Tile Scan Prepared by Myriam Gastard, PhD Application and Technical Support Group, Leica Microsystems, Inc. In this issue of our Confocal Application Notes, proper set up of the Tile function enables you to scan multiple partial images from a specimen and stitch them together to form a complete image. By extension, this feature makes possible to scan a much larger field of view than a high magnification objective alone would be able to give you, and still keeping the high resolution of the objective. In order to have all the images aligned in x, y and possibly z (when z-stack are acquired simultaneously), and to avoid any gap and/or misalignment of the resulting tile, you must precisely adjust the tile scan. One important tool supporting this feature is the Tile Scan Calibration. First requisite is to have the stage initialized at startup of LASAF. Place a sample onto the stage and setup your system in order to be able to image the sample (laser, PMT, gain etc ). To assure a precise calibration, an oil objective with a magnification of at least 40x, and high numerical aperture, is recommended. If for any reason an oil objective cannot be used, then a 20x dry is appropriate. You should zoom in order to have enough details to adjust accurately the different structures of interest. 1- In acquisition mode select the Tile Scan function. Acquire Acquisition Select the Tile Scan function 2- When Tile Scan function is selected, a drop down window named Tile Scan appears: Tile Scan window interface 1
3- Click on the Calibration function. 4- A new interface window Tile Scan Calibration appears. 2
5- Activate the Live imaging and click on the preview image in order to activate the marked line. This will be used as reference in order to adjust the rotation. 6- While in Live mode zoom in to find a structure in your sample that you can use as a horizontal reference point. Reference structure 3
7- Using the Smartmove and xy precise move the structure along the line and pay attention to its relative position to the marked line. Reference structure moved on the other side of the field of view. If the sample needs to have some rotation adjustment (like shown in this example), the structure will not be parallel when moved from side to side of the field of view. 4
8- Adjust the rotation by moving the rotation cursor or by adjusting the degrees numbers in the window while in Live mode. Check the reference point by moving the structure back and forth along the marked line and be sure that it is parallel to the marked line. 5
9- Once the new rotation position is found, stop the live scanning and click on the Run Test tab, then click on the Run Test button. If a region of the resulting tile scan is showing some tiling discrepancies (like in this image noted in yellow), then go to Set parameters. After each change, the Run Test needs to be activated and a full tiling is taken. Adjust then the parameters in order to compensate accordingly. 6
Once the Tiling parameters have been correctly modified, you should be able to get a very good tiling. 10- Once the Set parameters is chosen, the Stage Configuration window appears: 1 2b 2a 7
In this window, 2 different areas may need adjustments: 1- Tile Scan: set overlap sizes to: X % and Y% The percentage of overlap needed to have the best tiling can be adjusted by entering positive values corresponding to the X and Y axes overlap. Try to change one parameter at the time in order to understand which way it will go. 2a- Tile scan: set merge settings to: Factory and Custom Here are the settings needed to assemble the tiling. It is essential to change only one parameter at the time. Factory: It is by default on Factory settings. Custom: If the factory settings are not compensating completely (or the tiled image seems to be scrambled), then these settings must to be adjusted. You can flip the X, Y or even swap XY axes in order to reorder the tiles. 2b- Tile scan: set merge settings to: Smooth, Multi frame Search, Auto Stitching The stitching process can be started either automatically right after the tile scan acquisition, or any time post acquisition in Process Mosaic Merge tab. To enable the stitching right after acquisition, check the Merge box in Tile Scan interface. In this case, the Auto-Stitching/Smooth parameters are set in Configuration Stage tab. Smooth: It enable the stitching of the images together with a function which will even the sharp grid lines (after stitching), in order to make it disappear from human eyes. The Smooth function can be turned on or off during Auto-Stitching and it will not affect the image alignment. Multi frame Search: This function applies the smooth and/or Auto Stitching functions to every sections in x, y and z allowing a better stitching at every level of the merged sections. The calculations will make this step slower than if smooth and/or Auto Stitching functions are selected alone. Auto Stitching: When this box is checked prior the Tile Scan acquisition, it allows having a full tiled image without having to request it in Process Mosaic Merge and hence acting as a short cut for this post-processing method. 8
11- Tricks and Tips: To enable the stitching right after acquisition, check the Merge box in Tile Scan interface. If the Merged Tile Scan is poor, try to open a little bit the pinhole to 1.5 or 2 Airy Units. When using a low magnification objective (5x or 10x) the corners of each tile scan can appear less illuminated, which can be very noticeable once the Tile Scans are merged. To resolve this, please use the zoom in function (~ 1.7 zoom in) to obtain a better field uniformity. 9