Running ASHLAR
This page serves as a reference guide for ASHLAR syntax. Refer to the quick start guide for step-by-step instructions.
Description
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.
Input
An .ome.tiff file of unstitched* images
Many BioFormats-supported microscope vendor file formats are supported. Further information on how to determine if your file format is compatible will be added soon.
*Ashlar requires unstitched individual “tile” images as input - it is not suitable for microscopes or slide scanners that only provide pre-stitched images. A list of microscopes that we know are compatible with ASHLAR will be coming soon.
Output
A pyramidal, tiled .ome.tif
Usage:
Stitch and align one or more multi-series images
ashlar FILE [FILE ...] [OPTIONS]
Required arguments
| Name | Description |
|---|---|
FILE |
an image file to be processed (one file per cycle) |
Optional arguments
| Name; Shorthand | Description | Default |
|---|---|---|
--help; -h |
Show this help message and exit | |
--output DIR; -o DIR |
Write image files to DIR | Current directory |
--align-channel CHANNEL; -c CHANNEL |
Align images using channel number CHANNEL | Numbering starts at 0 |
--flip-x |
Flip tile positions left-to-right to account for unusual microscope configurations | |
--flip-y |
Flip tile positions top-to-bottom to account for unusual microscope configurations | |
--flip-mosaic-x |
Flip mosaic image horizontally | |
--flip-mosaic-y |
Flip mosaic image vertically | |
--output-channels CHANNEL [CHANNEL...] |
Output only channels listed in CHANNELS | Numbering starts at 0 |
--maximum-shift SHIFT; -m SHIFT |
Maximum allowed per-tile corrective shift in microns | |
--filter-sigma SIGMA |
Width in pixels of Gaussian filter to apply to images before alignment | Default is 0 (which disables filtering) |
--filename-format FORMAT; -f FORMAT |
Use FORMAT to generate output filenames, with {cycle} and {channel} as required placeholders for the cycle and channel numbers | default is cycle_{cycle}_channel_{channel}.tif |
--pyramid |
Write output as a single pyramidal TIFF | |
--tile-size PIXELS |
Set tile width and height to PIXELS (pyramid output only) | Default is 1024 |
--ffp FILE [FILE ...] |
Read flat field profile image from FILES | If specified must be one common file for all cycles or one file for each cycle |
--dfp FILE [FILE ...] |
Read dark field profile image from FILES | If specified must be one common file for all cycles or one file for each cycle |
--plates |
Enable mode for multi-well plates (for high-throughput screening assays) | |
--quiet; -q |
Suppress progress display | |
--version |
Print version |
Note: Detailed information on how to tune these parameters will be added soon.