Crosstalk CorrectorThe Huygens Software is extended with a tool to measure and correct for crosstalk (a.k.a. bleedthrough or crossover). Crosstalk is the recording of some signal as a certain dye when it really comes from a different one. This effect can be visualized with the 2D histogram.
Correcting for crosstalk is advisable before performing deconvolution. If you are interested in multi-channel analysis like for example colocalization, we highly recommend to use the Crosstalk Corrector first and then perform deconvolution. In this line, you may also want to consider correcting chromatic aberrations between different channels with the Chromatic Aberration Corrector.
WindowThe Crosstalk Corrector can be found in both Huygens Essential and Professional under Deconvolution.
At startup, the Crosstalk Corrector window shows a 2D histogram of the original data, and previews of the histogram and MIP. Options at the bottom of this window, allow you to:
- set the channels for visualization.
- toggle between 2D histogram and crosstalk coefficient matrix
- toggle between corrected and original MIP view.
|Estimate crosstalk values & Reduce Noise
When you start the Crosstalk Corrector, nothing is estimated yet. The crosstalk is estimated upon pressing the "Estimate" button. If you have more than four channels and/or the image is large, it might take time for the estimation to finish.
The 2D histogram is important to verify if the estimation was correctly calculated and if it should be adjusted manually. In general, the estimation is very accurate. The preview windows show the corrected 2D histogram and MIP according to the position of the two slopes in the main 2D histogram. If you are satisfied with the estimation, press the "Correct" button to create a crosstalk-corrected image. In case you have an images with a lot of noise you can first press the "Reduce Noise" button. A median filter is applied to reduce the noise from the 2D Histogram. This is only for the estimation of the crosstalk, and the original data are not affected
|Crosstalk Coefficient Matrix
If you already know the crosstalk coefficient for your image, you can set the number within the matrix. The values should be inserted as follows: if you know the crosstalk of channel 0 within channel 1, you should adjust the value in the row of channel 0 in the column of channel 1. You can "read" the matrix row-wise as "Channel 0 bleeds 0.2774 times through channel 1 and....".
Images were used with permission of Dr. Dimitra Thomaidou group, Neurobiology department and Light Microscopy Unit, Hellenic Pasteur Institute, Greece.