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Huygens Object Stabilizer

Measure and correct for any type of sample movement



Huygens Object Stabilizer can measure and correct for any type of sample movement that is not desired. Such unwanted movement can occur during image acquisition because of stage drift or vibration, or can simply be the motility of a cell or organism that you like to undo. Huygens Object Stabilizer can stabilize 2D and 3D time series, movement in x, y and z, axial rotation, and also mis-allignment of slices within a 3D stack. Both the measurement and subsequent stabilization are done in 3D and at sub-pixel level. Since object stabilization is a crucial step for deconvolving STED data, this option is integrated in the STED (3D) optical option. Object stabilizaton can be applied to a batch of images, using Huygens Batch Processor or Huygens Remote Manager/Core.

Image description:
Time acquisition of this widefield image suffered from a persistent drift and sudden bumb of the stage, preventing proper tracking of the red lysosomes/endosomes. Both stage issues were corrected with Huygens Object Stabilizer. Image shows a mouse embryonic fibroblast (MEF) in which Fascin1 (protein binding to actin, green) and Lamp1 (lysosomal membrane protein 1, red) have been transfected. Courtesy of Prof. Fumio Matsumura, Department of Molecular Biology and Biochemistry, Rutgers University, NJ, USA

Stabilized
Not Stabilized


Easy to-use Wizard

Wizard which offers the most optimal stabilization method, correction of rotational movement, graphs presenting the measured displacements, and advanced cropping tools

All-round Methods

Versatile toolbox for correcting stage drift and movement during acquisition (like with 3D STED imaging), or natural yet unwanted movement of objects such as cells or organisms.

Deskewing Light Sheet

De-skew (de-shear) option for 3D Light Sheet setups where the excitation and emission axis are oriented at an oblique angle relative to the sample stage, which is scanned horizontally to move the focus (see below).


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Stabilization of 3D time series

Stabilization over time works best if the 3D time series has been deconvolved first. Because deconvolution enhances resolution, reduces noise, and improves contrast, the analysis of motion is more accurate. Stabilization in time series refers to the image as a whole and not to individual objects that move over time. If you are interested in analyzing tracks of the natural movement of individual objects over time, we like to refer you to the Huygens Object Tracker Option.

For time series there are four stabilization methods available:

  • Cross correlation. This can be considered an 'all-round method'. It can correct for both x-y-z translation and axial rotation. Adjacent time frames are compared to find the best alignment by maximizing structural overlap.
  • Model-based correlation. When the geometry of the imaged object did not change much during the acquisition, the time series can be stabilized using a model of the object. The stabilizer creates the model automatically.
  • Multi object tracking. When a image contains well-defined objects, i.e. nuclei or small particles, the image can be stabilized using object tracking. Objects are tracked over time and their average movement is used to stabilize.
  • Center of mass alignment. This method works best if the image contains a single large object. No objects should cross the image borders, and the contrast between object and background should be high.

Shaking Stable Small
Images show the sum of four time frames of an apoptotic cell stained with fluorescent H2B nuclear protein. Time interval between slices is 300 seconds. Courtesy of M. Varecha and M. Kozubek, Masaryk University, Czech Republic.


Alignment of slices in 3D stacks (no time series)

3D alignment can be applied prior to deconvolution if the stack suffers from "severe" misalignment between adjacent slices (e.g. due to temperature changes altering the Z-stepper position). In those cases the Object Stabilizer improves the deconvolution of 3D stacks and is applied beforehand. In all other cases applying deconvolution first is preferable.

Align Comparison
An x-z view of a misaligned z stack (left), and result after alignment withh Huygens Object Stabilizer (right), The additional chromatic shift in this 3D image can be corrected using the Huygens Chromatic Aberration Corrector option.


STED Thermal Drift Correction

This method is optimized for correcting thermal drift in STED imaging systems, and is very straightforward since no rotation correction or additional user input is needed. Following the Preprocessing stage, you will be taken directly taken to the final Stabilization stage.

Deskewing (shearing correction) of Light Sheet images

In some Light Sheet Fluorescence Microscopy (LSFM) setups, the excitation and emission axis are oriented at an oblique angle relative to the sample stage. In such systems, the specimen mounted on the stage is scanned horizontally to move the focus through the sample. However, since the scanning does not occur parallel to the optical axis, this leads to a movement in multiple spatial directions. The effect is a shear (skew) in the recorded 3D image stack. This complicates any type of deconvolution since the PSF is sheared accordingly. Therefore, the image must first be corrected by shifting all the z-slices back into their correct position. The shifts between consecutive z-slices is constant and depends on the step distance of the stage and the objective angle. Huygens Object Stabilizer (version 18.04 and later) has the option to correct for the skewing within light sheet images. For more information please see the CMBF, Harvard Medical School FAQ: here

Image description:
Skewing and de-skewing of LSFM images. Some LSFM setups have their excitation and emission objectives positioned at an oblique angle relative to the sample stage. The sample mounted on the stage is scanned horizontally to move the focus through the sample (see arrow), which leads to a skew in the recorded 3D image stack an also in the PSF. The Huygens Object Stabilizer option can de-skew all z-slices to their correct position.

Deskewing



Available for Huygens Essential, Professional and Core

The Huygens Object Stabilizer can be tested with a free test license. The product is commercially available as a licensed option for Huygens Essential, Professional and Core.

Why wait? Try out this option by downloading Huygens and request a test license, or receive pricing information.

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