FBI-AT 2022
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FBI-AT 2022 – Description

The course will have plenary lectures given by experts in the microscopy development field. These seminars will be advertised as a series and will be broadcasted for a large audience.  In addition, specific techniques will be introduced.

Hands-on practicals will train attendants on these techniques every afternoon in different sites in Paris including Institut Curie, Institut Pasteur, Institut Cochin, Institut Jacques Monod, Institut de Psychiatrie et Neurosciences de Paris and ENS Paris. Access to this part of training will be restricted to selected and registered trainees.

To guaranty access to set-ups and proper training, each practical session will host only 3-4 persons. The sessions will be run in parallel.

Register now, attendance will be limited to 25 participants! 

FBI-AT is ideal for researchers with a basic training in microscopy willing to become familiar with advanced techniques to answer their specific biological questions, or to be exposed to new developments that will allow them to tackle new questions in their project. We will consider applications from early career researchers (PhD students, post-docs), technical staff members and more senior scientists.

List of practicals

Practical 1: Combining micro UV-irradiation and Single Particle Tracking in living cells
OrganisersDescriptionLectures
Judith Miné-Hattab, David Mazaud, Patricia Le Baccon, Fabiola Garcia Fernandez
In this module, we will learn how to perform Single Particle Tracking (SPT), a powerful technique allowing the observation of individual molecules in living cells. We will learn: i) how to prepare samples, ii) how to perform the acquisition iii) and how to analyze trajectories of individual molecules. Moreover, we have combined our SPT system with a UV-laser micro irradiation module, allowing us to induce DNA damages and follow the dynamics of individual molecules as early as a few seconds after irradiation. We will use this module in human cells during the workshop.
Lectures associated to the practical session will be given by:

● Invited speaker 1: Joerg Bewersdorf (Yale School of Medicine, USA)
● Invited speaker 2: Christophe Leterrier (Institut de Neurophysiopathologie, France)
● Specific-module lecture 1: Judith Mine-Hattab (Institut Curie, France)

Practical 2*: Spectral detection multiphoton microscopy, signal unmixing with FLIM contrast & SMLM multi-color: from sample preparation to quantification
OrganisersDescriptionLectures
Thomas Guilbert, Julie Lesieur, Pierre Bourdoncle, IMAG’IC Institut Cochin
In this module, we will have a look on how to explore thick tissues with multiphoton microscopy. The advantages and disadvantages of this technique will be reviewed in the context of the exploration of thick, fixed or living tissue imaging. Tips and tricks will be shared and a focus will be made on fluorescence lifetime imaging (FLIM) to separate auto fluorescence from specific fluorophore.
In parallel, we will cover the theoretical and practical basis in super-resolution microscopy based on the detection of individual molecules using the dSTORM (direct STOchastic Reconstruction Microscopy) method. For this we will go from the preparation of the sample to the quantification of the images acquired in 2D and 3D
Lectures associated to the practical session will be given by:

● Invited speaker 1: Gaëlle Recher (Institut d’Optique d’Aquitaine, France)

Practical 3: FRET-based molecular tension sensors and FLIM
OrganisersDescriptionLectures
Nicolas Borghi, Xavier Baudin
The aim of the module is to show our recent developments in the field of FRET-based molecular tension sensors, and generally FRET-based biosensors and imaging. We will address biosensor design, fluorophore choices, acquisition modalities and analysis.
The attendees will learn hands-on how to perform FRET-based imaging in spectral and FLIM modes of one, two or more biosensors simultaneously, and to analyze their data.
Lectures associated to the practicle session will be given by:
• Invited speaker 1: Giulia Bertolin (IGDR)
• Invited speaker 2: Marie Erard (U Paris Sud)
• Specific-practicle lecture 1: Nicolas Borghi (IJM)
• Specific-practicle lecture 2:  Nom (affiliation)
Practical 4: Imaging of cellular ultrastructures with expansion microscopy
OrganisersDescriptionLectures
Juliette Azimzadeh
In this module, we will explain how to process and image samples using expansion microscopy. Students will learn hands-on the key steps in sample preparation, including the preparation and handling of the polymer hydrogels used for this technique. In the context of this training, the protocol will be applied to mammalian culture cells. Students will then perform high-resolution imaging of expanded samples using a confocal microscope equipped with an Airyscan detector.
Lectures associated to the practical session will be given by:
• Invited speaker 1: TBA
• Invited speaker2: TBA
• Specific-practical lecture 1: Juliette Azimzadeh (IJM, France)


Practical 5*: SIM, STED or STORM ? : from sample prep to 3D imaging & 3D STED : Comparing flat cells vs thick samples

OrganisersDescriptionLectures
Philippe Bun, David Geny, Julie Nguyen, Lydia Danglot , Institut de Psychiatrie et Neurosciences de Paris
The aim of the module is to illustrate what are the consideration to have in mind before planning an experiment of super resolution microscopy. Through international invited speakers and 4 practicals on NeurImag imaging facility, we will expose what are the tips and tricks to improve and take the most benefit of each kind of super-resolution microscopy modality (SIM, STED and STORM). The module will address both thin cells in culture and also thick samples like organoïdes or thick tissue slices.
Lectures associated to the practical session will be given by:

  • Invited speaker 1: BEWERSDORF Joerg (Yale School of Medicin, USA) – Expansion microscopy and Single Molelule Localization Microscopy
  • Invited speaker 2: LETERRIER Christophe (Marseille, FR) – STORM microscopy in neuronal cell culture
  • Invited speaker 3: Sandrine LEVEQUE-FORT (Orsay, FR) – STORM in thick tissue through structured illumination
  • Specific-practical lecture 1: DANGLOT Lydia (IPNP, FR)


Practical 6*: Culturing and imaging multicolour 3D live brain organoïds & Combining fast imaging on 3D live sample with Z resolution preservation

OrganisersDescriptionLectures
Philippe Bun, David Geny, Julie Nguyen, Lydia Danglot , Institut de Psychiatrie et Neurosciences de Paris
The aim of the module is to focus on imaging of 3D live samples using various microscopy approaches, as Light Sheet microscopy, SO-SPIM microscopy, or 3D fast Airyscan. Imaging of both cell culture or thick organoïdes will be presented by speakers and within practicals with a focus on multiscale visualization.
Lectures associated to the practical session will be given by:

  • Invited speaker1: SIBARITA Jean-Baptiste (Bordeaux, FR) – SOSPIM technology
  • Invited speaker2: Gaelle RECHER (bordeaux, FR) – STORM microscopy in neuronal cell culture
  • Invited speaker 3: Eduardo QUINTAS (Basel, CH) – Light Sheet microscopy and multiscale visualization


Practical 7*: Biological structures imaging in 3D using double helix-STORM and 3D-SIM

OrganisersDescriptionLectures
Audrey Salles, Mickael Lelek
During this module, we will present to the attendees the process to image a biological structure using 3D-STORM and 3D-SIM. Using the same sample, we will show the gain in spatial resolution between using STORM method but also the temporal gain using SIM microscopy.  During the workshop, the trainees will work on a home made system STORM and commercial SIM microscope (Elyra 7 – Lattice SIM from Carl Zeiss). Concerning the STORM experiments, 2D and 3D acquisitions will be acquired. In the 3D acquisitions, the axial positions of the single molecule will be encoded in a non common PSF (Double Helix PSF). ZOLA-3D software will be used to localize single PSFs and to visualize the 3D high resolution image of the imaged samples. Some important notions seen during the lecture will be introduced in order to get the best high resolution image.  Concerning the SIM experiments, the same biological structure will be used. For this modality, a detailed explanation of the advantages and disadvantages of structured illumination will be given as well as a focus on time resolution and live sample imaging. Part of the session will be devoted to the artefacts that can be generated in super-resolution imaging followed by a discussion of the methods and tools for diagnosing them. Then the trainees will appreciate the gain in resolution between the both systems.
Lectures associated to the practical session will be given by:

  • Invited speaker 1: Lothar Schermelleh
  • Specific-practical lecture 1:  Audrey Salles and Mickael Lelek 



Practical 8: Light sources for optogenetics

OrganisersDescriptionLectures
Isabelle Aujard, Ludovic Jullien, Aliénor Lahlou, Thomas Le Saux
The aim of the module is to implement simple and reliable protocols to measure light intensity and characterize maps of light intensity at the sample in one photon microscopy. It will be relevant for end-users, who would like to implement optogenetics in their research group without any previous experience in the field.

Lectures associated to the practical session will be given by:

  • Invited speaker1: DEDECKER Peter (KU Leuven, BE) 
  • Invited speaker 2: DEO Claire (EMBL, D) 
  • Specific-module lecture 1: JULLIEN Ludovic (ENS, FR) 
  • Specific-module lecture 2: GAUTIER Arnaud (Sorbonne University, FR) 



Practical 9: Non-classical genetically modified fluorescent probes for biological imaging

OrganisersDescriptionLectures
Arnaud Gautier, Ludovic Jullien, Thomas Le Saux, Marie-Aude Plamont
The aim of the module is to demonstrate the opportunities provided by new fluorescent probes and imaging protocols in biological imaging. More specifically, this module will illustrate the possibilities provided by (i) the emergent chemogenetic FAST system, which enables for reversible and tunable fluorescent labeling in a wide range of emission wavelengths, and (ii) the reversibly photoswitchable fluorescent proteins, which enable to overcome limitations such as autofluorescence, ambient light, or multiplexing in fluorescence imaging.

Lectures associated to the practical session will be given by:

  • Invited speaker1: DEDECKER Peter (KU Leuven, BE) 
  • Invited speaker 2: DEO Claire (EMBL, D) 
  • Specific-module lecture 1: JULLIEN Ludovic (ENS, FR) 
  • Specific-module lecture 2: GAUTIER Arnaud (Sorbonne University, FR) 



*Two-fold practical