Mosaic Images

Bringing Together What Belongs Together

October 05, 2011

Confocal laser scanning microscopes are widely used to create highly resolved 3D images of cells, subcellular structures and even single molecules. Still, an increasing number of scientists are extending their focus of bio-research from single cell studies to entire organs and organisms, analyzing the complex interactions within whole animals. One solution to observe large specimens in 3D would be the use of a confocal large scale imaging system based on macroscopic zoom optics, but in some cases the required resolution can only be achieved using a microscopic system. However, here the recorded field of view is relatively small, in particular when using higher magnifications to reach subcellular resolution: e. g. the recorded area of a 63x objective is in the range of 200 x 200 µm, therefore larger specimens cannot be entirely observed and analyzed.

Large specimen albeit small scanning area

To solve this problem – large specimen, but small scanning area at high resolution the mosaic function can be used, assembling single images to form one large super image of the specimen. The prerequisite for such a stitched super image is a tile scan, which records a defined number of adjoining single images of the sample (the "tiles"). The sample is generally moved using motorized stages with high precision. To perfectly match the tiles a small percentage (2–10 %) of overlapping pixels between single images is necessary. Most application software provides helpful functions where only two reference points at opposite corners of the specimen must be defined or the area of the specimen is automatically detected; in turn the number and position of the scan fields to cover the specimen are calculated, including predefined overlapping regions. Following the scanning process, the mosaic image is assembled using specifically designed stitching algorithms. 

Fig. 1: Scheme of a 2D mosaic scan. Drosophila melanogaster (eye section); Red: F-Actin, Cy3; Blue: Nuclei, DAPI; Green: pigmented cells, GFP; Courtesy of Anne Galy, IGBMC, Strasbourg-Illkirch, France.

3D mosaic images

The mosaic function is not only restricted to xy images, but can also be used to create large 3D images. Hence the tile scan must be performed not only in one focal plane, but in different z-planes, creating a 3D mosaic of adjacent image stacks. However, a fundamental prerequisite is a synchronized z-distance between the focal planes.

Fig. 2: Scheme of a 3D mosaic scan. Drosophila melanogaster (eye section); Red: F-Actin, Cy3; Blue: Nuclei, DAPI; Green: pigmented cells, GFP; Courtesy of Anne Galy, IGBMC, Strasbourg-Illkirch, France.

Time lapse movies

As mosaic scans can be repeated in loops, this function can be used to create time lapse movies of living samples. The only restriction would be the velocity of changes in the specimen, as tile scanning takes time, particularly in three dimensions.

Fig. 3: Scheme of a 3D mosaic scan over time. Drosophila melanogaster (eye section); Red: F-Actin, Cy3; Blue: Nuclei, DAPI; Green: pigmented cells, GFP; Courtesy of Anne Galy, IGBMC, Strasbourg-Illkirch, France.

Multi position function

In addition, the mosaic function can be carried out at different positions of the stage. This allows the recording of several specimens in one experimental setup (e.g. different slices on a slide or several samples in a multi-well plate). As the recording conditions will vary from specimen to specimen, the system used should provide the possibility to define different scanning setups for each position or even for each tile of the scan.

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