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Manual slide scanning works best, if the camera sensor is aligned as good as possible to the motion directions of the stage.
If the camera is not aligned well, when moving the specimen in X-direction, the computer observes e.g. 95% motion in X-direction and 5% motion in Y-direction.
This can be seen easily by observing the motion of the grid lines.
Fine-tuning the camera rotation can save scanning time, because it allows to reduce the amount of overlap between the stripes.
In theory, an overlap greater than one tile between the current row/stripe and the last row/stripe should be enough when acquiring horizontal stripes.
The vertical component of the motion results in reducing the overlap below one row of tiles. This results in stitching errors and bad image quality.
Therefore the overlap should be at least two or three rows of tiles to ensure proper stitching of the images.
Rotation of the camera can be tricky. Note that the camera is mounted tight on the camera adapter and cannot be rotated itself. Instead, the camera is rotated by rotating the camera adapter.
The camera adapter usually is fixed using a clamp. Adjust the rotation in very small steps. Be careful when tightening the clamp.
Some microscopes have rotatable stages. On these microscopes you can adjust the rotation by rotating the stage, too.
Preferred alignment method: Adjust the camera rotation by aligning the stage or camera to the ground edge of a slide.
1. select a slide with excellent ground edges like (Leica SnowCoat (TM)) An empty slide is ok, too
2. remove all dust from the slide and the slide holder
3. place that slide onto the stage. make sure it is aligned perfectly with the slide holder
4. engage a low magnification objective lens (4x or 10x magnification)
5. move to the upper edge of the slide
6. bring the glass slides ground edge into focus (be careful not to focus on the cover glasses edge!)
7. the closer the edge is located to the top of the cameras field of view, the better angular errors can be seen.
8. align the camera to the edge of the slide by rotating the camera adapter or the stage
9. move the slide from left to right and observe the motion of the glass slides edge
10. if the glass slides edge does not move up and down significantly the slide should be perfectly adjusted
11. in the unlikely case that the slides edge is moving up or down significantly there could be following errors
11.a. if the edge is moving up AND down while the slide is moved, the slide seems not to be suitable for calibration
11.b. if the edge is moving up OR down while the slide is moved, the slide holder of the stage could be misaligned to the stage. This can easily happen if the slide holder can be unmounted with two screws by hand. To resolve the issue unmount the slide holder, clean the slide holder and the stage from dust and remount the slide holder while pushing it towards the screws to align it properly.
Watch a video demonstration on camera alignment using the edge of a slide:
Alternative (precise, but difficult) alignment method: use the image stitching process of the manualWSI software to align the slide.
Perform these steps to fine-tune the camera rotation
1. select a specimen that allows to perform a very long horizontal motion
2. engage a high magnification objective lens (e.g. 40x)
3. prepare as if a slide scan should be conducted
4. as starting location, choose the left edge of the sample
5. start scanning and move to the right for approximately 10 camera field of views
6. observe the motion of the horizontal grid lines (they either move up or downwards)
6.a. if they moved less than the size of one tile upwards or downwards, the rotation is fine-tuned
6.b. if the horizontal lines move upwards, the camera has to be rotated clock-wise to reduce the drift
6.c. if the horizontal lines move downwards, the camera has to be rotated counter-clock-wise to reduce the drift
7. Press F10 to reset the scan and repeat the procedure until condition 6.a. is satisfied
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