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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.