Summary of

3rd Euro-Bioimaging Stakeholder Meeting WP7 Break-out session Heidelberg Germany, 30-31 January 2012

Chairs: R. Pepperkok & B. Geiger

WP7 Access to Innovative Technologies-ALM Chairs: R. Pepperkok & B. Geiger

3rd Euro-Bioimaging Stakeholder Meeting WP7 Break-out session

•  Major results of Euro-BioImaging Survey related to WP7

•  Major results of Euro-BioImaging open call for Proof-of-Concept Studies

related to WP7

•  Bottlenecks for providing access to innovative light microscopy technologies

•  Emerging imaging technologies – update

Topics to be discussed

WP7 Access to Innovative Technologies-ALM Chairs: R. Pepperkok & B. Geiger

Major results of Euro-BioImaging Survey related to WP7

•  Community wishes to have access to survey data (put on web)

•  Significant need for WP7 technologies exists

•  Instrument, teachers and data analysis is mostly required

•  Personnel and instruments are most limiting for providing access

WP7 Access to Innovative Technologies-ALM Chairs: R. Pepperkok & B. Geiger

Major results of open call for Proof-of-Concept Studies related to WP7

Bottlenecks for providing access to innovative light microscopy technologies

•  Several applications did not target the right technology (questionaire for applicants,

e.g. describe biological problem first then decide on technology),

Better information on technology on Web, training of users

•  Users expectations go too far (discussion by phone before application, questionaire for

applicants), better information on technology on Web, training of users.

•  Project will last for longer than POC period, general problem (science never ends)

Project milestone program may help to terminate when needed

WP7 Access to Innovative Technologies-ALM Chairs: R. Pepperkok & B. Geiger

15.15 - 15.35 General status report of WP7 WP7 ELMI satellite meeting 2011, survey, proof of concept studies, plans for 2012

15.35-16.30 Status of proof of concept studies in WP7 Rainer Pepperkok, high throughput microscopy Judith Klumperman, correlative microscopy Lars Kastrup, superresolution microscopy Sunil Kumar, functional imaging

16.30-17.15 Stakeholder presentations (emerging technologies)

Pavel Hozak: Digital holographic microscopy

Dorus Gadella: Intravital microscopy

Francesco Pavone: New light microscopy technology to image morpho-functional intact neural networks

3rd Euro-Bioimaging Stakeholder Meeting WP7 Break-out session

WP7 Access to Innovative Technologies-ALM

The goals of WP7 are:

1.  To search for innovative light microscopy technology developments, which may be considered for inclusion in the forward planning of Euro-BioImaging infrastructure.

2.  To define the needs of the scientific community with regard to instrument developments and access to the identified innovative technologies.

3.  To search for existing European infrastructure enabling innovative light microscopy technologies.

4.  To determine the bottlenecks for providing access to innovative light microscopy technologies.

5.  To explore the feasibility of providing access to the identified innovative technologies.

6.  To develop a plan for the implementation of European access to innovative technologies.

ELMI Satellite Meeting 2011

Meeting agenda:

1.  Review of current and future innovative technologies in EUBI.

2.  Identification of potential European facilities and infrastructures that could be

able and would be willing to provide access to innovative technology.

3. WP7 proof-of-principle studies.

ELMI Satellite Meeting 2011

Defined already at the EUBI application level: 1. Super resolution microscopy. 2. Functional imaging (e.g. FRAP, FRET, FCS/FCCS). 3. Correlative microscopy. 4. High throughput microscopy for systems biology.

Technologies discussed at the EUBI 2010 stakeholder meeting: 5. Light sheet based fluorescence microscopy. 6. Correlative light/MRI microscopy. 7. Differential polarization laser scanning microscopy. 8. Non-linear-microscopy techniques (e.g. CARS, SHG, THG and SRS). 9. Photothermal heterodyne imaging. 10. Single molecule imaging techniques.

Review of current and future innovative technologies in EUBI :

Additional technologies suggested during the ELMI satellite meeting are: 11. Biomolecular imaging mass spectroscopy (BIMS) 12. Raman/ IR microscopy 13. In vivo super-resolution microscopy 14. Nano-antenna-based techniques

ELMI Satellite Meeting 2011

Meeting agenda:

1.  Review of current and future innovative technologies in EUBI.

2.  Identification of potential European facilities and infrastructures that could be

able and would be willing to provide access to innovative technology.

3. WP7 proof-of-principle studies.

WP7 POC Technology Responsible Super resolution microscopy S. Hell, Göttingen, Germany Correlative microscopy J. Klumperman (Netherlands) High throughput microscopy R. Pepperkok (Germany)

B. Geiger (Israel) Functional imaging P. French (Imperial College, UK)

National coordinators should identify potential candidate facilities being able and willing to conduct such studies.

Euro-BioImaging

Strategic Inventory Map

DRAFT

Results from 136 users and 213 providers

I have access

0 10 20 30 40 50 60 70 80 90

Photothermal Heterodyne Imaging

4PI Microscopy

Single Plane Illumination Microscopy (SPIM)

Non-linear Microscopy Techniques (e. g. CARS, SHG, THG and SRS)

Stochastic Optical Reconstruction Microscopy (STORM)

Stimulated Emission Depletion Microscopy (STED)

Photoactivated Localization Microscopy (PALM)

Correlated Light and Electron Microscopy

Single Molecule Imaging Techniques

Fluorescence Correlation Spectroscopy (FCS)

High-throughput Microscopy

Fluorescence Lifetime Imaging Microscopy (FLIM)

Electron Microscopy

Functional Imaging of Living Cells (e.g. FRAP, FRET)

Laser Scanning Confocal Systems

Series1

I need access

0 5 10 15 20 25 30

Photothermal Heterodyne Imaging

4PI Microscopy

Single Plane Illumination Microscopy (SPIM)

Non-linear Microscopy Techniques (e. g. CARS, SHG, THG and SRS)

Stochastic Optical Reconstruction Microscopy (STORM)

Stimulated Emission Depletion Microscopy (STED)

Photoactivated Localization Microscopy (PALM)

Correlated Light and Electron Microscopy

Single Molecule Imaging Techniques

Fluorescence Correlation Spectroscopy (FCS)

High-throughput Microscopy

Fluorescence Lifetime Imaging Microscopy (FLIM)

Electron Microscopy

Functional Imaging of Living Cells (e.g. FRAP, FRET)

Laser Scanning Confocal Systems

Series1

I need access

Examples of others mentioned:

•  Atomic force microscopy

•  Structured illumination/ 3D SIM

•  Virtual slide imaging scanner

•  (raster) image correlation spectrsocopy, (R)ICS

•  SNOM

•  Laser Scanning Confocal Systems: in which samples are vizualised vertically and alive

•  Interference microscopy

•  Raman microspectroscopy,

•  Electron Paramagnetic Resonance imaging

I need access (industry, n=11)

0 10 20 30 40 50 60 70 80

Correlated Light and Electron Microscopy Electron Microscopy

Non-linear Microscopy Techniques (e. g. CARS, SHG, THG and SRS)

4PI Microscopy Optical Coherence Tomography (OCT) Optical Projection Tomography (OPT)

Bioluminescence Imaging (BLI) Fluorescence Correlation Spectroscopy

(FCS)

Stimulated Emission Depletion Microscopy (STED)

Stochastic Optical Reconstruction Microscopy (STORM)

Multiphoton Systems Single Molecule Imaging Techniques

Laser Scanning Confocal Systems Photoactivated Localization Microscopy

(PALM)

Single Plane Illumination Microscopy (SPIM)

Fluorescence Lifetime Imaging Microscopy (FLIM)

Functional Imaging of Living Cells (e.g. FRAP, FRET)

High-throughput Microscopy

Series1

Which resources do you need?

0 10 20 30 40 50 60 70 80 90 100

Instruments

Data processing and analysis

Technical assistance to run instrument

Training in infrastructure use

Server Space

Wet lab space

Animal facilities

Methodological setup (e.g. design of study protocol)

Probe preparation

Animal preparation

Guest house for facility users

Training workstations

Training seminar room

Series1

Willingness to travel

20

12

14.4 20

33.6 >500 km

<500 km

<250 km

<100 km

Local infrastructure

Limitations for providing access

0 10 20 30 40 50 60 70 80 90

Not enough office space and lab space capacity

Not in line with institute`s access policies

Not in line with requirements of existing funding source policies

Not enough instrument capacity

Not enough personnel capacity

Series1

Conclusions

•  For all WP7 technologies except “functional imaging” less than 40% of participants have access.

•  The need for access to WP7 technologies is broadly distributed.

•  Access to instruments, analysis and technical assistance most required.

•  About 70% of participants wish to travel, 20% more than 500km.

•  Instruments and personnel are the most limiting factors for providing access.

Euro-BioImaging

Proof-of-Concept Studies – Evaluation of Open User Call

DRAFT

Timeline: October 1st – November 30th 2011, open user call

December 2011: evaluation of proposals (experts and facility heads)

January – July 2012: visit of users

PCS – Open User Call

Evaluation Euro-BioImaging PCS – Open user call

•  How many applicants want to visit which type of facility?

•  How many applicants request which type of technology?

•  How many applicants are willing to cross borders?

Biological Imaging

How many applicants wish to apply for which type of technology in WP7

Total number of facilities WP7: 15 Total number of PCS applicants WP7: 91

Correlative Light and Electron Microscopy (4 facilities): 11

High-throughput Microscopy (3 facilities): 8

Super-resolution Microscopy (4 facilities): 52

Functional Imaging (4 facilities): 20

How many applicants cross borders for visiting the facility of their choice?

PCS – Open User Call

Apply for transnational access

Apply for national access

PCS – HTM

HTM POCS facilities: WIS-AUTOSCOPE, Weizmann Institute of Science, Rehovot, Israel.

Multimodal Imaging Core, Kuopio, University of Eastern Finland,Finland.

Advanced Light Microscopy Facility (ALMF); EMBL Heidelberg, Germany.

Current status: Preparation of visits is ongoing.

All applicants will be accepted.

All applicants will conduct the visit.

Duration of most HTM projects will exceed POCS time.

WP7 Access to Innovative Technologies-ALM Chairs: R. Pepperkok & B. Geiger

15.15 - 15.35 General status report of WP7 WP7 ELMI satellite meeting 2011, survey, proof of concept studies, plans for 2012

15.35-16.30 Status of proof of concept studies in WP7 Rainer Pepperkok, high throughput microscopy Judith Klumperman, correlative microscopy Lars Kastrup, superresolution microscopy Sunil Kumar, functional imaging

16.15-17.15 Stakeholder presentations (emerging technologies)

Pavel Hozak: Digital holographic microscopy

Dorus Gadella: Intravital microscopy

Francesco Pavone: New light microscopy technology to image morpho-functional intact neural networks

3rd Euro-Bioimaging Stakeholder Meeting WP7 Break-out session

Proof of Concept sites Correlative light electron microscopy • Inventory of possible POCs; 13 approached; 10 applied In consultancy with the EuroBioImaging staff 4 sites were selected based on: • technological excellence, leading in Europe • ability to provide open access for transnational users • robustness of running the facility to meet user needs • complementary in techniques and expertise to the already identified PCS site(s) • ability to offer access for minimally one PCS project in kind • Geographical distribution

Name core

facility

Location Scientific

Director

website contact Specialization

Cell Microscopy

Centre &

Department of

Molecular Cell

Biology, Section

Electron

Microscopy

University Medical

Centre Utrecht, The

Netherlands &

Leiden University

Medical Center, Leiden,

The Netherlands

Judith

Klumperman

&

Bram Koster

www.cmc-utrecht.nl

&

http://www.lumc.nl/con

j.klumperman@umcutr

echt.nl

&

A.J.Koster@lumc.nl

live cell imaging – immunoEM; thin

section LM – immunoEM;

membrane trafficking

&

Fluorescence – cryoET; iLEM

(integrated LM-SEM); technique

development

Wolfson

Bioimaging Facility

University of Bristol,

United Kingdom

Paul Verkade http://www.bristol.ac.uk/bioc

hemistry/research/pv.html

p.verkade@bristol.ac.u

k

live Cells –HPF-FS-electron

tomography. Cell Biology

Telethon Electron

Microscopy Core

Facility

Institute of Protein

Biochemistry, National

Research Council (IBP-

CNR)/ Telethon

Institute of Genetics

and Medicine

Napoli,

ITALY

Alberto Luini

& Roman

Polishchuk

http://www.tigem.it luini@tigem.it

polish@tigem.it

Live cell imaging – pre-embedding –

TEM; membrane trafficking

Imaging Center -

IGBMC

IGBMC-CERBM

Strasbourg-Illkirch

France

Yannick Schwab http://www.ici.u-strasbg.fr yschwab@igbmc.fr Live imaging, F-techniques.

HPF- FS- Lowicryl/EPON

Immuno EM, 3D EM.

Developmental and Cell Biology.

Applicants who wish to apply for which type of technology in WP7 – Innovative Technologies: • Total number of applicants applying for WP7 technology: 91 • Correlative Light and Electron Microscopy: 11 requests, 12% of total

=> 4 to 6 projects will be executed

Bottlenecks for open access to CLEM 1. Users are not always aware what the technique can do or add to their

research question => too limited use of possibilities; asking for the wrong applications => Information & education, Website

2. Users often lack specific technical expertise. There is not one standard

approach => substantial input from expert personnel provider is required to do key experiments => organize on-site user courses;

Providers should be facilitated to hire dedicated personnel 3. - Preliminary studies sometimes too limited (e.g. testing antibody

dilutions). - Questions should be limited to those for which the technique is

absolutely required (not: 5 conditions, in 3 cell types, using 2 fixations) => Develop very detailed Questionnaire!!!

4. Not always clear how costs are distributed between users and providers

(e.g. antibodies, constructs). Providers have limited resources.

5. How to stop a project? Science is never finished…

July 15-20th 2012: EMBO Practical Course Correlative light electron microscopy Bristol, UK Org: Paul Verkade

Lars Kastrup Dept. of NanoBiophotonics,

MPI for Biophysical Chemistry, Göttingen

WP7 Superresolution

3rd Stakeholder Meeting 2012–01–30/31

EMBL Heidelberg

Superresolution it the most popular innovative light microscopy technique judging by the number of applications submitted

Source: Euro-BioImaging applications, Department of NanoBiophotonics, MPI Göttingen

0 50 100 150 200 250 300

1

2

Application submissions to Euro-BioImaging

Total

Super- resolution

Applications per PCS site in superresolution

1

2

3

4

52

228

Barcelona

Bordeaux

Turku/ Stockholm

Göttingen

Maria Garcia-Parajo 7

Daniel Choquet 20

Pekka Hänninen Hjalmar Brismar

18

Stefan W. Hell 7

~ 23%

PCS site PCS director # of applications

Submitted applications cover a broad field of topics including structural imaging, dynamics studies and colocalization

Source: Euro-BioImaging applications, Department of NanoBiophotonics, MPI Göttingen

NOT EXHAUSTIVE

Photothermal heterodyne imaging?

Selected examples

Structure imaging

Dynamicimaging

Colocaliz-ation studies

▪ Imaging of cytoskeletal proteins relevant for cancer research ▪ Study and movement of microfilaments ▪ Structure and dynamics of synaptic vesicles ▪ Organization and structure of proteins in skeletal muscle cells

▪ STED-FCS to uncover different nanoclustering behaviour ▪ Temporal dynamics of membrane proteins in living E. coli ▪ Stress fibre stability and dynamics on the nanoscale

▪ Colocalization study of proteins involves in synaptic transmission ▪ Colocalization of proteins within lipid domains ▪ Concentration an colocalization of mitochondrial proteins ▪ Colocalization study of cell adhesion proteins and cell-cell

adhesion proteins

Topic

Review of the applications identified several challenges in setting up open access for superresolution techniques

Source: Euro-BioImaging applications, Department of NanoBiophotonics, MPI Göttingen

Wrong expec-tations

Selection of method

Impact/ implica-tion of research

▪ Wrong expectations on applicability of superresolution techniques in general

▪ Low awareness on the usage of specifically optimized fluorophores/markers

▪ Overly/too optimistic on timeline and potential outcomes ▪ Too demanding requirements regarding superresolution

e.g. request for simultaneously applying live-cell, multicolor, video-rate, whole animal imaging, deep-tissue imaging etc.

▪ Sometimes wrong matching of proposed problem and superresolution technique (e.g. suggestion of temporal dynamics study with SMS technologies)

▪ Sometimes unclear, if superresolution imaging is really key to solve the proposed problem

▪ Authors often unclear in the advantages of superresolution over common microscopy techniques

▪ Applicants often not precise enough in defining the potential outcomes and implications on their research field (assuming success of the project)

▪ Elaborate guiding of users in the phase of designing the experiment needed

▪ Guidance on selecting the correct method

▪ Guidance in selecting the right fluorophores/markers

▪ Guidance on the overall challenge of the suggested project, i.e. high risk vs. high chance of feasibility

▪ Permanent, structured, organized educational program needed within the facilities

Example of challenge Topic

WP7 Functional Imaging PCS sites

• University of Amsterdam (Facility 15 - also WP6)

• LENS Firenze (Facility 35)

• Imperial College London (Facility 36)

• University of Konstanz (Facility 37)

• MARS -University of Montpellier (Facility 38)

Functional Imaging Imperial College London (Facility 36)

• Advertising

₋ initially we relied on the EuroBioImaging advertising but this was not productive within short time frame

₋ when we used our BioImagingUK network , we rapidly received 5 applications with a few more interested.

• Evaluation

− For this PCS, since there is no significant funding for the research projects, it should be up to hosts to decide with whom they will work

− Clearly an objective and transparent evaluation process is required when projects are to be supported by EU funds and when demand exceeds capacity

• Expertise

− Our expertise and capabilities include, e.g. super resolution, mesoscopic imaging and in vivo clinical imaging – we suggest it is too constraining to limit EuBI sites to specific imaging modalities

Functional Imaging Imperial College London (Facility 36)

We develop and apply advanced fluorescence imaging instrumentation with a particular emphasis on fluorescence lifetime imaging (FLIM) applied to FRET readouts for cell biology and label-free contrast in biological tissue. Our fluorescence imaging and FLIM instrumentation ranges from confocal and multiphoton microscopes to automated FLIM multiwell plate readers, FLIM confocal endomicroscopes and FLIM optical projection tomography and diffuse tomography systems. For the EuroBioImaging Proof of Concept studies, we are particularly aiming to share our expertise in the following areas: Automated FLIM multiwell plate readers for High Content Analysis, particularly for FLIM FRET studies of protein-protein interactions. Optical Projection Tomography (OPT) with FLIM. We have developed a FLIM OPT platform that can provide 3-D absorption and fluorescence intensity and lifetime information in samples ranging from ~1-10 mm in scale

Functional Imaging PCS studies at Imperial College London

• University of Dundee

− High speed FLIM FRET of kinetochore-microtubule interactions in live cells

• Beatson Institute of Cancer Research

− Global Analysis of TCSPC FLIM-FRET data

• National Institute of Medical Research Mill Hill

− Screening for Rdh54’s kinetochore interaction partners using automated multiwell

plate FLIM FRET

• National Heart & Lung Institute, Imperial College London

− Automated FLIM FRET applied to spatio-temporal regulation of RhoA activation states by Rho Guanine Nucleotide Exchange Factors (GEFs) during cell-cell contact formation

• Syngenta

− Using FLIM to determine how chemical structure affects movement on and in plants and plant cuticles.