are focused in different points of the image plane.

Recording Beads is a good method to calibrate the color shift to correct for it during the Image Restoration . When you use a multicolor bead you can simultaneously distill a Multi Channel Point Spread Function (PSF) and calibrate for the color shift.

TetraSpeck 0.2 µm beads (fluorescent blue/green/orange/red, cat. ref. T-7280) from Molecular Probes can be used, for example. These microspheres are stained throughout with four different fluorescent dyes, yielding beads that each display four well-separated excitation/emission peaks: 365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange) and 660/680 nm (dark red).



Two Channel image of three multicolor beads (two of them stick to each other), as recorded in the microscope. This is a ZX slice, with the optical axis in horizontal position.



The restored image, after Doing Deconvolution . The color shift is still present.

Color shift calculation

The Psf Distiller in the Huygens Professional extracts the PSF's for the different channels all aligned in their centers of mass. Therefore, the PSF itself does not correct the color shift while Doing Deconvolution. Still, the color shifts are reported at the end of this distillation, so you can use these values to apply the correction later. This must be done after restoration, and prior to any data analysis that is affected by it.

You can also use the Object Analyzer present in the Huygens Software to measure the local color shift in a multichannel bead image. A special experiment preset 'Calculate color shift' is available that reports directly, for each object in one pipe, the distance to the nearest neighbor in the other pipe (distance between Center Of Mass ). Assuming that, in this image, nearest neighbors across channels are always the same bead, simply with a slight color shift, this shift vector provides a local estimation of the color shift. All you have to do is to put two different channels of your image through the two available analysis pipes. If your image has more than two channels, you can repeat the analysis in pairs (channel 0 can be the reference, always in the Primary analysis pipe, while you put other channels in the Secondary pipe).

You can then use the average of all vector components to estimate a global color shift correction, if you don't want to complicate the analysis by using a local remapping. To calculate average values of columns in the Object Analyzer statistics table, simply select the columns you want (in this case, the three columns 1NP.CMCMx 1NP.CMCMy 1NP.CMCMz that report the vector coordinates), right click on them and select 'Stats' in the pop-up menu. Read more in Object Analyzer Expert Tutorial and Object Analyzer Neighbors .

Like that you can calculate the color shift with a bead calibration image, but now you want to apply it to other images acquired in the same microscope.

Once the shift vector is known, the command shift can be used in Huygens Core and Huygens Professional to align the images. The shift operation is freely available in these programs, you don't need a license to use it. See the Huygens Core Programmer Guide. Because the shift command works on a single-channel image, it may be tedious to do the correction manually, first splitting the channels, shifting them, and joining them at the end to build the corrected image.

To simplify the procedure of applying a shift (and make it available also in Huygens Essential , that has not access to the shift command) you can also use an applet that extends the basic interface capabilities of the Huygens Software and allows you to do this operation interactively. See Color Shift Correction Applet .

Alternative ways

With a simple Tcl Huygens script you can accurately calculate the shift vector between Two Channels with voxel resolution: see Correlate Dot Tcl . This procedure uses some functions that require a License String to work, but you can estimate the shift vector by some other means, for example by looking at position of the maximum in each channel.



The same restored image as above, after channel alignment with the output of the correlate script. The shift is corrected, but some Chromatic Aberration is noticeable that makes the beads to have slightly different shapes in the two channels.

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