Microscopic images often suffer from various acquisition artifacts such as noise, unwanted movement, crosstalk, bleaching, hot pixels, and channel shifts, all of which can seriously impact subsequent image analysis. While it's possible to visually inspect images to identify and correct these issues, this process requires significant time and expertise.
Huygens Image Quality Control option streamlines this process by automatically identifying problems within your microscopy images, providing detailed explanations of any detected issues, and offering direct links to the necessary options and tools for correction. This page lists all these restoration options that Huygens offers to prepare your images for reliable analysis.

Deconvolution

A microscopy image is not an exact copy of the object under the microscope: a so-called convolution of the object light leads to blurring in the resulting image. The inverse process, deconvolution, reverses this blurring and brings the image closer to the actual object. The Huygens deconvolution options thus provides noise correction, deblurring, and increased resolution.

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Chromatic Aberration Correction

Chromatic aberration results in misalignment and other distortions between different channels in a microscope image. It can be caused by the physical properties of the used objectives, misalignment in the microscopy set-up, or by a combination of both. Nearly all multi-channel images show some degree of chromatic aberration. The Huygens Chromatic Aberration Corrector can estimate and corrects these aberrations.

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Crosstalk & Autofuorescence Correction

Crosstalk (a.k.a. optical crosstalk, bleed-through or crossover) is a phenomenon in multi-channel microscopy where signal from one dye is collected as signal from another dye, i.e., signal seems to come from one dye when it really comes from another. The Huygens Crosstalk & Autofuorescence Corrector can quickly estimate and correct crosstalk between multiple channels simultaneously.

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Hot & Cold Pixel Removal

Hot pixels and cold pixels are produced by individual sensors on the CCD camera with higher than normal rates of charge leakage or lower than normal sensitivity. Hot and Cold pixels appear in the image as bright and dark pixels, respectively. They can be effectively corrected with the Huygens Hot & Cold Pixel Remover.

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Object stabilization

Object stabilization is needed in cases of undesired sample movement, caused by for example stage drift or vibration or simply by the motility of a cell or organism. The Huygens Object Stabilizer can measure and correct this movement by stabilizing 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.

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Image Stitching and Vignetting Correction

Many images suffer from uneven illumination of the field, with poorer illumination at the image periphery. This so-called vignetting problem is due to factors in the microscope light path, such as limitation in the lenses or in the camera. The vignetting effect becomes particularly evident when 2D or 3D tile images are stitched together. The Huygens Stitcher can correct for vignetting and create an uniform reconstruction of the large field of view.

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Light Sheet image Fusion

Common in Light Sheet Fluorescence Microscopy (LSFM) is the acquistion of multiple (opposing or rotational) views, and fuse these to compensate for light absorption and scattering issues. Typically, interest points (e.g. beads) are included to facilitate this fusion process. Huygens Fuser does not require the use of beads. Instead, each view can be optimally positioned by shifting, rotating, and scaling it using the interactive scene. Advanced correlation algorithms then fine-tune the alignment. Real-time visual feedback during the alignment process gives you full control over the fusion!

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Bleaching Effects Correction

Bleaching Effects are practically unavoidable phenomena in fluorescence microscopy. Because the image planes are acquired sequentially, bleaching will vary along the Z direction and/or over time. Assuming it is not strong it will not affect deconvolution results of confocal images. But in widefield microscopy deconvolution bleaching is more of a problem. Fortunately, the bleaching in most of WF images can be corrected quite easily. Huygens will do so automatically. If the bleaching is too severe, the correction might not be perfect resulting in lower quality deconvolution results. From Huygens version 17.04 onwards, there is a new Bleaching Corrector tool available.

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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.