LABEX INFORM A Focus on Imaging January 12th -16th, 2015 Training Objectives: This training is an introductory course to different microscopy techniques, dedicated to PhD students willing to explore the most advanced and popular techniques used for biological applications. The course is being held, in Luminy, in the laboratories (AFM, CIML, IBDM). It is composed of theoretical courses on the techniques basics and principle and hands-on practical in the laboratory in small groups (4 students). By the end of the training the students should have a good understanding of Basic optics, Wide Field Microscopy, Confocal Microscopy, F-techniques (FRAP), Electron Microscopy, Atomic Force Microscopy, Dyes, Detectors, Single molecule imaging, Super-resolution, Image processing and Quantitative analysis. Teachers : Aïcha Aouane – IBDM CNRS Sophie Brustlein – CIML INSERM Claire Chardes – IBDM CNRS Jean Paul Chauvin –IBDM CNRS Frederic Eghiaian – AFM INSERM Mathieu Fallet – CIML CNRS Yanick Hamon –CIML CNRS Sébastien Mailfert –CIML CNRS Cédric Matthews –IBDM CNRS Pierre Recouvreux –IBDM CNRS Fabrice Richard– IBDM CNRS Félix Rico – AFM INSERM Simon Scheuring – AFM INSERM aicha.aouane ’at’ univ-amu.fr brustlein ’at’ ciml.univ-mrs.fr claire.chardes ’at’ univ-amu.fr jean-paul.chauvin ’at’ univ-amu.fr frederic.eghiaian ’at’ inserm.fr fallet ’at’ ciml.univ-mrs.fr hamon ’at’ ciml.univ-mrs.fr mailfert ’at’ ciml.univ-mrs.fr cedric.matthews ’at’ univ-amu.fr pierre.recouvreux ’at’ univ-amu.fr fabrice.richard ’at’ univ-amu.fr felix.rico ’at’ inserm.fr simon.scheuring ’at’ inserm.fr Length : 5 days Theoretical courses: 11h30 Practical Works: 19h Requirements: Bring personal computer with spreadsheet software and ImageJ (download here: http://rsb.info.nih.gov/ij/download.html ) __________________________ _________________ LABEX INFORM ● IBDM UMR 7288 ● Case 907 Parc Scientifique de Luminy _________________ 13288 Marseille Cedex 9 France ● Téléphone : (+33) 491 26 93 95 Fax : (+33) 491 26 97 26 ___________________________ Mail: [email protected] ● ___________________________ _________________ LABEX INFORM COURSES Course 1: General Optics and wide field microscopy, Sophie Brustlein Light mater interaction, diffraction, fluorescence, sources and objective lenses. Course 2: From sensor to image, Cédric Matthews Photodetectors: rationale and implementation. Point detectors (PMT, APD) 2D detectors (CCD, CMOS), reading modes. Image formats (open /proprietary access) will be presented, as well as image reading/processing plateforms. Course 3: Confocal microscopy and F-techniques (FRAP), Mathieu Fallet General principles, resolution, image formation, single-point scanning, spinning disc, FRAP principles and fitting models. Course 4: Atomic Force Microscopy , Simon SCHEURING Introduction to operation principles and to Imaging and force measurement modes. Course 5: Electron Microscopy, Fabrice Richard The electron microscopy is a way to observe samples with nanometric resolution. In this course, we will present the different types of electron microscopy techniques and their possible applications. Course 6: Dyes and staining, Yannick Hamon The purpose of this course is on the one hand to remind the audience the specificities of the different types of fluorescent probes (fluorescent proteins, organic dyes, quantum dots). The second objective is to raise their awareness on the experimental tips enabling to avoid artifacts because of their indiscriminate use. Course 7: Super-resolution and Single molecule imaging, Pierre Recouvreux The resolution of an optical imaging system is fundamentally limited by diffraction. However, recent technological advances made it possible to "break" the diffraction limit (STED, PALM/STORM,...). In this course we will present these different techniques and show in which context they can be used. We will also present recent methods that allow the localization and counting of single fluorescent molecules. __________________________ _________________ LABEX INFORM ● IBDM UMR 7288 ● Case 907 Parc Scientifique de Luminy _________________ 13288 Marseille Cedex 9 France ● Téléphone : (+33) 491 26 93 95 Fax : (+33) 491 26 97 26 ___________________________ Mail: [email protected] ● ___________________________ _________________ LABEX INFORM Practical Work 1: Optical Bench, Sébastien Mailfert, Claire Chardes (4 groups of 4 students) On optical bench: principles of image formation and resolution (Abbe). Acquisition of spectra from different sources (Xenon fluorescence lamp, lasers), spatial spectrum enlargement by diffractive prism, transmission characterization of fluorescence filters, fluorescence detection of solutions, crosstalk, Stokes shift. Practical Work 2: Confocal Microscopy, Sophie Brustlein+ Sébastien Mailfert (4 groups of 4 students) How to set-up and tune a confocal microscope. Channel crosstalk, Nyquist-Shannon sampling, photobleaching, gain and offset, laser power vs. Detector gain, scanning speed, resolution. Practical Work 3: F-technique (FRAP), Mathieu Fallet + Cédric Matthews (4 groups of 4 students) FRAP experiments will be done in living cells to extract diffusion properties of components in the cytosol and in the plasma membrane. Interpretation of curves will be discussed and simple fitting will be implemented. Practical Work 4: Image processing, and quantification Cédric Matthews + Pierre Recouvreux + Claire Chardes? In this session we will work on how to handle images (open, display, manage N-dimensions), understand image quality, calibration and filtering, 2D event counting and segmentation using ImageJ software. Advanced Image J users will work on a case study: how to measure the cortical distribution of a polarity factor in fission yeast cells. We will analyze a large number of cells, imaged in different conditions of illumination, background intensity and protein expression, in order to extract valuable statistical information from the measurements. Requirements: ImageJ and your favorite software for graph creation. Practical Work 5 : AFM, Frederic Eghiaian, Félix Rico (4 groups of 4 students) The AFM can work in different modes and at different length scales. We will image two different samples each using a different AFM mode: contact mode and PeakForce tapping mode. In contact mode we will image purple membrane, a native membrane from an organism from the Dead Sea and rich in bacteriorhodopsin proteins organized in 2D crystals. We will be able to observe individual proteins at nanometer resolution. In PeakForce tapping mode we will image living cells, from the micrometer scale of the whole cell, down to the tens of nanometer of the cell's cytoskeleton. We will discuss about limitations, possible artifacts, sample preparation and the versatility of the AFM technique. Practical Work 6 : Electron Microscopy, F. Richard – J.P. Chauvin- A. Aouane The sample preparation is a vital step in Electron microscopy. How to prepare sample by chemical fixation, High Pressure Freezing and Negative staining. Then, you will compare the ultramicrotomy and the cryoultramicrotomy in order to see the advantage and difficulty of each technique. You will focus on Imaging. You will see your sample on Transmission Electron microscope and compare different imaging method as TEM, STEM, Electron Tomography and MEB. The imaging software is now an important tool to extract the essential information of imaging. __________________________ _________________ LABEX INFORM ● IBDM UMR 7288 ● Case 907 Parc Scientifique de Luminy _________________ 13288 Marseille Cedex 9 France ● Téléphone : (+33) 491 26 93 95 Fax : (+33) 491 26 97 26 ___________________________ Mail: [email protected] ● ___________________________ _________________ LABEX INFORM th Monday January 12 Welcome – S. Courtès -‐ IBDM room 032 8h45 9h15-‐ 10h45 C1 : General Optics and wide field microscopy (S. Brustlein) – IBDM room 032 11h00-‐ 12h30 PW 1A : Optical bench -‐ Spectrum S. Mailfert -‐ Kourilsky RoomXXXX, CIML PW 1B : Optical bench -‐ Image formation C. Chardes – Room 936, IBDM PW 1B : Optical bench -‐ Image formation C. Chardes – Room 936 IBDM PW 1A : Optical bench -‐ Spectrum S. Mailfert -‐ Kourilsky Room, CIML Group 3 -‐ 4 15h15-‐ 16h45 Lunch with Experts, CROUS Group 1 -‐ 2 12h30-‐ 13h30 13h30-‐ 15h00 Coffee break, IBDM room 032 C2 : From the sensor to the image (C. Matthews) – IBDM room 032 th Tuesday January 13 8h00-‐ 9h30 9h45-‐ 12h45 12h45-‐ 14h00 14h00-‐ 17h00 C3 : Confocal microscopy and advanced techniques (FRAP) (M. Fallet) – IBDM room 032 Coffee break, IBDM room 032 Group 4 PW 2 : Confocal S. Brustlein -‐ Confocal Leica SP5-‐ Room 112, R-‐1, CIML Group 3 PW 2 : Confocal S. Mailfert -‐ Confocal Zeiss LSM780-‐ Room 113, R-‐ 1, CIML Group 1 PW 3 : FRAP C. Matthews -‐ Spinning disk -‐ Room 928, IBDM Group 2 PW 3 : FRAP M. Fallet -‐ Spinning disk-‐ Room 165, R-‐1, CIML Lunch with Experts, CROUS Group 1 Group 2 Group 3 Group 4 PW 2 : Confocal S. Brustlein -‐ Confocal Leica SP5-‐ Room 112, R-‐1, CIML PW 2 : Confocal S. Mailfert -‐ Confocal Zeiss LSM780-‐ Room 113, R-‐1, CIML PW 3 : FRAP F. Michel -‐ Spinning disk -‐ Room 928, IBDM PW 3 : FRAP M. Fallet -‐ Spinning disk-‐ Room 165, R-‐1, CIML __________________________ _________________ LABEX INFORM ● IBDM UMR 7288 ● Case 907 Parc Scientifique de Luminy _________________ 13288 Marseille Cedex 9 France ● Téléphone : (+33) 491 26 93 95 Fax : (+33) 491 26 97 26 ___________________________ Mail: [email protected] ● ___________________________ _________________ LABEX INFORM th Wednesday January 14 9h30-‐ 10h00 10h00-‐ 12h00 Welcoming coffee, IBDM room 032 C4 : AFM (S. Scheuring) – IBDM room 032 12h00-‐ 13h30 13h30-‐ 16h30 Lunch with Experts, CROUS Group 1 and 3 PW 7 : Electronic Microscopy – A. Aouane, JP. Chauvin,F. Richard – IBDM groud floor 22-‐27 Group 2 PW 6: AFM – F. Rico – Room in AFM lab Group 4 PW 6:AFM -‐ F. Eghiaian – Room in AFM lab th Thursday January 15 9h30-‐ 10h00 10h00-‐ 12h0 Welcoming coffee, IBDM room 032 C5 : Electronic microscopy (F. Richard) -‐ IBDM room 032 12h00-‐ 13h300 13h30-‐ 16h30 Lunch with Experts, CROUS Group 1 PW 6: AFM – F. Rico – Room in AFM lab Group 3 PW 6:AFM -‐ F. Eghiaian – Room in AFM lab Group 2 and 4 PW 7 : Electronic Microscopy – A. Aouane, JP. Chauvin,F. Richard – IBDM groud floor room 22-‐27 th Friday January 16 9h00-‐ 10h30 C6 : Dyes and staining (Y. Hamon) – IBDM room 032 Coffee break, IBDM room 032 11h00-‐ 12h30 C7 : Super-‐resolution and Single molecule (P. Recouvreux) -‐ IBDM room 032 12h30-‐ 14h00 Lunch with Experts, CROUS 14h00-‐ 17h00 Group 1 and 3 (beginners) PW5 : Image processing C. Matthews – IBDM XXX 17h00-‐ 17h30 PW5 : Quantification, case study P. Recouvreux – Room XXX ROUND TABLE and feedback -‐ IBDM Cafeteria __________________________ _________________ Group 2 and 4 (Image J users) LABEX INFORM ● IBDM UMR 7288 ● Case 907 Parc Scientifique de Luminy _________________ 13288 Marseille Cedex 9 France ● Téléphone : (+33) 491 26 93 95 Fax : (+33) 491 26 97 26 ___________________________ Mail: [email protected] ● ___________________________ _________________
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