Image Registration for Multiplexing Experiments in arivis Pro

How to: Align multiple 3D images based on the nuclei staining

This guides goal is to guide the user in the correct installation of the Anaconda3 Python package and the image registration workflow. This script will read multiple .czi image files (or SIS imagesets) in a given folder in a tile-wise manner and create the SIS files for each of the original files. During the registration process, the new SIS file will be created with each tile as a new imageset. The first cycle image will be copied from the original file and the image stacks from the following cycles will be registered to the first cycle image based on the indicated channel, typically, nuclei staining. In the last step, the Tile Sorter tool needs to be run to assemble the registered mosaic stack together. The resulting image can be used for the following image analysis.

Overview

To use image registration script in arivis Pro we need the following:

Install Anaconda Python for Vision4D
Import the registration environment in the Anaconda Navigator
Configure arivis Pro to install the arivis libraries into the registration environment
Define the required parameters and run the image registration script
Assemble the resulting image using the Tile Sorter tool in arivis Pro for the following analysis

Introduction

Image registration is the process of transforming different sets of data to overlay images from different experiments, taken at different times, positions or angles, or with different imaging modalities. The algorithm attempts to discover the matching areas and align them together. Image registration methods can be divided into two large groups: rigid and non-rigid transformations. For the multiplexing experiments, we will use the nuclei staining in each of the experiment cycles to register the subsequent experiments to the first image. This method is applicable both to 2D and 3D images.

The application workflow starts from importing the original image files (for example, CZI) in arivis Pro and registering the subsequent experimental cycles to the first one based on the indicated nuclei staining channel (i.e. DAPI or Hoechst). The images will be imported as a stack of individual tiles with all the channels from all the experiment cycles, each registered to the nuclei staining in the first experiment cycle. After the registration, the mosaic image should be stitched using the Tile Sorter tool.

The images used in this application note were published in https://www.sciencedirect.com/science/article/pii/S0165027022001807

Image registration in a nutshell

Application Workflow

The application workflow starts by importing the original image files in arivis Pro. For each staining/hybridization cycle the new SIS file will be created. All Z planes from all the tiles will be imported separately into a single stack (single imageset). The import step is optional. Alternatively, the user can start the registration workflow by simply opening the already stored SIS files.

The registration workflow starts from creating the new SIS file to store the registration results. The first cycle image (fixed) will be identified automatically based on the filename. This image will remain untransformed. The 3D stack for each subsequent cycle (moving) will be transferred as new channels in the corresponding tile in the registered image file. The number of tiles should be consistent for all staining cycles.

Preliminary Remarks

arivis Pro runs multiple applications (image registration) using external and independent Python libraries and tools produced by third parties. These tools must be installed by the user under its responsibility, strictly following the instruction on this document. arivis has tested the setup protocol on several computers, however, due to the different and not predictable hardware and software configuration of each computer, the results can be different case by case. Therefore, arivis declines any responsibility concerning the correct tools installation and setup on the user computer. arivis cannot be blamed about any malfunctioning or failure of the deep learning environment setup. arivis will not give technical support on the setup task. Both activities are totally on the user charge. arivis also declines any responsibility about the scientific results gathered from this application.

Installing the prerequisites

To use the registration script for multiplexed data in arivis Pro, we need 3 configuration steps:

Install Anaconda Python.
Import the registration environment.
Configure the arivis Pro Scripting preferences to use the environment.

Installing Anaconda

Instructions for downloading, installing, and configuring anaconda for arivis Pro can be found here.

Setting up the image registration environment

Application note

Download Full How to: Install and run predictions with Cellpose PDF