Loading...
 
Skip to main content
Scientific Volume Imaging B.V.

Scientific Volume Imaging

Deconvolution - Visualization - Analysis


™Huygens Chromatic Aberration Corrector

Correct xyz shifts, rotations, scaling and radial differences between channels

Nearly all multi-channel images show some degree of chromatic aberration. Chromatic aberration appears as focal shifts and possible scaling, rotational, and radial (2nd, 4th, 6th degree) distortions between fluorescent channels. In addition, misalignment between light paths during acquisition can result in lateral (xy-direction) shifts between channels. These aberrations clearly affect any type of multi-channel analyses like colocalization and object-based analysis, and should therefore be fixed beforehand. The versatile Huygens Chromatic Aberration Corrector includes an easy-to-use Wizard that accurately estimates, visualizes and corrects chromatic aberration. With just a few mouse clicks, you can correct for shifts and - if needed - for higher order distortions including up to 14 different chromatic aberration parameters.

Image description

HeLa cells stained with two different fluorophores. This image shows a shifts between the two different channels which has been corrected with Huygens Chromatic Aberration Corrector. Raw image data was provided by Dr. Yury Belyaev, ALMF, EMBL Heidelberg, Germany

Before
Corrected

Easy-to-use Wizard

Apply automated estimation and correction with just a few mouse button clicks.

Accurate and complete

Visually inspect the estimated distortions using the orthoslicer and interactive 3D wireframe MIP.

Batch correction

Save correction templates for replicability and batch processing in the Workflow Processor.

Testimonials

The deconvolution and chromatic aberration corrector have improved our bead representation. Chromatic shift was a problem with our objective but now it seems much reduced, so we are happy. Dr. Dominic Waithe, researcher at Wolfson Imaging Centre, University of Oxford, UK.
I have been a Huygens user for 8-9 years now and the support provided by you guys are fantastic! Dr. Priyam Banerjee, image analysis researcher and instructor, MD Anderson Cancer Center, USA.

Reliable analysis

Chromatic aberration significantly influences the results of multichannel analysis like colocalization, or distance measurements as part of object-based analysis. Huygens Chromatic Aberration Corrector offers an easy-to-use wizard that allows you to correct for shifts, and also scaling, rotational, and radial distortions.

Image description

Chromatic aberration correction reveals colocalization between labeled Mis12 (green) and Aurora B kinase (red) in a U2OS cell. Data from Livio Kleij en Martijn Vromans, Center for Molecular Medicine, UMC Utrecht, The Netherlands.

ChromAb U2OS

Distortions corrected for large field of view

CAC14Params

Chromatic aberrations cause a broad set of distortions in addition to an xyz-shift, as exemplified in the TetraSpeck (Thermo Fisher Scientific Inc.) bead image below. A 3 parameter xyz-shift cross-correlation based correction is sufficient to effectively correct chromatic aberrations in the center of the image (compare panels g,h,i,j). However, higher order distortions can be observed when inspecting the image edges, as the green channel is shifted to the left in area 1, and to the right in area 3 (compare panels c and k). It is clear that a shift is not sufficient to correct for this. While additional correction of z-rotation and xy scale (5 parameters) already pose an improvement, the full set of 14 parameters is needed to fully account for the chromatic aberrations (compare panels d,e,f and l,m,n). Hence, while an xyz-shift is sufficient for a small field-of-view image, higher distortions must be accounted for in images with a larger field of view. Uniquely, Huygens offers the option to first determine the higher corrections from a bead image, save them to a template and then apply them to biological image data, even in batch mode.

Further note that with Huygens unique cross-correlation approach, an xyz-shift can often be estimated using the biological content of the image, averting the need for a separate bead calibration. When using this approach, it is advised to inspect the results carefully as a structural relationship between the content of each channel with the selected reference channel is assumed.

Image description

Tetraspeck bead images on a glass slide recorded in green (emission peak 530 nm) and far-red (emission peak 686 nm) in Widefield mode. a) The xz-view and b) xy-view show that all beads lie in a plane. c-n) Zoom-in of the areas 1,2 and 3 for increasing number of correction parameters. Area 2 in the center of the image can be corrected with an xyz-shift, but areas 1 and 3 require 14 parameters for full correction. Data courtesy of Say-Tar Goh, Caltech university.

Wireframe 3x2 Shiftbead

Broad set of distortions corrected

Huygens Chromatic Aberration Corrector Wizard helps you with selecting the right set of corrections to address misalignments (shifts), scaling and rotational differences, and radial (2nd, 4th, 6th degree) distortions between channels. Radial deformations are caused by optical aberrations and appear as a pincushion or barrel distortion, or as a combination of both, also known as mustache distortion.

Image description Besides a 3D shift between channels, shown within this axial view of a SFP-rendered 4-channel TetraSpeck® bead (Thermo Fisher Scientific), your image may also suffer from other distortions. These distortions are visualized as a 3D rendered wireframe by the Wizard. Example wireframes (top views) show typical distortions found within microscopy data. Beads were imaged with a confocal by Dr. Ron Hoebe, Cellular Imaging, Core Facility Dept. of Medical Biology, Amsterdam UMC, The Netherlands

Use in research

Ivanova, D., Imig, C., Camacho, M. et al. CtBP1-Mediated Membrane Fission Contributes to Effective Recycling of Synaptic Vesicles. Deconvolution and Chromatic Aberration Correction were performed with Huygens. Cell Rep. 30, 2444–2459 (2020).

Dumbović, G., Sanjuan, X., Perucho, M. et al. Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells. Huygens Chromatic Aberration Corrector was used to estimate shift between channels. Methods 187, 68-76 (2021).

For more, see Scientific Publications

Multi-channel images often suffer from crosstalk (a.k.a. bleed-through), which should be corrected before performing analyses. Huygens offers a wide range of analysis options, for example the assessment of the colocalization between different molecules.

Crosstalk Corrector Colocalization Analyzer

More information

Point Spread Function Huygens Deconvolution Restoration examples Chromatic Aberration

mip_beforealignment+alignment_3.png
Recording Beads is a good method to calibrate the existing chromatic aberration. The result of the analysis and correction delivered by the Chromatic Aberration Corrector on a beads image can easily be saved to a template and applied to any image taken under similar conditions. Left: MIP rendered projection of a Huygens deconvolved 3D widefield stack of 100nm 4-channel multicoloured beads (TetraSpeck - Life Technologies). Chromatic Aberration between channels can be more clearly measured and corrected after deconvolution. Right: MIP projection of the same image after using the Huygens Chromatic Aberration Corrector. Insets show a larger view of a single XZ slice of one bead.
Not aligned
Aligned
HeLa cells stained with two different fluorophores. This image shows a shifts between the two different channels which has been corrected with the Chromatic Abberation Corrector . Raw image data was provided by Dr. Yury Belyaev, ALMF, EMBL Heidelberg, Germany.
CAC WindowsRightpart
Huygens Chromatic Aberration Corrector shows a table with the values defining the XYZ shift, scaling and degrees of rotation.
Nups CAC
Huygens Chromatic Aberration Corrector can also be used to align channels. Figure shows both a confocal (green) and STED (red) channel of the nuclear pore complex. The left image shows the deconvolved and chromatic aberration corrected image, the middle shows the deconvolved image before chromatic aberration correction and the right image shows the raw data. Before correction, a clear shift is visible between the confocal and the STED channel. Image courtesy: Leica Microsystems.