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Huygens Object Stabilizer

Measure and correct for sample movement and 3D misalignment




Huygens Object Stabilizer can measure and correct for any type of undesired sample movement, caused by for example stage drift or vibration or simply by the motility of a cell or organism. Huygens Object Stabilizer can stabilize 2D and 3D time series, movement in xyz, axial rotation and mis-alignment of slices within a 3D stack. Both the measurement and the subsequent stabilization are done in 3D and at sub-pixel level. Object stabilization can be applied to a batch of images, using Huygens Batch Processor or Huygens Remote Manager/Core.

Since object stabilization is a crucial step for deconvolving STED data, this option is integrated in the STED (3D) optical option.

Stabilize (3D) time series

Align slices in 3D stacks

Correct for thermal drift in STED

Deskew Light Sheet images


Stabilized
Not Stabilized
Persistent drift and a stage bump during acquisition complicate tracking of the red lysosomes/endosomes (left). Both issues were corrected with Huygens Object Stabilizer (right). This widefield time series of a mouse embryonic fibroblast (MEF) is courtesy of Prof. Fumio Matsumura, Rutgers University, USA.


Easy-to-use Wizard

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


All-round toolbox

Versatile toolbox for correcting stage drift and movement during acquisition and/or natural yet unwanted movement of live objects.


Z-stacks and time series

Correct misalignment between z-stack slices as well as movement across time series.




Testimonials

Huygens object stabilizer is doing a very good job to fix drift of any kinds.

Dr. Juraj Kabat and Dr. Olena Kamenyeva, staff scientists at the Biological Imaging Section, NIAID-NIH, USA.


these stacks require some time to collect, often with some lateral drift. The Object stabilizer seems to be a game changer for what we are doing.

Prof. Gregory I. Frolenkov, Department of Physiology, University of Kentucky, USA.



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


3D alignment can be applied prior to deconvolution if the z-stack suffers from misalignment between adjacent slices, for example due to temperature changes altering the Z-stepper position. By fixing the spatial alignment, the Object Stabilizer improves the deconvolution of 3D stacks. Note that when stabilizing time series, applying deconvolution first is preferable as it will improve the stabilization.

Align Comparison
An x-z view of a misaligned z stack (left) and the result of alignment with Huygens Object Stabilizer (right); red box highlights region where misalignment is particularly apparent. The additional chromatic shift in this 3D image can be corrected using the Huygens Chromatic Aberration Corrector option.


STED thermal drift correction


STED PSF

STED images typically have a much higher resolution in the lateral (x and y) direction than in the axial (z) direction. The corresponding PSF then has a pencil-like shape that extends through multiple slices in a 3D stack. Because of the high ratio of the lateral to the axial resolution, any stage shift or thermal drift during 3D acquisition causes significant misalignment between the slices. This misalignment must always be corrected before performing deconvolution and subsequent analysis. It is therefore included in the STED Deconvolution Wizard: at the dedicated Stabilization step, reliable and fully automated drift correction is performed without the need for user input.


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 on (de)skewing, visit the CMBF, Harvard Medical School FAQ.

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



Use in research

M.K. Oklinski, H.-J. Choi, T.-H. Kwon, Peripheral nerve injury induces aquaporin-4 expression and astrocytic enlargement in spinal cord
Huygens Object Stabilizer was used to stabilize 3D confocal images.
Neuroscience 311, 138-152 (2015).

B.-J. Zandt, A. Losnegård, E. Hodneland et al., Semi-automatic 3D morphological reconstruction of neurons with densely branching morphology: Application to retinal AII amacrine cells imaged with multi-photon excitation microscopy,
Huygens Object Stabilizer was used to align images along the z-axis.
J Neurosci Methods. 279, 101-118 (2017).

For more, see Scientific Publications

After correcting for unwanted global movement, motion of objects within the sample can be tracked more reliably with the Huygens Object Tracker.

Object Tracker

More information

Live Cell for information on live cell imaging
Support page for practical information on the Object Stabilizer
Stabilizer webinar for a video tutorial on using the Object Stabilizer