| Dimitra Thomaidou, principal investigator
Tel. +30 210 6478 833
|Haralabia Boleti, principal investigator
Tel. +30 210 6478879
The Light Microscopy Unit (HPI-LMU) of the Hellenic Pasteur Institute (HPI) is one of the most modern and well-equipped Imaging Units in Greece and is supported by investigators with strong experience in Imaging Techniques. Aim of the Unit is to provide advanced Light microscopy tools and services to in-house scientists and to visiting scientists. The Unit is stuffed by two experienced researchers: Dr. Dimitra Thomaidou specialising in the field of Neurobiology-Stem Cells and Dr. Haralabia Boleti specialising in the field of Cellular Microbiology. It is also supported by a Research Assistant, Dr. Evangelia Xingi. The HPI-LMU is equipped with a Confocal microscope and a wide field live cell imaging system. Both instruments are widely used by scientists of the HPI who perform studies in the fields of Microbiology, Cell Biology and Neurobiology. The instruments allow double and triple co-localization of molecules in both cellular and sub-cellular levels and imaging of live cells in 4D and 5D.
- The methods currently used in the Unit are the following:
- Multi-channel fluorescence and transmission microscopy
- Multi-color 3D imaging
- In vivo time-lapse experiments
- Fluorescence resonance energy transfer (FRET)
- Spectral unmixing of fluorescent dyes
- Deconvolution and deblurring
- Particle tracking
- Colocalization analysis
- Calcium imaging
The researchers of the Unit have co-ordinated a Greek Secretariat of Research and Technology “Human Netwoks of R&D” grant on “Light Microscopy Applications in Biomedical Research”. Through this grant two series of workshops on Light Microscopy techniques were organized in the Unit during 2004 and 2005, where a significant number of internationally known experts participated as tutors and lecturers. In addition, the Unit closely collaborates with the Platform for Dynamic Imaging of the Pasteur Institute-Paris, for the transfer of knowledge of cutting-edge imaging approaches to HPI imaging users. The HPI Light Microscopy Unit is a member of the European Light Microscopy Initiative (ELMI ) and the Euro-BioImaging Network . The equipment of the HPI Light Microscopy unit is available for use by external users from other Greek Research centers and Universities under a symbolic price of 20 € / hour of use.
Haralabia Boleti, Principal investigator
Laboratory of Molecular Parasitology
Tel. +30 210 6478879
Dimitra Thomaidou, Principal investigator
Laboratory of Cellular and Molecular Neurobiology
Tel. +30 210 6478 833
Evangelia Xingi, PhD
Tel. +30 210 6478834
Current equipment in the light microscopy facility includes:
1. Leica TCS-SP5II Two-Photon Confocal Microscope with Spectra Physics Mai Tai infrared source (http://www.leica-microsystems.com/products/confocal-microscopes/) Acquired through 7th EU Framework Neurosign/Regpot grant. A detailed description of the system will be available shortly.
2. Leica TCS-SP Confocal Microscope
2. Wide-field Time-lapse Olympus IX-81 Cell-R imaging system ( - link - ) A Live Cell Imaging System, composed of a wide field, inverted, fully motorized Olympus IX-81 microscope equipped with:
- Olympus IX8S1F-ZDC stage (inverted) for DIC and phase contrast imaging.
- a Hamamatsu CCD ORCA/AG cryocamera
- a fast excitation filter wheel
- Temperature and CO2 chamber
- hardware for FRET
- the CellR imaging software
- air conditioning unit
This system allows fast multicolor 3D imaging, transmission microscopy (phase-contrast and Nomarski-DIC), in vivo time-lapse experiments (4D imaging), spectral unmixing of fluorescent dyes, FRET and 3D-dencovolution analysis (with the following softwares: No neighbor, Nearest neighbor, Inverse filter and 3D blind). The system is also equipped with video editing softwares (AVS video remaker, AVS video converter).
|Objective||Numerical Aperture(ΝΑ)||Type depending on the immersion medium|
|10x Ph1 UPlanFLN||0.3||dry|
| 40x LUCPLanFLN
4. Workstations and software for image processing
The Light microscopy unit is also equipped with two Image analysis workstations with RAM memory > 3G and softwares for analysis of the digital data obtained by the Confocal microscope and the Olympus Cell R system.
The workstations are equipped with the following image processing and analysis softwares:
- Olympus Cell R
- Image-pro Plus
- Image J/Fiji
- Adobe Photoshop
5. Cell-culture facility
A small tissue culture facility consisting of a BSL2 laminar flow hood, a centrifuge and a CO2 incubator is also set up within the imaging unit.
New users of the Light Microscopy Unit (LMU) of HPI as well as current users, who are starting a new project should submit a project submission form at the Light Microscopy Unit of HPI. We need all necessary information to organize on time introductory sessions for using the instruments, as well as for teaching and supporting sample preparation techniques and new imaging protocols. You can download the LMU Project Submission Form, fill it in and send it to firstname.lastname@example.org.
The use of all the equipment of the HPI Light Microscopy Unit is free of charge for HPI scientists. HPI users are currently booking the instruments through sending an e-mail to the group e-mail address: email@example.com. Each user can book a microscope for a maximum of 4 hours per day twice a week. In the case that the microscopes are available this time can be extended.
The HPI’s imaging systems can be used by external users, who should contact the responsible scientists of the Unit in order to arrange the exact time of the microscope booking. There is a charge of 20 € / hour for the use of either one of the two systems.
New users’ training
The new users of the two imaging systems must follow a training session by the responsible technician before they are allowed free entrance to the Unit. More specifically, the new users should:
• Initially get informed by the responsible scientists about the booking system and get trained on the basic principles of the systems’ use, as well as, the possibilities they offer for the application he/she needs them.
• For the first three sessions of use of the particular microscopes the user should be accompanied and supervised by the Unit’s technician.
All work published that includes results obtained using the microscopes and/or the image analysis software of the LMU of HPI must be acknowledged and a copy of the publication should be sent in paper or pdf to the LMU of HPI .
Scientific projects can be performed in collaboration with the members of the LMU of HPI. Collaboration is typically characterized by intensive scientific input, preparation and analysis of samples and/or development of innovative imaging protocols. The members of the LMU of HPI involved in collaboration must be authors on resulting publication.
Τraining in the use of microscopes and in image analysis applications
1. Confocal Microscopy (Guide in the use of the Leica TCS-SP Confocal microscope)
2. Wide Field microscopy and live-cell imaging (Guide in the use of the Olympus IX-81 Cell-R imaging system)
4. Image processing and analysis
- Basics of Quantitative Image Analysis
- Detect Information Loss
- Nyquist and PSF calculator
- Image pro plus- Quantification of fluorescence intensity
- Quantitative Imaging for Colocalization Analysis
- Image pro plus- Quantitative colocalization analysis
- Fiji- Colocalization Analysis using Coloc_2
- Tips for colocalization studies (Colocalization hardware setup and image acquisition)
- Imaris- 3D reconstruction
- Image J- 3D Viewer
- Olympus Cell R- 3D Deconvolution
- Olympus Cell R- Spectral unmixing
- Image J- (Particle tracking, Particle Analysis, MTrack J)
2013 RIIP (Institut Pasteur International Network) Regional Course
“Digital image processing/analysis tools in Light Microscopy:
From the basics and beyond”
Course date: June 10-17 2013
Application deadline : 28th February 2013
- Haralabia Boleti, firstname.lastname@example.org
Dept. of Microbiology & Light Microscopy Unit
Hellenic Pasteur Institute, GREECE
Tel. +30 2106478879
- Jean Christophe Olivo-Marin, email@example.com
Quantitative Image analysis Unit, Institut Pasteur, FRANCE
Tel + 33 (0)1 45 68 85 06
- Dimitra Thomaidou, firstname.lastname@example.org
Dept. of Biochemistry & Light Microscopy Unit
Hellenic Pasteur Institute, GREECE
2004 & 2005
Workshops in Light Microscopy and Image Analysis
The scientists of the HPI Light Microscopy unit organized (2004 & 2005) a series of workshops on Light Microscopy and image analysis and coordinated the activities of the “greek Light Microscopy network” formed by scientists from HPI, the University of Ioannina, the Institute of Biomedical Research at Ioannina, and the University of Crete. The workshops were sponsored by the Greek Secretariat of Research and Technology in the context of the Operational program “COMPETITIVENESS” (http://www.antagonistikotita.gr/) action 8.3.6- Creation of communication channels for academics, research/technology entities and enterprises with common scientific interests- “HUMAN NETWORKS”.
Organizers of the HPI Workshops : Drs H.Boleti & D.Thomaidou
Project coordinator: Dr Haralabia Boleti
Information for the HPI Unit equipment:
Websites with useful information for microscopy:
- Olympus Microscopy Resource Center (http://www.olympusmicro.com/)
- Zeiss Campus
- Molecular expressions microscopy primer
- Introduction to Light Microscopy
- Confocal laser scanning microscopy
- Image processing and analysis
Databases of fluorochromes, dyes, their spectra and appropriate filter combinations:
Useful information for Deconvolution:
Useful information for FRET:
Practical Microscopy courses: