• Stitch image folders that contain a .gci file using Keyence BZ-X800 Analyzer and Wide Image Viewer software.
• Output: The stitched acquisition in uncompressed and compressed tifs.
• Reduce image size by up to 50% using Fiji/ ImageJ, Image, Color, Stack to RGB.
• Add metric information (image width in microns) to the output image using Fiji/ ImageJ, Analyze, Set Scale.
• Compress a copy of the raw image folders.

• Autohotkey version 1.x (Open source)
• Keyence Analyzer Analysis Software, incl. stitching add-on (Proprietary)
• Fiji/ ImageJ (Open source)
• 7-zip (Freeware)

• Microscope: Keyence BZ series
• Software: BZ-X800 Viewer
• Image type: Still image
• Sample holder: Slide
• Channels: CH1, DAPI; CH2, GFP; CH3, RFP; CH4, Brightfield. Modifiable in the script or via microscope set up
• Overlay: Deactivated, later generated by the Analyzer software if multiple channels were used for acquisition
• Capture method: Multi-Color (Single color can be acquired via Multi-Color setting)
• Capture area settings: Multi-Point supported

• The order of the channels during acquisition defines the output name. The output of CH1 is generated first, in our setting that image name will be output_dapi.tif.
• Channel dependent extensions and overlay: CH1, _dapi; CH2, _gfp; CH3, _rfp; CH4, _bf; Overlay, _ovly
• Uncompressed and compressed tifs, .tif and _small.tif, respectively

The microscope allows the user to image up to three slides in one run (Multi-Point acquisition). When using Multi-Point acquisition, however, only one common name can be entered in the Capture-window (example: myExp).
The Analyzer output for three slides that are imaged together is the following:
A folder myExp that contains up to three subfolders, XY01, XY02, XY03. The names XY01-3 correspond to the position of the slides in the slide holder.

This script allows the user to define up to three slide names by using #.

Example: Slide positions XY01-3 are used. Slide names can be specified individually if these names are preceeded by the # symbol.

Script output for three imaged slides, example shown for slide 2:

Input option 1. Common name entered, myExp. Output for slide 2: myExp_slide2
Input option 2. Common and slide names entered, myExp#Cond1#Cond2#Cond3. Output for slide 2: myExp_Cond2
Input option 3. Slide names entered, #Apples#Pears#Oranges. Output for slide 2: Pears

An example for the entire output for slide 2 (XY02) using Input option 2, acquired with CH2 (here: GFP) and CH4 (here: Brightfield):

1. Uncompressed overlay: myExp_Cond2_ovly.tif
2. Uncompressed brightfield: myExp_Cond2_bf.tif
3. Uncompressed gfp: myExp_Cond2_gfp.tif
4. Compressed overlay: myExp_Cond2_ovly_small.tif
5. Compressed brightfield: myExp_Cond2_bf_small.tif
6. Compressed gfp: myExp_Cond2_gfp_small.tif

Error handling: If an unrecognized input name, e.g. the subfolder name XY04 appears, the output name generated with Input option 2 would be myExp#Cond1#Cond2#Cond3_XY04.

To stitch images with this script the Keyence BZ-X800 Analyzer software needs to be installed, including the locally plugged USB dongle that enables Keyence's stitching add-on. If the installation is for some reason incomplete, the Load a Group button will not respond and stitching is not possible. Autohotkey, Fiji/ ImageJ, and 7-zip need to be installed.

• Download this script and save it to a folder. This will be your installation folder, for example:

C:\Tools\AutoStitch-Keyence\

• Edit ahkStitch.ahk, set the path to the installation folder (lines 6 and 9) and to the external programs (lines 63, 64 and 65):

6   STITCH_BASE_DIR := "C:\Tools\AutoStitch-Keyence\"
9   #include C:\Tools\AutoStitch-Keyence
...
63	    options["keyenceAnalyzer"] := "C:\Program Files\Keyence\BZ-X800\Analyzer\BZ-X800_Analyzer.exe"
64	    options["imageJ"] := "C:\Program Files\fiji-imageJ\Fiji.app\ImageJ-win64.exe"
65	    options["gzip"] := "C:\Program Files\7-Zip\7zg.exe"

• Edit run-instructions\runStitch.ahk,set the path to the installation folder (line 3):

3   #include C:\Tools\AutoStitch-Keyence\ahkStitch.ahk

• Copy the folder run-instructions on your Desktop and follow the instructions of the included README.txt file.