Ashlar performs fast, high-quality stitching of microscopy images. It also co-registers multiple rounds of cyclic imaging for methods such as CyCIF and CODEX. Ashlar can read image data directly from BioFormats-supported microscope vendor file formats as well as a directory of plain TIFF files. Output is saved as pyramidal, tiled OME-TIFF.
Note that Ashlar requires unstitched individual "tile" images as input, so it is not suitable for microscopes or slide scanners that only provide pre-stitched images.
Visit labsyspharm.github.io/ashlar/ for the most up-to-date information on ASHLAR.
ashlar [-h] [-o PATH] [-c CHANNEL] [--flip-x] [--flip-y]
[--flip-mosaic-x] [--flip-mosaic-y]
[--output-channels CHANNEL [CHANNEL ...]] [-m SHIFT]
[--stitch-alpha ALPHA] [--filter-sigma SIGMA]
[--tile-size PIXELS] [--ffp FILE [FILE ...]]
[--dfp FILE [FILE ...]] [--plates] [-q] [--version]
FILE [FILE ...]
Stitch and align multi-tile cyclic microscope images
positional arguments:
FILE Image file(s) to be processed, one per cycle
optional arguments:
-h, --help Show this help message and exit
-o PATH, --output PATH
Output file. If PATH ends in .ome.tif a pyramidal OME-
TIFF will be written. If PATH ends in just .tif and
includes {cycle} and {channel} placeholders, a series
of single-channel plain TIFF files will be written. If
PATH starts with a relative or absolute path to
another directory, that directory must already exist.
(default: ashlar_output.ome.tif)
-c CHANNEL, --align-channel CHANNEL
Reference channel number for image alignment.
Numbering starts at 0. (default: 0)
--flip-x Flip tile positions left-to-right
--flip-y Flip tile positions top-to-bottom
--flip-mosaic-x Flip output image left-to-right
--flip-mosaic-y Flip output image top-to-bottom
--output-channels CHANNEL [CHANNEL ...]
Output only specified channels for each cycle.
Numbering starts at 0. (default: all channels)
-m SHIFT, --maximum-shift SHIFT
Maximum allowed per-tile corrective shift in microns
(default: 15)
--stitch-alpha ALPHA Significance level for permutation testing during
alignment error quantification. Larger values include
more tile pairs in the spanning tree at the cost of
increased false positives. (default: 0.01)
--filter-sigma SIGMA Filter images before alignment using a Gaussian kernel
with s.d. of SIGMA pixels (default: no filtering)
--tile-size PIXELS Pyramid tile size for OME-TIFF output (default: 1024)
--ffp FILE [FILE ...]
Perform flat field illumination correction using the
given profile image. Specify one common file for all
cycles or one file for every cycle. Channel counts
must match input files. (default: no flat field
correction)
--dfp FILE [FILE ...]
Perform dark field illumination correction using the
given profile image. Specify one common file for all
cycles or one file for every cycle. Channel counts
must match input files. (default: no dark field
correction)
--plates Enable plate mode for HTS data
-q, --quiet Suppress progress display
--version Show program's version number and exit
Ashlar can be installed in most Python environments using pip:
pip install ashlarIf you don't already have miniconda or Anaconda, download Anaconda and install. Then, run the following commands from a terminal (Linux/Mac) or Anaconda command prompt (Windows):
Create a named conda environment with python 3.12:
conda create -y -n ashlar python=3.12Activate the conda environment:
conda activate ashlarIn the activated environment, install dependencies and ashlar itself:
conda install -y -c conda-forge numpy scipy matplotlib networkx scikit-image scikit-learn tifffile zarr pyjnius blessed
pip install ashlarThe docker image of ashlar is on DockerHub at labsyspharm/ashlar and should be suitable for many use cases.