FBI-AT 2024 – Description
Light sheet fluorescence microscopy is a rapidly expanding imaging technique that has been elected method of the year in 2014 by the Journal “Nature Methods”. Thanks to its abilities unique among fluorescence microscopy methods, LSFM is revolutionizing a large number of areas of research: biology of development, neuroscience, plant imaging, cell biology, oncology, virology, immunology….
This technology makes it possible to image three-dimensional samples on very vast scales ranging from the entire animal to the individual cell and the single molecule, and this, in a very minimally invasive way. Its very high imaging speed combined with excellent spatial resolution makes possible the rapid acquisition of large volumes with a high level of detail.
This advanced training aims to (1) present the theoretical bases, (2) clarify and synthesize the different existing approaches to both sample and instrumental preparation and (3) provide an overview of treatment and management solutions of acquired data. These objectives will be addressed through the prism of two important biological fields of application: Neurosciences and 3D Cell Cultures.
The training program will be structured around 4 main thematic tracks, enabling us to address the issues of imaging sample preparation and data analysis for given samples. Participants will have the choice of following one of these tracks, or navigating between them according to their skills and interests. The tracks are :
- P1: Large sample imaging – Clearing & Expansion
- P2: 3D cellular models Culture & Imaging
- P3: Neuronal network imaging
- P4: Image Analysis
It will take the form of lectures and seminars in the morning, providing a theoretical grounding in the different areas covered (sample preparation, imaging, image processing) and presenting the latest developments in these fields, and workshops in the afternoon on the various sites of the Bordeaux node (IINS, BIC, VoxCell).
Apply now, attendance will be limited to 25 participants!
Workshops description
Thematic courses 1: Cleared or expanded large sample 3D imaging
Workshop P1-1: Whole brains imaging by Ultramicroscopy
The aim of the workshop will be to present the practical considerations for large mouse brain clearing, and then to carry out the imaging of these samples by ultramicroscopy. Finally, we will present a 3D analysis pipeline of the acquired data using the clearmap tool developed by Nicolas Renier .
Sample: Cleared whole mouse brains
Instrument: Ultramicroscope II (Miltenyi)
Workshop P1-2: 3D imaging of neuronal expanded samples by Ultramicroscopy
This workshop aims at presenting the practical considerations of sample expansion. Expanded neurospheres will then be imaged by Ultramicroscopy. Finally, considerations on 3D data-set handling, management and analysis will be discussed.
Sample: Expanded Neurospheres
Instrument: AxL (3i)
Workshop P1-3: 3D entire small animal imaging
In this workshop we will discuss the practical consideration for the clearing of entire embryonic mouse. We will then present their fast imaging by ultramicroscopy to study the primordial lymph nodes constitution at the early embryonic stages. An analysis pipeline using the software Imaris will finally be demonstrated in order to quantify the presence, the size and the location of cluster of Lymphoïd Tissue Inducer cells which lead to the formation of functional lymph nodes.
Instrument: Blaze (Miltenyi)
Thematic courses 2: 3D Cellular models – Culture & Imaging
Workshop P2-1: 3D Cellular models culture and imaging using the soSPIM technology
During this workshop, the soSPIM 3D screening platform will be presented. This technology allows to parallelize both the culture and the imaging of 3D cellular models. First, the culture of spheroids will be demonstrated using cancerous cell lines. Then, we will perform the automatic screening of hepato-organoids in 3D and multi-color. Finally, an automated analysis pipeline dedicated to the imaged samples will be presented that integrate both Deep-learning based segmentation and conventional image analysis.
Sample: Oncospheres and hepato-organoids
Instrument: soSPIM (Home-made)
Workshop P2-2: Micro-niche creation for 3D cell culture and 3D imaging using the HS-ISM technique
This workshop aims at demonstrating the creation of customizable micro-niches for the culture of 3D cellular models, such as Neurospheres using the 3D patterning technology developed by the company Alvèole. Those neurospheres will then be imaged in 3D and multi-color using an Hyper-spectral Instant Scanning Microscope (HS-ISM) that allows to image in depth and in multi-color at high- and Super-resolution 3D samples.
Sample: Neurospheres
Instrument: The PRIMO system (Alvéole) & a HS-ISM (home-made)
Workshop P2-3: Neurospheres culture and imaging using the MuViSPIM
This workshop will present a method to culture 3D neurospheres from rat primary neurons in ultra-low attachment multi-well plates. Considerations about the different labelling approaches will be discussed and demonstrated in particular to image the neuronal connection that has formed and their functional activity. The neurospheres will then be imaged (live or fixed) in 3D using the TrueLive3D system from Brucker. Finally, the HIVE storage and analysis solution will be presented.
Sample: Neurospheres from rat primary neurons
Instrument: TrueLive3D & HIVE (Brucker)
Thematic course 3: Neuronal network imaging
Workshop P3-1: Brain slices imaging using a Lattice Light Sheet Microscope
This workshop aims at demonstrating the capacity to image brain slices, either fixed or live, with the Lattice Light Sheet Microscope at high temporal and spatial resolutions. Considerations for mounting the sample for LLSM imaging will first be discussed. Then fixed and live brain slices will be imaged at high spatial and temporal resolution.
Sample: Fixed and living brain slices from rodents
Instrument: Lattice Light Sheet Microscope (Home-made)
Workshop P3-2: Single Cell electroporation for Brain slices labelling
This workshop aims at discussing and demonstrating the different strategies that exist to label brain slices for neuronal network imaging and functional imaging. First, the preparation and the culture of brain slices will be presented. Then, the different methods for brain slices labelling will be discussed (electroporation, virus infection, single cell electroporation, …) with a presentation of their range of applications. Finally, the single cell electroporation technique will be demonstrated on an electrophysiology microscope. This technique allows sparsely to label living neurons in brain slices enabling their accurate imaging without being impaired by important background signals using different 3D imaging approaches such as Lattice Light Sheet Microscopy (Workshop P3-1).
Sample: living brain slices from rodents
Instrument: Electrophysiology microscope (home-made)
Workshop P3-3: Functional neuronal network imaging in ZebraFish
Neuronal network functional light sheet imaging allows to monitor the activity of a neuronal network in 3D and real time. Such an approach will be demonstrated on a small animal model, the Zebrafish, using a line genetically modified to express a calcium activity reporter dye. The different imaging method to perform functional imaging within a Zebrafish will be discussed before to be presented using the MuViSPIM light-sheet system.
Sample: Living ZebraFish
Instrument: MuViSPIM or TrueLive3D (Bruker)
Thematic course 4: Image analysis
Workshop P4-1: Orchestrating complex bioimage workflows using the Arkitekt solution
In this workshop, we will explain the challenges of modern bioimage workflows, especially real-time data analysis and management. Furthermore, we will introduce our solution to this problem: Arkitekt – a powerful middleman between users and bioimage apps for building and orchestrating real-time analysis and microscopy workflows. The workshop will rely on conventional bioimage software (ImageJ, Napari, Micro-Manager) and modern deep learning frameworks (CARE, StarDist) to build advanced data pipelines that can go from acquisition to statistics. It will also demonstrate Arkitekt’s capability to set up a “Smart Microscopy” workflow by modifying microscope acquisition parameters in real time according to on-the-fly detection of specific events.
Instrument: Computers
Workshop P4-2: Napari for 3D data handling
Big 3D data-sets easily generated by Light-Sheet Fluorescence Microscopy raise several problems regarding their manipulation and analysis. Recently several solutions have been proposed to handle such big data-sets amongst them the Python-based solution Napari. This workshop aims at presenting the Napari solution, how to install it and use it to visualize and perform image analysis steps in this environment through the many plugins developed.
Instrument: Computers
Workshop P4-3: How to segment a 3D dataset in just a few clicks?
In this workshop, we propose to train participants to a new open-source tool dedicated to 3D and 3D+t microscopic images. We have developed a new way to interact with 3D images using the surface meshes of the segment objects. We use the powerful rendering and interactivity of meshes while each segmentation plugin is directly applied to the original images.
During this workshop we will first introduce the concept of the MorphoNet platform and explain classical usage of 3D (or 4D) interactions. And secondly train the participants to several plugins of 3D segmentation that we integrated in the MorphoNet software. Moreover we will show how to simply develop and add a new plugin to MorphoNet. At the end of this workshop the participants will be able to load their original microscope images in MorphoNet in order to perform interactive segmentation.
Instrument: Computers