Huygens Re-scan Confocal Deconvolution Option

Specially suited for deconvolving Re-Scan

Raw Re-scan Confocal
Huygens Deconvolved

Huygens deconvolution for re-scan based confocal imaging can be added to Essential and Professional as a separate option, and improves the resolution, signal, and contrast within re-scan images significantly. The Re-scan Confocal Microscope (RCM) hardware is an add-on that can turn any fluorescent microscope into a 3D confocal system. The RCM is developed and sold by Confocal.nl.

Image Description
XY and XZ (bottom) slices show raw and restored versions of a Re-scan Confocal Microscopy (RCM) image. Huygens was used to correct for Hot Pixels, crosstalk, and noise and out-of-focus signal (more details in the Gallery below). Also note the resolution improvement (1.5-2x) in the Z dimension. Image shows microtubules (Alexa 488-labelling, green) and TOMM20 protein (Alexa 568-labelling, red). Image courtesy: Dr. Tobias Schwarz, ScopeM, ETH Zurich, Switzerland

Use in research

Lucidi M, Hristu R, Nichele L, Stanciu G.A et al., Characterization of Acinetobacter baumannii Filamentous Cells by Re-Scan Confocal Microscopy and Complementary Fluorometric Approaches.
Huygens was used to deconvolve Confocal and Re-Scan Confocal (RCM) images.
IEEE J. Sel. Top. Quantum Electron 27, 1-7 (2021)

For more, see Scientific Publications

The best deconvolution results can be achieved by using a measured PSF extracted with the Huygens PSF Distiller. After deconvolution, images can be used for reliable image analysis, for example with the Huygens Object Analyzer.

PSF Distiller Object Analyzer

More information

Introduction to deconvolution
Huygens Deconvolution
Deconvolution images

Re-scan image restoration with Huygens

1. Raw image

Huygens is able to restore a wide variety of imaging artifacts. Besides the noise, blur and background - present in all fluorescent microscopy data, this specific raw Re-scan confocal example of TOMM20 mitochondrial staining also suffers from several hot pixels (see arrowheads in first image) and crosstalk (as indicated by the arrow in second image). The first image shows the raw Re-scan image with all the above-mentioned artifacts, which are one-by-one addressed in the images that follow. The first restoration step involves the successful removal of hot pixels using Huygens Hot & Cold Pixel Remover.

2. Hot & Cold Pixels Remover

This second image shows the effective correction of all hot pixels (see arrowheads in previous image). There is however still crosstalk, which can be seen in this TOMM20 channel (red) as bleedthrough of signal originating from the microtubular staining (see green channel in figure at the top of this page). This crosstalk seriously poses a problem when performing multi-channel analyses like colocalization or object-based analysis. With Huygens CrossTalk Corrector you can easily and quickly estimate, and correct crosstalk between many channels simultaneously.

3. Crosstalk Corrector

In this third image the crosstalk has been corrected. The image was further restored with Huygens specific Re-scan deconvolution option. Deconvolution is a mathematical operation used in image restoration to recover an image that is degraded by a physical process which can be described by the opposite operation, a convolution. Huygens renowned deconvolution with its advanced MLE algorithms involves the effective correction of background, blur and noise, typically leading to significant increase in resolution and signal (contrast).

4. Deconvolved

Lastly, this fourth image shows the completely Huygens restored Re-scan confocal microscopy image of the TOMM20 channel from the image that is also shown at the top of this page.

Image Description
Click on image to zoom and enlarge. Image showing three restoration steps with tools available in the Huygens Software of a re-scan confocal image of TOMM20 protein (Alexa 568-labelling). Image courtesy: Dr. Tobias Schwarz, ScopeM, ETH Zurich, Switzerland

Confocal RCM Decon Meiosis Highres WithText
Images show individual chromosomes of a nuclear spread from fixed mouse spermatocytes, immunostained for SYCP3 a component of the synaptonemal complex (Alexa 488-labelling). Raw images were acquired with RCM without re-scanning (left) and with re-scanning (middle). The raw RCM image was deconvolved with the Huygens Re-scan Deconvolution Option (right). Sample courtesy of A. Agostinho and C. Höög, Karolinska Institutet, Sweden; imaging and Huygens Deconvolution performed by Erik Manders, Confocal.nl, Netherlands.