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.
The Huygens Software gives the flexibility to the user to decide between two correction modes: manual and automatic. The manual correction needs a 2D flat field image as input and also allows the use of a dark frame, while the automatic correction does not require these additional images. The automatic correction can be optimized by adjusting the setting for the vignetting model. Both correction modes can be applied to 2D and 3D stack images.
Manual Vignetting correction
Requires a 2D flat field image acquired with the same dimensions and using the same optical path as the other tiles, but from a region on the coverslip or slide without any sample. Ideally this is done for all channels independently as they might contain different vignetting patterns. Although not mandatory, it is also possible to load a darkfield image to recover the true signal even more precisely. This dark frame is acquired without a light source or with closing the shutter, and will compensate for residual signal from the internal temperature, electric currents, etc. The vignetting correction works on 2D & 3D tile images. For more information about acquiring flat field correction images of fluorescence images see this protocol.
Automatic Vignetting correction
Vignetting model: the vignetting model can be set to circular (default) and ellipsoidal. This model describes the shape of the shading of the image, as illustrated in these examples in Fig.1:
Examples of uneven illumination for a circular (A) or a ellipsoidal (B and C) vignetting model.
Vignetting adjustment: with the vignetting adjustment controls the user can adjust the model to the severity of vignetting . Moving the slider left and right will adjust the correction near the edges. Towards the left (towards -1) the correction is following a steeper curve, whereas towards the right (towards +1) the correction will be more gradual (see Fig. 2). In the center (at 0) the correction is more focused near the image center.
Examples of line profiles (A) across an image suffering for vignetting (B), with the respective settings for the vignetting slider.
If there is not enough information for vignetting correction because there are not enough tiles, the wizard will skip the vignetting correction step and proceed with creating the mosaic.
Automatic Vignetting correction and Stitching
No Vignetting Correction
Auto Vignetting correction
Widefield (Leica) fluorescent Leica LIF data were stitched with the Huygens Stitcher. The same data is shown with and without Huygens automatic vignetting and shading correction. Image represents a developing mouse cortex (P30) stained for Tbr1 and reelin.