Summary and Schedule
This lesson provides an overview of the fundamentals of imaging data that has been acquired through light microscopy experiments. This course is aimed at all researchers who are looking to work with light microscopy imaging data, or to refresh their knowledge on some of the key fundamentals.
Prerequisites
Some prior experience with acquiring/working with light microscopy data would be useful, but isn’t necessary. This course is aimed at anyone who is looking to work with light microscopy imaging data in their research projects.
No python experience is necessary or assumed, but you will get more out of the course if you know some basics e.g. from the online Software Carpentry python course or an in-person course at UCL.
Please bring a laptop (Windows, Mac or Linux) to the lesson. Before the start of the course, please follow the setup instructions so that your laptop is ready to run the material in the lessons.
Setup Instructions | Download files required for the lesson | |
Duration: 00h 00m | 1. Imaging Software |
What are the different software options for viewing microscopy
images? How can Napari be used to view images? |
Duration: 00h 55m | 2. What is an image? | How are images represented in the computer? |
Duration: 01h 50m | 3. Image display | How are pixel values converted into colours for display? |
Duration: 02h 45m | 4. Multi-dimensional images | How do we visualise and work with images with more than 2 dimensions? |
Duration: 03h 45m | 5. Filetypes and metadata | Which file formats should be used for microscopy images? |
Duration: 04h 40m | 6. Designing a light microscopy experiment |
What are the key factors to consider when designing a light
microscopy experiment? |
Duration: 05h 35m | 7. Choosing acquisition settings | What are the key factors to consider when choosing acquisition settings? |
Duration: 06h 30m | 8. Quality control and manual segmentation |
What is segmentation? How do we manually segment images in Napari? |
Duration: 07h 25m | 9. Filters and thresholding |
How can thresholding be used to create masks? What are filters and how do they work? |
Duration: 08h 25m | 10. Instance segmentation and measurements |
How do we perform instance segmentation in Napari? How do we measure cell size with Napari? How do we save our work to create re-usable workflows? |
Duration: 09h 25m | 11. Additional resources | Where can I find further information? |
Duration: 09h 35m | Finish |
The actual schedule may vary slightly depending on the topics and exercises chosen by the instructor.
Download Data
During these lessons we will be using two microscopy images and
creating images of our own. Create an empty folder somewhere on your
computer to use during the course, this will be your
working directory
. You can name it anything you like but we
recommend microscopy-ideas
.
Download 00001_01.ome.tiff (CC BY 4.0 Licence) from the open microscopy environment pages to your working directory.
Download Plate1-Blue-A-12-Scene-3-P3-F2-03.czi (CC BY 4.0 Licence) from the open microscopy environment pages to your working directory.
Software Setup
During these lessons we will use the napari image viewer and the napari-aicsimagio plugin to load imaging data files of various file formats. For beginners we recommend using Anaconda to install and manage these packages. The first step is to download and install Anaconda.
The remaining instructions are written assuming you will be installing and running the software from a terminal. Follow the instructions below to open a terminal on your operating system.
- Windows: Click Start > Search for Anaconda Prompt > Click to Open
- macOS: Launchpad > Other Application > Terminal
- Linux: Open a terminal window
For more information on starting and running Anaconda you may visit the ‘Starting Conda’ section of the Anaconda getting started pages.
Run the following commands in the terminal (lines starting with # are comments and do not need to be run). You can run the commands by copy and pasting them into the terminal and pressing the Enter key.
BASH
# Make sure conda is up to date
conda update -n base conda
# We need to use a python program called pip to install
# the napari-aicsimagio plugin, so we need to configure
# conda to work well with pip
conda config --set pip_interop_enabled True
# This line will create a "virtual environment" (called napari-env)
# that will contain all of the software that will be used in the lessons
conda create -y -n napari-env -c conda-forge python=3.10 bioformats_jar
# Activate the napari-env virtual environment.
# Running this should change the terminal prompt to '(napari-env)'.
conda activate napari-env
# Install napari and plugins using pip
pip install "napari[all]" napari-aicsimageio
If you’re new to running commands in the terminal you may be alarmed if running some of these commands results in a lot of text output to the screen. This is normal and usually helpful for checking things are working OK. For our purposes you can ignore it. If you see the word ERROR appear near the end (often highlighted in a different colour) it is possible something has gone wrong. In this case the best place to start is any messages in the text expanding on the source of the error.
If you are using an M1/M2 mac, you may encounter errors when running the commands above. If so, try the alternative commands below:
BASH
# Remove the old environment
conda remove --name napari-env --all
# This line will create a "virtual environment" (called napari-env)
# that will contain all of the software that will be used in the lessons
conda create -y -n napari-env -c conda-forge python=3.10 napari pyqt bioformats_jar napari-aicsimageio
# Activate the napari-env virtual environment.
# Running this should change the terminal prompt to '(napari-env)'.
conda activate napari-env
# Install czi file reader
pip install aicspylibczi>=3.0.5 fsspec>=2022.7.1
Post Setup Checks
Before starting the course it is important to check that your setup is working.
Check napari opens a tiff image.
Any napari installation should be able to open a tiff image. Start
napari. Note that if you’re already in the napari-env virtual
environment from the installation steps it is not necessary to run
conda activate napari-env
.
You should now see the napari viewer like this …
![A screenshot of the default Napari user interface](../fig/blank-napari-ui.png)
Open a file with
File > Open file(s)...
- Navigate to the directory where you saved
00001_01.ome.tiff
earlier on. - Select
00001_01.ome.tiff
and click open. - If you see a
Choose reader
dialog, selectnapari builtins
.
![A screenshot of freshly opened 00001_01.ome.tiff](../fig/ome_00001.png)
Close napari: File > exit
Check napari opens a czi image.
During the course we’ll be working with czi images. To open these the napari-aicsimageio plugin is needed. Let’s check the plugin is working.
Start napari. Note that if you’ve already done the previous test
(tiff image) then it should not be necessary to run
conda activate napari-env
. However, it is necessary to
restart napari to ensure the image display is correctly formatted.
File > Open file(s)...
- Navigate to the directory where you saved
Plate1-Blue-A-12-Scene-3-P3-F2-03.czi
to earlier on. - Select
Plate1-Blue-A-12-Scene-3-P3-F2-03.czi
and click open. - If you see a
Choose reader
dialog, selectnapari-aicsimageio
.
![A screenshot of freshly opened Plate1-Blue-A-12-Scene-3-P3-F2-03.czi](../fig/plate1-blue.png)
Close napari: File > exit