Hands-on

Rotation of the Hands-on:

 Brief description 

Marie Erard (ICP)

Characterization of spectroscopic properties of a probe 

• How to choose the right fluorescent protein for your experiment? What are the important parameters?
• Spectra acquisition et properties (absorption, fluorescence)
• Lifetime measurements
• Photobleaching observation and characterization (if possible) 

A few key words on methods :

• Probes : Fluorescent proteins
• Biological question : none 
• Instrument : Spectrometer, time resolved fluorescence, wide field microscope
• Data Analysis : none

Location (adress and meeting point): Institute of Physical Chemistry, building 350 on the Orsay campus

Dounia Zamiati (ICP)

Detection of FRET by FLIM to probe protein interactions at membrane contact sites

Forster resonance energy transfer (FRET) between two fluorescent proteins (a donor and an acceptor) fused to proteins of interest is an efficient approach to monitor their interaction. We assess the FRET efficiency with the direct measurement of donor fluorescence lifetime (FLIM). 

This hands-on will present how to design such a FRET-FLIM experiment to monitor membrane contact sites. We will discuss the choice of FPs, the required controls and the data processing.

A few key words on methods :

• Probes : Fluorescent proteins
• Biological question : identification of protein interactions membrane contact sites
  
• Instrument : Fluorescence lifetime Imaging (FLIM)
  
• Data Analysis : SymphoTime64, Picoquant

Location (adress and meeting point): Institute of Physical Chemistry, building 350 on the Orsay campus

Karen Perronet (LuMIn)

2D single-particle tracking using Total Internal Reflection Fluorescence Microscopy

The goals of this labwork are :

• To explain and show how TIRFM works
•  Acquire data from basic samples
• Introduce how we measure intraneuronal transport in cultured neurons
• Explain the analysis pipeline developed in the lab to extract pertinent parameters of this transport  

A few key words on methods :

• Probes : organic dyes or nanoparticles (fluorescent nano-diamonds)
• Biological question : intraneuronal transport
• Instrument : TIRFM
• Data Analysis : Modified Intraneuronal Nanoparticle Tracking (MINT)

Location (adress and meeting point): ENS Paris-Scalay 4 avenue des Sciences 91190 Gif sur Yvette – wait in the main lobby.

Antoine Pallandre (ICP)

Microfluidic and optical detection  

This practical training will allow you to start learning about microfluidic. Interfacing with Maltlab software to perform synchronization of optics, fluidic and electrical instruments will be explained in details. We will also build a microfluidic chip from a 3D printer before using it for microscopic observations. The use of different cameras (CMOS, CDD and modified webcam) will help you to better determine your need for your future microfluidic experiments.

A few key words on methods : coding, microscopy, 3D printing, fluorescence, microfluidic and data acquisition

• Probes : rhodamine and derivatives
• Biological question : how to store and stimulate living systems ?
• Instrument : homemade set-up based on a Zeiss inverted microscope
• Data Analysis : Matlab

Location (adress and meeting point): Institute of Physical Chemistry, building 350 on the Orsay campus

Marie-Noëlle Soler (Institut Curie Centre de Recherche)

Proteins recruitment after DNA damages on living cells, imaging and analysis

• To understand, depending on the biological question, which imaging modality should be used.
• To know how to highlight and/or follow cellular processes/markers thanks to the different methods and techniques of light imaging
• To know the limits of the imaging techniques used

After DNA damages with 2 different lasers wavelengths, an acquisition timelapse allow to measure the recruitment of PARP2 protein in the damaged region.

A few key words on methods :

• Probes : PARP2-GFP
• Biological question : PARP2 recruitment after DNA damage
• Instrument : Laser Scanning Confocal Microscope and FRAP technique
• Data Analysis : ImageJ Macro 

Location (adress and meeting point): Institut Curie, Centre de Recherche sur le Centre Universitaire, building 110, Rue Henri Becquerel, 91401 Orsay

Sandrine Lecart (I2BC)

Investigation of endolplasmic reticulum – mitochondria contact sites using structured illumination microscopy (S.I.M.) 

Mitochondria–endoplasmic reticulum contact sites are unique environments within cells where important processes occurs (calcium homeostasis / lipid metabolism / autophagosome formation …). It is essential to understand its structure and identify the proteins recruited at those specific locations. To investigate this question a high level of resolution is required. In this workshop we will use a super resolution light microscopy technic (SIM) to image the ER-Mitochondria contact sites.

A few key words on methods :

• Probes : Conventionnal probes (BFP / MitoTracker / Alexa)
• Biological question : Anderstanding the organization of the membrane contact sites within the cell
• Instrument : Sructured Illumination Microscopy (Elyra7 Lattice SIM / Zeiss)
• Data Analysis : Zen Black (Zeiss)

Location (adress and meeting point): 1 Avenue de la terrasse, building 21 (ground floor of the Hall), 91198 Gif-sur-Yvette cedex

Monitoring of intracellular pH with a ratiometric biosensor in living cells  

The legume-rhizobium symbiosis is characterized by the formation of symbiotic root nodules, in which the bacteria differentiate into nitrogen-fixing bacteroids. In order to identify novel features of terminal bacteroid differentiation, we tested on free-living bacteria and bacteroids undergoing TBD (Sinorhizobium meliloti in Medicago nodules) a novel genetically encoded ratiometric fluorescent biosensor (pHP, derived from GFP) to estimate intracellular pH. A complementary approach in flow cytometry and imaging makes it possible to calibrate the biosensor, and to combine the statistical power of cytometry and the resolving power at the tissue level of imaging.

A few key words on methods :

• Probes : PHP sensor (derivative of GFP)
• Biological question : Measuring intracellular pH in Sinorhizobium meliloti bacteria to understand involvement of acidification in differentiated nitrogen-fixing bacteroids
• Instrument : Cytometer Cytoflex S
• Data Analysis : Cytexpert software and Rstudio

Location (adress and meeting point): 1 Avenue de la terrasse, building 21 (ground floor of the Hall), 91198 Gif-sur-Yvette cedex

 

Sophie Dupré (ICP) 

Dynamic analysis of lipids in cell membranes  

AIM : Imaging phospholipid dynamics during stimulation of leukocytes 

Cells : A leukocyte cell line (neutrophil-like) transfected by one or two lipid biosensors  

Method : 4D video microscopy or TIRF microscopy will be performed in order to follow the localization of the lipid biosensors (phosphoinositide biosensors) at the cell membranes during the stimulation by particle phagocytosis or frustrated phagocytosis. 

A few key words on methods :

• Probes : lipid binding domain coupled to fluorescent proteins
• Biological question : How to follow phospholipid dynamics in cell membrane during cell stimulation ? 
• Instrument : spinning disk confocal microscopy (Imagerie-Gif platform)
• Data Analysis : image analysis with metamorph software and/or ImageJ/Fiji

Location (adress and meeting point): 1 Avenue de la terrasse, building 21 (ground floor of the Hall), 91198 Gif-sur-Yvette cedex

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Measuring bacteria internal pH using PHP biosensor and light microscopy 

The legume plants - bacteria symbiosis is characterized by the formation of symbiotic root nodules, in which the bacteria differentiate into nitrogen-fixing bacteroïds. In order to identify novel features of bacteroïd differentiation, we tested a novel genetically encoded ratiometric fluorescent biosensor (pHP, derived from GFP) to estimate intracellular pH.  To exploit this biosensor we need to master multiple steps:

• Generate a calibration curve of the PHP biosensor on the microscope.
• Measure the fluorescence of the biosensor in the different conditions of interest
• Analyze the images to retrieve pH values form the ratiometric fluorescent signals acquired.

A few key words on methods :

• Probes : PHP biosensor (derivative of the GFP)
• Biological question : Measuring intracellular pH in the Sinorhizobium meliloti bacteria to understand modifications occurring during the symbiosis.
• Instrument : Confocal Laser Scanning Microscope SP8-X (Leica)
• Data Analysis : FIJI (custom scripts)

Location (adress and meeting point): 1 Avenue de la terrasse, building 21 (ground floor of the Hall), 91198 Gif-sur-Yvette cedex

Single Molecule Localization Microscopy (Super-resolution imaging)

Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution ofSingle Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. In this workshop, we will demonstrate how to perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples.

A few key words on methods :

• Probes : immunofluorescence (dSTORM, DNA-PAINT)
• Biological question : optimization of an SMLM image
• Instrument : Single Molecule Localization Microscope
• Data Analysis : 2D/3D reconstruction

Location (adress and meeting point): Institut des Sciences Moléculaires d’Orsay (ISMO, building 520, rue André Rivière)