Hellenic Pasteur Institute » Research » Technological Infrastructure / Core Facilities » Light Microscopy Unit

Light Microscopy Unit

Responsible Researchers:

Dimitra Thomaidou, principal investigator
Email: thomaidou@pasteur.gr
Tel. +30 210 6478 833		 Haralabia Boleti, principal investigator
Email: hboleti@pasteur.gr
Tel. +30 210 6478879

Overview

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.

Members

Researchers:

Haralabia Boleti, Principal investigator
Laboratory of  Molecular Parasitology
Email: hboleti@pasteur.gr
Tel. +30 210 6478879

Dimitra Thomaidou, Principal investigator
Laboratory of  Cellular and Molecular Neurobiology
Email: thomaidou@pasteur.gr
Tel. +30 210 6478 833

Research assistant:

Evangelia Xingi, PhD
Email: exingi@pasteur.gr
Tel.   +30 210 6478834

Equipment

Equipment

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 

A DMR upright microscope, equipped with Argon and Helium-Neon Lasers and 3 fluorescence detector PMTs (spectral detection option) as well as 1 transmission light PMT (phase contrast). The Confocal system operates with the Image acquisition and analysis Leica Confocal Software LCS, which allows for 2D measurements as well as for 3D image reconstructions. The microscope is also equipped with FITC and Rhodamine filter sets for conventional epi-illumination Μονάδα Φωτονικής Μικροσκοπίας

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
Μονάδα Φωτονικής Μικροσκοπίας Μονάδα Φωτονικής Μικροσκοπίας

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
 20x Ph2LUCPLanFLN   0.45   dry
 40x LUCPLanFLN
   0.6   dry
 60x PlanApo   1.42   oil
 60x UPLSApo   1.2   water
 100x UPLSApo   1.4   oil

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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:

  • Imaris
  • Olympus Cell R
  • Image-pro Plus 
  • Image J/Fiji
  • Adobe Photoshop

Μονάδα Φωτονικής Μικροσκοπίας

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.

 

 

 

 

 

Regulations

Regulations

Registration

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 exingi@pasteur.gr.

HPI users

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: imagingusers@pasteur.gr. 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.

External users

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.

Publications

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 .

Collaboration

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.

Training

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)

3. FRET

4. Image processing and analysis

Courses

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

Download: Course Application Form , Poster, Course Information , Course Tentative Program

Organizers:

Dept. of  Microbiology &  Light Microscopy Unit
Hellenic Pasteur Institute, GREECE
Tel. +30 2106478879

Quantitative Image analysis Unit,  Institut Pasteur, FRANCE
Tel + 33 (0)1 45 68 85 06

Dept. of Biochemistry & Light Microscopy Unit
Hellenic Pasteur Institute, GREECE
Tel. +302106378833

 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

Group photos