Light Microscopy Unit & FACS-Facility
LIGHT MICROSCOPY UNIT
Responsible Scientists
Dimitra Thomaidou
Research Director, Laboratory of Cellular and Molecular Neurobiology
Tel.: 210 6478 833
Email: 
@
CV
Haralabia Boleti
Principal investigator, Laboratory of Molecular Parasitology
Tel.:210 6478879
Email:
@ CV
OVERVIEW
The Light Microscopy Unit (LMU) of the HPI is one of the most modern and well-equipped Imaging Units in Greece and a member of the Greek Bioimaging-GR Consortium and the European Light Microscopy Initiative (ELMI- www.embl.org/elmi/). LMU is an open-access platform aiming to provide advanced Light microscopy and image analysis services to HPI, national or international academic scientists.
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The LMU has created a wide network internationally with top imaging groups in the EU and the Institut Pasteur Paris that have largely contributed to the development and establishment of cutting-edge technologies in the Unit. European and National grants such as BIOIMAGING-GR – Ministry of Education Infrastructure Grant (2017-2020), Stavros Niarchos Foundation Grant: (2016-2020), KRHPIS I-II (2015-2018), NEUROSIGN EU FP7 Program (2010-2013) have all supported the significant upgrade of the LMU during the period 2013-2017. LMU is the hub of 2-photon Intravital Imaging in the Bioimaging-GR Consortium, while it is currently up-graded for fast live cell imaging.
Τhe main state-of-the art methodologies currently in place in the LMU are: 2 photon intravital imaging in the CNS of transgenic mice, short and long time live cell imaging and digital image processing using commercially available and open-access image processing software.
The LMU has emerged through the Institut Pasteur International Network as a node for advanced imaging training and scientific collaborations (see Courses). Additionally, the LMU has organized significant training activities, including organization of international hands-on training workshops in digital image processing/analysis (2013) and Advanced Imaging and electrophysiology in the mammalian CNS (2013). In the time interval 2013-2017, several publications with participation or acknowledgement of the HPI-LMU has been published in peer review journals.
Our strategic goal is to develop the appropriate tools and technology that will allow us to monitor in real time disease progression in a wide range of communicable and non-communicable disease models, to develop live cell Calcium imaging, as well as multiple parameter middle-throughput and high-throughput screening, aiming to the understanding of biological processes and screening for novel therapeutics.
EQUIPMENT
Current equipment in the Light Microscopy facility includes:
(Click to see details)
The Leica TCS-SP8 confocal system is appropriate for imaging of fixed samples and live cell imaging. In the near future the system will be upgraded with a tandem scanner ideal for fast live cell imaging, an environmental chamber with controlled temperature, humidity and CO2 concentration, and a navigator for imaging unlimited number of positions for multi-well projects. The upgrading is funded by the Bioimaging-GR program. More specifically, the technical characteristics of the system are:
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
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 
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External users
The HPI’s imaging systems can be used by external users as well they should contact the responsible scientists of the Unit in order to arrange the exact time of the microscope booking. There is a charge of 30 € / hour for the use of either one of the two systems (Leica SP8 and Leica SP5) and 20 € / hour for the wide field Olympus Cell R and the confocal TCS SP.
TRAINING
Τraining in the use of microscopes and in image analysis applications
Training in the use of the microscopes and the image analysis software are conducted by the members of the Unit. Guides in the use of the Leica TCS-SP Confocal microscope and the wide-field Olympus IX-81 Cell-R imaging system can be found in the greek version of the website.
Leica SP5 manual, Leica_SP8 manual.
Educational videos for SP8 and Tandem (resonant Scanner)
Navigator sofware
Image processing and analysis
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- 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)
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, 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.
COURSES
(Click to see details)
A 5-day hands-on workshop was organized in the Hellenic Pasteur Institute in Athens Greece in collaboration with the Image Analysis hub of the Institut Pasteur Paris with the objective to provide theoretical and hands-on training in image analysis and automation tools.
July 4-8 2016
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The workshop was organized in the Hellenic Pasteur Institute in Athens, Greece. The aim of this workshop was to provide post graduate and postdoctoral scientists with a conceptual and practical understanding of CNS live cell imaging in whole animal level, as well as electrophysiology.
A 8-day International course was organized in the Hellenic Pasteur Institute in Athens, Greece. The course objective was to provide theoretical and practical training in basic and advanced concepts and methods of digital image analysis and in the use of several open source and commercial software for quantitative assessment of fluorescence microscopy data through.
The scientists of the HPI Light Microscopy unitorganized (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” (LINKS
Click to see the links
Information for the HPI Unit equipment:
- Leica Microsystems
- Olympus microscopy
- Imaris (Bitplane)- Web Training
- Image J– Plugins
- icy.bioimageanalysis.org/
Websites with useful information for microscopy:
- Olympus Microscopy Resource Center
- Zeiss Campus
- Molecular expressions microscopy primer
- Introduction to Light Microscopy
- Confocal laser scanning microscopy
- Image processing and analysis
Useful information for Deconvolution:
Databases of fluorochromes, dyes, their spectra and appropriate filter combinations:
- http://probes.invitrogen.com/resources/education/
- http://www.cyto.purdue.edu/flowcyt/software/Spectra
- http://home.earthlink.net/~mpmicro/
- http://www.mcb.arizona.edu/IPC/spectra_page
Useful information for FRET:
Practical Microscopy courses:
PUBLICATIONS
In the HPI-LMU, since its founding (2001), its users carried out projects that gave rise to a 39 publications in which members of the HPI LMU are listed as authors and/or the contribution of the HPI-LMU personnel is acknowledged.
FUNDING
The Scientists supporting the HPI-LMU have participated either as members or as Scientific responsibles in several grant applications succeeding to attract funding for the Unit either for organizing courses or for upgrading the equipment and development of new methodology from the following grants:
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- ΒΙΟΙΜAGING GR 2018-2020: “Hellenic Research Infrastructure for the Imaging and Observation of Fundamental Processes in Biology and Medicine” (MIS 5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund, coordinator Dr. Tavernarakis, IMBB Forth).
- Stavros Niarchos Foundations Sponsorships 2016-2020: «Development of innovative Biological products and health services for infectious and neurodegenerative diseases» (Coordinators Drs. R. Matsa and V. Myriagou)
- Ηellenic State Scholarship Foundation (Ι.Κ.Υ.) and German Burau for Academics’ Exchanges (DAAD): IKYDA program 2014-2015: Astroglia activation and reprogramming in brain injury and repair: analysis by in vivo imaging (2P-LSM) of the brain (Scientific Responsible Dr. D. Thomaidou).
- RIIP (Institut Pasteur International Network) International course 2016 :
” Cell Biology and infection: Digital Image Processing/Analysis Tools for Quantitative Light Microscopy Imaging”. Scientific Responsible Dr. H. Boleti - FP7 EU program 264083 NEUROSIGN 2010-2013: Development of Centre of Excellence in Neurosciences (cordinators. Dr.S. Tzartos– Dr. R. Matsa & Dr. L. Probert (Dr. D. Thomaidou Responsible for WP of purchasing the Infrastructure equipment for in vivo imaging).
- RIIP (Institut Pasteur International Network) Regional course 2013:
“Digital image processing/analysis tools in Light Microscopy: From the basics and beyond”. Scientific Responsible Dr. H. Boleti - Competitive grant from the Greek General Secretariat of Research and Technology in the context οφ European program “Human Networks for training in Research and Technology” 2004-2006 –Establishment of the Greek Light Microscopy Network Title: “Applications of Light Microscopy in Biomedical research and Diagnosis” In colaboration with the U. of Ioannina & U. of Crete Coordinator of the project and of the Network, HPI-LMU. (Scientific Responsible ¨Drs H. Boleti & D. Thomaidou)
MEMBERS
Evangelia Xingi, PhD
Tel.: +30 210-6478834
Email: @
Petrina Koumarianou
Biologist – Cell Biologist (BSc, MSc, PhD)
Post-doctoral fellow, Sep. 2018-
Email: 
@
Funded by the BIOIMAGING.GR grant.
Irini Thanou
Predoctoral Fellow
Email: 
@
Funded by the BIOIMAGING.GR grant.
Nasia Antoniou
Predoctoral Fellow
Email: 
@
Funded by the BIOIMAGING.GR grant.
FACS-Facility
The HPI FACS-Facility is supported by a researcher and a part time technician who provide scientific consultation and technical assistance to the users. The instruments in the FACS-Facility are used by researchers of the Institute performing multiple studies, including the separation of cells (cell sorting), and the detection of several fluorescent and forward and side scatter parameters (FSC & SSC) that can be used in several applications including the characterization of immune cells and other cell populations, the detection of apoptosis, cell death, cell cycle analysis, cell division etc.
Responsible Researcher
Research Assistant
Despoina Smirlis, PhD
Researcher C
Tel.: +30 210-6478841, +30 210-6478827
Email: 
@
Evangelia Xingi, PhD
Tel.: +30 210-6478834
Email: @