Image Analysis

Introduction to digital images:

• How an image is obtained, sampling, monochromatic and color images, Fourier transforms

Introduction to Fiji and objects segmentation:

• Introduction to Fiji’s key functionalities: installation, basic options, opening of images (Bio-Formats Import), management of simple images, export of analysed images
• Measurements of images: image calibration, set measurements parameters, creation of selections and Region Of Interest
• Processing images: geometric transformations, filters, mathematical operations
• Binarization of images: image thresholding methods, processing of binary images
• Objects segmentation and detection: Analyse particles/Find Maxima functionalities

Hands-on part 1: Nuclear segmentation and spots detection:

• Step-by-step creation of an image analysis workflow for nuclear segmentation and nuclear spots detection

Hands-on part 2: Nuclear segmentation and spot detection:

• Step-by-step recording of macro commands within the analysis workflow

Introduction to Macro Language:

• Introduction to macro language: What is a macro? Why do we need macros?
• How can move from a set of recorded commands to a macro for batch processing?
• Built-in functions in FIJI: basics imaging analysis functions
• Basics of programming in Fiji macro language with examples: variables, arrays, loops, conditional statements
• Import and export of files: bio-formats and bio-formats macros extensions

Hands-on Macro Language:

• Hands-on: Generalization of a series of recorded commands to create a macro for batch nuclear segmentation

Introduction to Python/Jython programming languages:

• From ImageJ macro language to Jython programming language in Fiji
• Python basics: syntax, statements, expressions, list, loops, conditional statements, functions
• From Python to Jython: object oriented programming
• Jython main APIs and Classes for Fiji
• Python vs Jython: Pros and cons

Hands-on: practice on a nuclei segmentation macro and comparison with Jython language:

• Implementation of nuclei segmentation
• Focus on Regions Of Interest Manager
• Creation of customized Results table

More complex image analysis and Analysis of Multiple Images

Hands-on: nuclei segmentation with spots count and intensity measurement:

• Bio-formats handling
• User interaction
• Multiple channel handling
• Parent-children objects handling
• Multiple Results Table management
• Multiple images analysis

Segmentation in tissues with QuPath software:

• Introduction to QuPath for tissue sections analysis
• Cell segmentation in tissues

Hands-on: Cell segmentation in tissues using a pre-trained deep learning model and cell-type classification in different populations

Examples of Image Analysis in Fiji (Collective Motion and Tracking):

• Introduction to the concepts of collective cellular mobility and motivation
• Different ways and parameters to study collective cell motility:
  Wound Healing, Cell Streaming, Particle Image Velocimetry to evaluate speed of wound closure, velocity and coordination of movement

Images for publication and guidelines for best practices in image processing

Introduction to machine learning and deep learning in image analysis:

• Theoretical introduction about machine learning and deep learning
• Machine learning as tool for semantic segmentation/pixel classification
• Deep Learning as tool for image restoration and instance segmentation

Image restoration and instance segmentation using Deep Learning in Python:

• Introduction to Jupyter (Python) notebooks and their use in cloud with ZeroCostDL4Mic
• Deep Learning algorithms for image restoration and instance

Hands-on: Image restoration and nuclear segmentation with Deep Learning algorithms:

• Image restoration using Noise2Void algorithm; model training and application using Jupiter notebooks in ZeroCostDL4Mic
• Nuclear segmentation using 2D and 3D Stardist using Jupyter notebooks in ZeroCostDL4Mic

Cell segmentation using Cellpose software in Python:

• Introduction to conda environments and their utility for using local Jupyter (Python) notebooks
• Cellpose Deep Learning software for cell and nuclear segmentation

Hands-on: Segmentation of cells and nuclei using CellPose and evaluation of cytoplasmic signal using local Jupyter notebooks