20 - 24 February 2017
Maison du Savoir
Université du Luxembourg
Organisation:
Andreas GIROD
Aymeric d’HÉROUËL
Life Sciences Research Unit
7, avenue des Hauts-Fourneaux
L-4362 Esch-sur-Alzette
[email protected]
Luxembourg Centre for Systems Biomedicine
6, avenue du Swing
L-4367 Belvaux
[email protected]
Administrative support:
Caroline HERFROY
Annette BARNES
Life Sciences Research Unit
6, avenue du Swing
L-4367 Belvaux
[email protected]
Faculty of Science, Technology and Communication
2, Avenue de l’Université
L-4365 Esch-sur-Alzette
[email protected]
Information in a nutshell:
Venue
Maison du Savoir (MSA)
Université du Luxembourg
2, avenue de l’Université
L-4365 Esch-sur-Alzette
Rooms
Practicals
MSA 4.010, 4.020, 4.030, 4.180
4.190, 4.200, 4.300, 4.410
Lectures
MSA 4.510
Exhibition & breaks MSA 4.500
The
2nd
Hands-on Light Microscopy
Workshop
is made possible thanks to your participation
and kind contributions from our
industrial sponsors.
1
Sponsors
2
3
Campus map
N31
RU
Terrasse des
Hauts-Fourneaux
Maison du
Savoir
Square
Mile
AVENUE DE L’UNIVERSITÉ
S S C IE NC E S
HOLM
PO RT E D E FR A N CE
E
PORTE D
AV E N UE D U BLU ES
BO ULE VAR D DU JAZ Z
B O U L E VAR D DU JAZZ
AVENUE DU SWING
Hotel
ibis
ÉR UR G IS TE S
AVENUE DES HAUTS-FOURNEAUX
S ID
AV. D E S H AUT S -F.
E DU B R I L L
LIST
AVENUE DU ROCK’N’ROLL
ROUTE DE BELVAL
Gare
Belval
Université
4
Esch/Alzette
Maison du Savoir - 4th floor
4.300
4.010
4.500
4.410
4.200

Exhibition
&
Breaks
4.020 4.030
4.510
4.190 4.180

Public
Lectures
5
6
Programme
7
Schedule overview
For details and exact times, see daily schedules. Practical sessions take place
in parallel for 8 groups of 4 participants, and data analysis sessions for 3
groups of 10 participants each; see separate assignment leaflet.
The gala dinner takes place at Brasserie Mansfeld in the city of Luxembourg
and a coach will collect all dinner guests in front of the hotel ibis Belval at 19:30.
8
Monday 20/02/2017
08:00 Registration and coffee
09:15 Welcome
09:30 Thomas Gabler / LASOS
p. 16
09:55 Michael Sommerauer / AHF p. 18
10:20 Andreas Lotter / Leica Microsystems
p. 20
10:45 Emmanuel Pirson / Hamamatsu
p. 22
Light sources for microscopy
Optical filters for fluorescence microscopy
The true value of microscope objectives
Detectors and Smart pixels for high-performance microscopy
11:10 Coffee break
11:40 Philippe Baert / Nikon p. 24
Basics of the light microscope: how to become best friends?
12:05 Florian Eich / Olympus p. 26
The confocal principle - from a single point to a 3D image 12:30 Jelle Hendrix / Hasselt University *
Biomolecular dynamics and interactions
p. 28
13:00 Lunch
14:00 Peter Drent / confocal.nl p. 30
Re-scan Confocal Microscopy (RCM) for improved resolution and sensitivity
14:30 Bruno Combettes / Andor p. 32
15:00 Clément Laigle / Leica Microsystems
p. 34
Spinning Disc Confocal Microscopy, a technical overview
Beyond the diffraction limit: STED Nanoscopy
15:30 Coffee break
15:45 Alexander Egner / Laser-Laboratorium Göttingen e.V.
Tomographic and Isotropic STED Microscopy
p. 36
16:25 Ernst H.K. Stelzer / Goethe University Frankfurt
p. 38
Optical sectioning and the development of four-dimensional light microscopy
17:20 Coffee break
17:35 Erik Manders / University of Amsterdam **
Techniques of confocal microscopy
p. 40
18:05 Rainer Heintzmann / IPHT Jena p. 42
Structured Illumination and the Analysis of Single Molecules in Cells
Speaker kindly sponsored by *Zeiss, **confocal.nl, ***arivis
9
Tuesday 21/02/2017
09:00 Jürgen Mayer / Luxendo p. 44
09:30 Jochen Wittbrodt / Heidelberg University p. 46
10:05 Rainer Heintzmann / IPHT Jena
p. 48
High speed volume imaging with selective plane illumination microscopes
High throughput phenomics of juvenile and adult fish
Lightwedge and Lightsheet-Raman Microscopy
11:00 Coffee break
11:30 Christophe Zimmer / Institut Pasteur Paris
p. 50
12:05 Christophe Zimmer / Institut Pasteur Paris
p. 50
12:35 Arnaud Royon / ArgoLight p. 52
Localization microscopy…(Part I)
... and recent expansions (Part II)
Calibration and quality control of modern fluorescence microscopes
13:00 Lunch
14:00
Practical sessions p. 91
15:30 Coffee break
15:45
Practical sessions p. 91
17:15 Coffee break
17:30
Practical sessions p. 91
19:00 Posters & snacks
10
Wednesday 22/02/2017
09:00 Dmytri Kuchenov / EMBL Heidelberg
p. 54
09:35 Jerker Widengren / Royal Institute of Technology Stockholm
p. 56
10:10 Sandra Orthaus-Mueller / PicoQuant p. 58
10:35 Uwe Schröer / LaVision
p. 60
High content imaging platform for profiling intracellular
signaling network activity in living cells
Fluorescence fluctuation and super-resolution techniques:
fundamental biomolecular studies and towards clinical diagnostics
Principles and Applications of Fluorescent Lifetime Imaging Microscopy
and Foerster Resonance Energy Transfer
Clearing and light sheet imaging of murine tissue and organs
11:00 Coffee break
11:30 Anne Grünewald / Luxembourg Centre for Systems Biomedicine p. 62
Mitochondrial-nuclear interplay: Exploring the cause of
respiratory chain deficiency in single Parkinson’s disease neurons
12:00 Berta Cillero-Pastor / Maastricht University
p. 64
12:30 Rainer Pepperkok / EMBL Heidelberg p. 66
Unraveling the osteoarthritis disease with imaging mass spectrometry
High Throughput Microscopy for Systems Biology
13:00Lunch
14:00
Practical sessions p. 91
15:30 Coffee break
15:45
Practical sessions p. 91
17:15 Coffee break
17:30
Practical sessions p. 91
19:00 Posters & snacks
Speaker kindly sponsored by *Zeiss, **confocal.nl, ***arivis
11
Thursday 23/02/2017
09:00 Heinrich Leonhardt / Ludwig Maximilian University Munich
Visualization and Manipulation of the Invisible
p. 68
09:35 Corinne Lorenzo / ITAV Toulouse *** p. 70
Spatio-temporal dynamics of the bystander effects on cellular and tissular
levels in an integrated model: a study with light sheet microscopy
10:10 David Wiles / Arivis p. 72
10:35 Daniel Reisen / Bitplane p. 74
Introduction to arivis Vision4D
Imaris - The Ideal Solution to Interactively Analyze Microscopy Images
11:00 Coffee break
11:30 Sebastian Munck / VIB Leuven p. 76
12:00 Christel Genoud / Friedrich Miescher Institute Basel p. 78
12:35 Jens Rietdorf / Acquifer p. 80
Analyzing plasma membrane distribution patterns
by an inhomogeneity-based method
Correlative techniques for acquisition and analysis of large datasets
with serial-block face scanning electron microscopy following
light microscopy image acquisition
Re-thinking data flow at service facilities in times of
terabyte-scale research projects
13:00 Lunch
14:00 Lars Hufnagel / EMBL Heidelberg
p. 82
14:30 Konstantinos Kampas / Democritus University of Thrace
p. 84
Bioimaging across scales with light-sheet microscopy
NETs as a common pathogenetic mechanism - The paradigm of
autoinflammation in FMF
15:00 Individual demos
15:30 Coffee break
15:45
Practical sessions p. 91
17:15 Coffee break
17:30
Practical sessions p. 91
19:00 Finger food dinner
12
Friday 24/02/2017
09:00 Saskia Suijerbuijk / Hubrecht Institute Utrecht
p. 82
09:35 Jean-Marie Vanderwinden / Université libre de Bruxelles p. 84
10:10 Data analysis & coffee (session 1)
p. 95
11:10 Data analysis & coffee (session 2)
p. 95
12:10 Data analysis & coffee (session 3)
p. 95
High-resolution intravital imaging of cancer plasticity
Sample clearing, a clear(er) view in fluorescence microscopy deep imaging
13:10 Lunch
14:10 Closing remarks & group picture
Speaker kindly sponsored by *Zeiss, **confocal.nl, ***arivis
13
14
Lectures
abstracts & notes
15
Thomas Gabler
Lasos Lasertechnik GmbH
Monday 09:30
Light sources for Microscopy
Microscopy has challenging requirements, not only referred to the single laser source but also to
the overall system design considering aspects like easy handling, upgrade- and expandability
as well as easy service capability. An overview is given on the necessary laser characteristics
for different microscopy techniques regarding for example spatial and spectral properties. Laser
and laser system specifications are derived and various technological approaches are described
to fulfill these demands. Consequently a construction set out of laser sources, beam combining
systems, fiber optics and precision mechanics forming a versatile light source for microscopy
is described. This modular approach allows for different combinations of functional elements to
serve a variety of applications in microscopy.
16
Michael Sommerauer
AHF Analysetechnik AG
Monday 09:55
Optical filters and light sources for fluorescence microscopy
Optical filters are built into all fluorescence microscopes. This lecture will give a short overview
about filters and filter sets which are specially designed for fluorescence microscopy.
LED light sources are more and more substituting common arc lamps. Long term stability, ultra
fast switching time and adjustable intensities make LED light sources a perfect tool for any kind
of automated acquisition.
We often hear questions regarding existing filter sets. Do they fit to LED sources and is an excitation filter still necessary? This talk will show how filter sets influence the overall performance of
a fluorescence experiment especially when LED light sources are used for excitation.
18
Andreas Lotter
Leica Microsystems GmbH
Monday 10:20
The true value of microscope objectives
A large portfolio of microscope objectives is available to serve a multitude of imaging applications. To select an appropriate individual objective one has to know how to read its label and has
to rank the relevance of the given information.
The talk will address the value of a microscope objective in terms of image performance and
additional parameters. In this presentation, I will present the effort necessary in the optical design and the production process in order to achieve the desired specifications. Furthermore, it
will be demonstrated what the user has to consider in order to maintain the best performance a
microscope objective can provide.
20
Emmanuel Pirson
Hamamatsu Photonics SARL
Monday 10:45
Detectors and Smart pixels for high-performance microscopy
The recent evolution in the Photonics technology contributed in the development of new applications in the imaging microscopy. The single detectors (1D) mainly used in Confocal Microscopy
are currently driven by the PMT’s, the APD’s and the Hybrid Detectors, but the Si-PM can offer
new possibilities. Since more tha 30 years, the two dimensional sensors (2D) kept a continuous
evolution and contributed in significant breakthrough in imaging: the CCD sensor for the transition from the Analog to the Digital microscopy, the Deep Cooled CCD to achieve Ultra low Light
imaging and High Dynamic range, the EM-CCD for the High Sensitivity Fast Readout, the CMOS
sensors for High Resolution and small pixel sizes, and recently the sCMOS for Low Noise High
Frame imaging. The next “Smart Pixel” detector generation is coming to add new dimensions in
the imaging...and the future is already now.
22
Philippe Baert
Nikon Instruments Europe BV
Monday 11:40
Basics of the light microscope: how to become best friends?
This session will handle the very basics in light microscopy covering dia- and episcopic illumination. These illuminations can be found in different kind of microscopes, but emphasis during
this session will be on the upright microscope. We stand still at some fundamental knowledge
to come to what we really want: having the best image possible from the microscope. We will
take time in understanding the building parts of the microscope and how they affect what we
see through the binocular or camera. Understanding the nature of each objective is essential. It
allows us to look for the best configuration when looking into a specific sample. We will learn to
calculate the resolving power of the instrument. Several contrasting techniques will help us to see
what otherwise cannot be seen: phase contrast, polarization, darkfield, DIC and fluorescence
microscopy. From some of these, we see the emergence of microscopy based approaches to
study the biology in living cells.
24
Florian Eich
Olympus Europa SE & Co. KG
Monday 12:05
The confocal principle - from a single point to a 3D image
Confocal laser scanning microscopes are widely used in today’s research and confocal imaging
can be considered as a standard method to create multidimensional image data.
Starting from the general explanation of the underlying concepts, this lecture will highlight the
key components of a confocal microscope and explain their working principles. Exemplary applications from fixed cell 3D imaging to more complex applications like live-cell multimodal and
photomanipulation experiments will be used to get a more detailed understanding of technical
features translated into applicative needs.
26
Jelle Hendrix
Hasselt University, Belgium
Monday 12:30
Fluorescence fluctuation spectroscopy - the confocal as a molecular speedometer
Fluorescence fluctuation spectroscopy (FFS) (reviewed in [1]) groups a family of methods aimed
at quantifying molecular properties (diffusion rate, concentration, interaction affinities, stoichiometry…) from analysing fluorescence recordings or images (obtained mostly on a confocal microscope) by means of correlation analysis. I will talk about different popular FFS methods (panels A
& B), from the basics of autocorrelation to the combination of FFS with picosecond pulsed alternating excitation, and with fluorescence lifetime imaging microscopy (FLIM). In a second part of
the talk, I will focus on a biological application of FFS methods, in which we studied assembly of
the human immunodeficiency virus Gag protein in the cytosol of cell, prior to visible assembly at
the plasma membrane [2].
A:
B:
[1] Tetin, S.Y., Fluorescence Fluctuation Spectroscopy (FFS). Methods Enzymol. Vol. 518-9. 2013, Heidelberg: Academic Press.
[2] Hendrix, J., et al., Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers. J Cell Biol,
2015. 210(4): p. 629-46.
28
Peter Drent
Confocal.nl BV
Monday 14:25
An introduction to Re-scan Confocal Microscopy
The RCM (Re-scan Confocal Microscope) is a standard confocal microscope extended with a rescan detection unit. With a simple optical trick a lateral resolution of 170nm can be achieved for
any pinhole diameter. The pinhole is only needed for Z-sectioning. Since RCM has also a strongly improved sensitivity (4x better signal-to-noise ratio) here is no need for high laser power, being
friendlier for cells. RCM can work in multi-colour mode for different colour combinations. It is our
mission to go further by extending the spectral range into the IR for deep tissue imaging and the
use of NIR probes. Many more biological applications are on the horizon.
Further information:
De Luca, Giulia MR; Breedijk, Ronald MP; Brandt, Rick AJ; Zeelenberg Christiaan HC; De Jong BE; Timmermans W; Azal Nahidi
L; Hoebe RA; Stallinga S.; Manders Erik MM (2013) Re-scan confocal microscopy: scanning twice for better resolution. Biomedical Optics Express, 4 (11), 2644-2656
30
Bruno Combettes
Andor Technology Ltd
Monday 14:25
Spinning Disc Confocal Microscopy, a technical overview
In conventional widefield optical microscopy the specimen is bathed in the light used to excite
fluorophores (Figure 1a). The fluorescence emitted by the specimen outside the focal plane of
the objective interferes with the resolution of in focus features. As the sample increases in thickness, the ability to capture fine detail above out-of-focus signal becomes increasingly challenging. The confocal principle rejects out-of-focus light through the use of a pinhole, with the added
benefit of an improvement in lateral and axial resolution. It is these benefits that make confocal
microscopy so popular and why you find one in most core imaging facilities.
Confocal microscopy is now a routinely used optical technique across a wide range of biological
sciences, from plant science to mammalian models. Its use is popular because it has several
benefits over conventional widefield microscopy, namely in its ability to remove out-of-focus signal, capture information from a reduced depth of focus, image discreet optical sections in thick
samples and so create high contrast 3D image sets.The most common confocal technology is
that of confocal laser scanning microscopy (CLSM). In this format the sample is illuminated by a
single point of light from a laser. The laser beam is scanned point by point in a raster pattern and
signal is detected sequentially from each point by a photomultiplier tube until an entire image is
created.
Spinning disk confocal microscopy (SD) exploits the multiplex principle. The sample is illuminated, and so light is detected, at multiple points simultaneously.Unlike a conventional laser-scanning microscope, where a narrow laser beam sequentially scans the sample, in SDCLM an expanded beam illuminates an array of microlenses arranged on a (collector) disk. Each microlens
has an associated pinhole laterally co-aligned on a second (pinhole) disk and axially positioned
at the focal plane of the microlenses. The disks are fixed to a common shaft that is driven at high
speed by an electric motor. When the disks spin, and the scanner is coupled to a microscope
with the pinhole disk located in its primary image plane, an array of focused laser beams scan
across the specimen. The pinholes (and microlenses) are arranged in a pattern, which scans a
field of view defined by the array aperture size and the microscope objective magnification. The
scanning laser beams excite fluorescent labels in the specimen. Fluorescence emission will be
most intense where this array is focused - the focal plane. Some fraction of this light will return
along the excitation path where it will be preferentially selected by the same ‘confocal’ pinholes.
A dichroic mirror, which reflects emission wavelengths, is located between the two disks. This
separates the laser emission from any excitation light reflected or scattered from the microscope
optics. The geometry of the emission path results in a confocal fluorescence signal with extremely low background noise.
A limitation in conventional CSLM is the use of photomultiplier tubes (PMTs) whose quantum efficiency (QE, the probability of converting a photon to an electron) is rather low - typically 30-40%.
In contrast the SDCLM technique uses a camera as a detector that can have a very high QE; e.g.
an iXon+ 897 EMCCD has a peak QE of more than 90%, making it a near-perfect detector. This
combination of the Yokogawa dual spinning disk and high QE detector delivers a confocal instrument that can run at high speed and with unequalled signal-to-noise (SNR).
32
Clément Laigle
Leica Microsystems GmbH
Monday 14:55
Beyond the diffraction limit: STED Nanoscopy
Since its development in 1997 by Stephan Hell (Nobel Price 2014), STED nanoscopy has become a powerful imaging and localized excitation method, breaking the Abbe diffraction barrier
for improved spatial resolution in cellular imaging. It is now widespread available with different
modalities like gSTED or Pulsed STED. We will review the STED principle and its implementation,
the photophysics occuring in the fluorochromes, the various STED modalities and some biological applications.
34
Alexander Egner
Laser Laboratorium Göttingen, Germany
Monday 15:45
Tomographic and isotropic STED Microscopy
No abstract - more space for notes.
36
Ernst H.K. Stelzer
Goethe University Frankfurt, Germany
Monday 16:30
Optical sectioning and the development of four-dimensional light microscopy
The optical sectioning capability is fundamental for three-dimensional imaging. One of the very
microscopes, which claim this property is light sheet-based fluorescence microscopy (LSFM). In
general, fluorescence microscopy provides a high contrast, since only specifically labelled cellular components are observed while all other structures remain “dark”. Its fun-damental issues
are: 1) Excitation light degrades endogenous organic compounds and bleaches fluorophores.
2) Only a finite number of excitable fluorophores is available, which limits the quantity of collectable emitted photons. 3) Organisms are adapted to the solar flux of 1.4 kW/m2. Thus, irradiance
should not exceed a few mW/mm2 or nW/µm2 in live imaging assays.
In confocal epi-fluorescence microscopy, the same objective lens is used for the excitation of
fluorophores and the collection of emitted light. Since hardly any light is absorbed, the in-tegrated intensity is constant along the optical axis. Thus, for each acquired two-dimensional image,
all fluorophores in a three-dimensional specimen are excited. A spatial filter, i.e. a pinhole, is
required in the detection path to reject out-of-focus light. Hence, the ratio of the sample thickness
over the microscope’s depth of field indicates how many times fluorophores are exposed to the
illumination light when a three-dimensional stack of images is recorded.
LSFM makes a sincere effort to address this challenge by decoupling the excitation and emission
light pathways. The optical axis of the illumination objective lens is aligned with the focal plane of
the perpendicularly arranged detection objective lens. By design, only the fluorophores around
the focal plane are excited for each acquired two-dimensional image. Hence, each fluorophore
is exposed only once to the illumination light when a three-dimensional image is recorded. The
detection path resembles a conventional fluores-cence microscope with an objective lens, a
spectral filter, a tube lens and a camera. Neither a spatial filter nor a dichroic mirror are required.
The significance of the illumination-based optical sectioning property is that the viability and the
fluorescence signal of a living specimen are retained while millions of images are recorded for
days or even weeks.
Further benefits of LSFM are (i) a good axial resolution, (ii) imaging along multiple directions, (iii)
deeper tissue penetration due to the low numerical aperture of the illumination objective lens, (iv)
a high signal-to-noise ratio, (v) an unrestricted compatibility with fluorescent dyes and proteins,
(vi) reduced fluorophore bleaching and (vii) photo-toxicity at almost any scale, (viii) millions of
pixels are recorded in parallel and (ix) a dramatically improved viability of the specimen.
38
Erik Manders
University of Amsterdam, Netherlands
Monday 17:35
New modalities in Re-scan Confocal Microscopy (RCM)
In the last few years the Re-scan Confocal Microscope [1] has become a well-known and simple
technology to obtain images with an improved resolution (170 nm) as compared to standard
confocal microscopy (240 nm). The technology is based on a standard confocal microscope with
an additional scanner (the re-scanner) that directs the emitted light to a sensitive (CMOS or CCD)
camera. Precise control of the scanner (that “reads” the sample) and re-scanner (that “writes”the
image on the camera) allows super-resolution imaging without closing the pinhole to a minimum.
This highly photon-economical way of detection (no losses at the pinhole) and the use of a highly
sensitive camera reduce the noise level in RCM images. Apart from imaging with better resolution and less noise the RCM microscope has a lot more potentional.
In the last few years we have exploited the possibilities of the RCM technology. We have adjusted the microscope for sequential and simultaneous multi-color imaging, FRET imaging, pH-imaging, ratio-imaging and FRAP measurements [2]. Recently, we added to this list deep-tissue
imaging, SCIM-RCM (low-phototoxicity high-resolution imaging) [3,4], life-time imaging and other
techniques to further improve the resolution. In this presentation we present our latest results.
[1] De Luca, Giulia MR, et al. “Re-scan confocal microscopy: scanning twice for better resolution.” Biomedical optics express
4.11 (2013): 2644-2656.
[2] De Luca, Giulia MR, et al. Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications. 2016,
Journal of Microscopy (accepted)
[3] Hoebe, R. A., et al. “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell
imaging.” Nature biotechnology 25.2 (2007): 249-253.
[4] Krishnaswami, V., et al., Spatially-controlled illumination microscopy: For prolonged live-cell and live-tissue imaging with
extended dynamic range. Quarterly Reviews of Biophysics, 49 (2016).
40
Rainer Heintzmann
IPHT Jena, Germany
Monday 18:10
Structured Illumination and the Analysis of Single Molecules in Cells
In the past decade revolutionary advances have been made in the field of microscopy imaging,
some of which have been honored by the Nobel prize in Chemistry 2014. One high-resolution
method is based on transforming conventionally unresolvable details into measurable patterns
with the help of an effect most people have already personally experienced: the Moiré effect.
If two fine periodic patterns overlap, coarse patterns emerge. This is typically seen on a finely
weaved curtain folding back onto itself. Another example is fast moving coarse patterns on both
fences of a bridge above a motorway, when approaching it with the car. The microscopy method
of structured illumination utilizes this effect by projecting a fine grating onto the sample and imaging the resulting coarser Moiré patterns containing the information about invisibly fine sample
A:
detail (panel A). With the help of computer reconstruction
based on several such Moiré images, a high-resolution
image of the sample can then be assembled.
Another way to obtain a high-resolution map of the sample is to utilize the blinking behavior inherent in most molecules, used to stain the sample. Recent methodological
advances (Cox et al., Nature Methods 9, 195-200, 2012)
enable us to create pointillist high-resolution maps of
molecular locations in a living biological sample, even
if in each of the required many individual images, these
molecules are not individually discernible. Examples will
be shown as a film of a cell at 30 millionths of millimeter
resolution and 6 seconds between the individual movie
frames.
Podosomes of a cell image with structured illumination (SIM). F-actin shown in red and vinculin in green.
Image courtesy of Marie Walde, Gareth Jones and
James Moneypenny.
42
Jürgen Mayer
Luxendo GmbH
Tuesday 09:00
High speed volume imaging with selective plane illumination microscopes
No abstract - more space for notes.
44
Jochen Wittbrodt
Heidelberg University, Germany
Tuesday 09:30
High throughput phenomics of juvenile and adult fish: Imaging and population genomics
Jakob Gierten, Jochen Gehrig, Christian Pylatiuk, Venera Weinhard, Tilo Baumbach, David Hassel, Felix Loosli and Joachim Wittbrodt
We have been establishing a near isogenic resource for a each individual of a vertebrate population that will facilitate systematic genotype-phenotype correlations. Our collection of 150 inbred
strains is representing a vertebrate population genomics resource with millions of segregating
single nucleotide polymorphisms (SNPs). Due to the genetic uniformity within each line the genetic basis of quantitative traits concerning the heart will be captured on a continuous scale. We
are integrate multiple “ mics” datasets generated by high throughput larval and adult phenotyping and RNA-seq to comprehensively address multi-dimensional features of all lines. Taking
advantage of high-density SNP maps available for each line we aim at identifying and validating
associated polymorphisms with relevance for human health.
Heart disease is a leading cause of morbidity and mortality. Epidemiological data indicate substantial heritability but the vast majority of cases cannot yet be causally explained. Predicting
cardiac phenotypes from high-density genomic polymorphism maps is key for personalized
prevention and mechanism-based therapeutic approaches.
Genome-wide association studies (GWAS) in humans have generated a rapidly expanding list
of genetic polymorphisms associated with different heart pathologies but suffer from large background noise and uncontrolled environment, which limits the statistical power to detect low-penetrance variance. In contrast, GWAS in laboratory-grown inbred animals profit from controlled
external factors and facilitate functional validation.
I will present high throughput/content imaging approaches to systematically exploit this unique
resource.
46
Rainer Heintzmann
IPHT Jena, Germany
Tuesday 10:05
Lightwedge and Lightsheet-Raman Microscopy
Ulrich Leischner, Walter Müller, Michael Schmitt, Jürgen Popp, Rainer Heintzmann
Two recently developed modes of lightsheet imaging will be presented. Lightwedge microscopy
aims at mesoscopic imaging of fixed and optically cleared samples at 1 µm isotropic resolution
without the need for sample rotation. The key-idea is to focus a lightsheet at an unusually high
NA (thus the name “lightwedge”) and still obtain a large field of view due to refocusing of the
lightwedge and stitching the multiple small regions of thin illumination back together. This has
been simplified by electrical tunable lens technology which has become available recently.
The second mode is hyperspectral Raman imaging in a lightsheet illumination configuration [1].
To recover the spectral information a full-field Fourier-spectroscopic approach has been chosen.
The difficulty here is that in a Michelson approach, it would be technically very hard to maintain
the angular stability and common path approaches usually tolerate a relatively low product of
étendue and maximal optical path difference. We thus developed an optically stable Mach-Zehnder like scheme based on the use of retro-reflecting corner
A:
cubes, which is inherently stable. This enabled us to obtain
full spectrally-resolved Raman images consisting of over
four million spectra in about 10 minutes. Advantages over
the conventional Raman imaging are the reduced maximum
power on the sample and out of focus heating, the lightsheet-inherent good suppression of crosstalk from the illumiB:
nation side and the avoidance of glass close to the sample
mounting.
Light sheet illumination for Raman imaging at few specific
wavelengths was previously reported [2].
With a total laser power of 2 W at an illumination wavelength
of 577 nm, we obtained images (2048 × 2048 pixels) of
polystyrene beads (panel A), zebrafish and a root cap of a
snowdrop at a spectral resolution of 4.4 cm-1 with only few
minutes of exposure. The olefinic and aliphatic C-H stretching modes, as well as the fingerprint region are clearly
visible along with the broad water peak of the embedding
medium (panel B).
Spectrally resolved spontaneous Raman microscopy therefore promises high-throughput imaging for biomedical research and on-the-fly clinical diagnostics.
The work on hyperspectral Raman imaging was supported by a grant of the Carl-Zeiss-Stiftung.
[1] W. Müller, M. Kielhorn, M. Schmitt, J. Popp, R. Heintzmann, Light sheet Raman micro-spectroscopy, Optica 3, 452-457, 2016.
[2] I. Barman, K.M. Tan, G.P. Singh, “Optical sectioning using single-plane-illumination Raman imaging”, J. Raman Spectrosc.,
41, 1099–1101 (2010)
48
Christophe Zimmer
Institut Pasteur, France
Tuesday 12:05 & 12:35
Expanding single molecule localization microscopy in 3D and time
Super-resolution microscopy methods developed over the last decade can image biological
structures well below the diffraction limit of ~200-300 nm. Among them, techniques based on
single molecule localization such as PALM and STORM have become particularly popular, owing
to their superior resolution (~20-30 nm) and relative simplicity of implementation. Nevertheless,
important challenges remain, such as improving spatial resolution, facilitating 3D imaging, and
increasing throughput and temporal resolution. In this talk, I will present recent and ongoing
computational and experimental efforts of our lab to address these challenges and expand the
reach of single molecule localization microscopy in 3D and time.
50
Arnaud Royon
Argolight SA
Tuesday 12:35
Calibration and quality control of modern fluorescence microscopes
Although quality control of fluorescence microscopes is a topic that appeared more than fifteen
years ago in academic laboratories [1] and national regulatory agencies [2], it is still topical as
it was for example in the program of the Core Facility Satellite Meeting of the 15th international
ELMI meeting in 2015. Due to the increasing complexity of the instrumentation used for confocal
and high-end wide-field fluorescence imaging microscopy, national metrology institutes [3], microscope manufacturers [4], and more recently core facilities [5] have gotten involved in identifying, making and/or testing different tools, both hardware and software, to assess the numerous
aspects of fluorescence microscopes.
In particular, quality control is important: (i) for core facilities, to assure the performances of the
microscopes they make available to the end users; (ii) for microscope manufacturers, to guarantee the microscopes’ specifications and to improve maintenance; (iii) for end users to remove
the bias introduced by the microscopes in their experiments. In this talk, the context and issues
of quality control will be first outlined; the mostly used tools will be then presented and their advantages and drawbacks will be discussed; finally, examples of quality control of fluorescence
confocal and high-end wide-field microscopes with Argolight solutions will be shown.
[1] R. I. Ghauharali et al., “Fluorescence photobleaching-based shading correction for fluorescence microscopy,” Journal of
Microscopy 192, 99-113 (1998).
[2] R. M. Zucker and O. T. Price, “Practical Confocal microscopy and the evaluation of system performance,” Methods 18, 447458 (1999).
[3] U. Resch-Genger et al., “How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and
Suited Fluorescence Standards,” Journal of Fluorescence 15, 337-362 (2005).
[4] A. Dixon et al., Standardization and Quality Assurance in Fluorescence Measurements II, Chapter 1. (Springer-Verlag, Berlin
Heidelberg, 2008).
[5] R. W. Cole et al., “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure
quality control,” Nature Protocol 6, 1929-1941 (2011).
52
Dmitry Kuchenov
EMBL Heidelberg, Germany
Wednesday 09:00
High content imaging platform for profiling intracellular signaling network activity in living
cells
Essential characteristics of cellular signaling networks include a complex interconnected architecture and temporal dynamics of protein activity. The latter can be monitored by FRET biosensors at a single live cell level with high temporal
A:
resolution. However, these experiments are
typically limited to the use of a couple of FRET
biosensors.
Here, we describe a FRET-based multi-parameter imaging platform (FMIP) that allows simultaneous high- throughput monitoring of multiple
signaling pathways (panel A). We apply FMIP to
monitor the cross- talk between EGFR and IGF1R signaling, signaling perturbations caused by
pathophysiologically relevant EGFR mutations
and the effects of a clinically important MEK
inhibitor (selumetinib) on the EGFR network.
We expect that in the future the platform will be
applied to develop comprehensive models of
signaling networks and will help to investigate
the mechanism of action as well as side effects
of therapeutic treatments.
54
Jerker Widengren
KTH Stockholm, Sweden
Wednesday 09:35
Fluorescence fluctuation and super-resolution techniques - fundamental biomolecular
studies and towards clinical diagnostics
In this presentation it will first be presented how long-lived, non-fluorescent, photo-induced transient states of organic fluorophores and their dynamics can provide additional, to-date largely
unexploited, information about biomolecules, their interactions and their immediate environment. By two major approaches, where the transient state information is obtained either from
fluorescence fluctuation analysis or by recording the time-averaged fluorescence response to a
time-modulated excitation, it is possible to combine the detection sensitivity of the fluorescence
signal with the environmental sensitivity of the long-lived transient states. Proof-of-principle experiments, advantages, limitations and applications will be discussed, including live cell transient
state (TRAST) imaging of cell membrane fluidity and cellular metabolism.
Second, it will be shown how diffraction-unlimited imaging of cellular protein distribution patterns
using Stimulated Emission Depletion (STED) nanoscopy can potentially provide new diagnostic
parameters on the level of individual cells, and also give further insights into underlying disease
mechanisms. Examples including cultured cells, clinically sampled breast cancer cells and
platelets will be given.
56
Sandra Orthaus-Mueller
PicoQuant GmbH
Wednesday 10:10
Principles and Applications of Fluorescent Lifetime Imaging Microscopy and Foerster Resonance Energy Transfer
Sandra Orthaus-Mueller, Ben Kraemer, Tino Roehlicke, Michael Wahl, Hans-Juergen Rahn, Felix
Koberling, Rainer Erdmann
Over the last two decades, time-resolved fluorescence microscopy has become an essential
tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging
(FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime)
Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key
systems are available either as stand-alone units [1] or as upgrades for confocal laser scanning
microscopes (CLSM) [2]. We will discuss the underlying principles and actual instrumentation
based on Time- Correlated Single Photon Counting (TCSPC) as well as recent applications.
The fluorescence lifetime of a fluorophore is defined by its photophysical properties and can be
influenced by a wealth of environmental parameters such as pH, ion or oxygen concentration,
molecular binding, or the proximity of energy acceptors. Thus making methods based on its
measurement the technique of choice for functional imaging of many kinds. Such methods are
also more robust than intensity based ones, since fluorescence lifetime does not depend on
concentration, excitation intensity, sample absorption or thickness
Under certain conditions, fluorescence decay curves can act as fingerprints to differentiate dyes
. A novel FLIM method, called Pattern Matching, was developed based on this property. The
method allows for excellent separation of fluorophore types in FLIM images as well as from background autofluorescence.
Förster Resonance Energy Transfer (FRET) studies enable measuring intra- and intermolecular
distances on the scale of a few nanometers, allowing studying molecular interactions in vitro or
living cells. Additionally, FRET sensors can be used to monitor environmental conditions such
as pH and ion concentration. When working with biological systems like cells where fluorophore
concentration can often not be accurately determined, FRET measurements are further improved
by observing changes in donor fluorescence lifetime instead of intensity, as the former is mostly
concentration independent. These so-called FLIM-FRET experiments can further reveal sub-populations, allowing to determine the fraction of free donors compared to associated donor molecules within a complex in addition to the lifetime distribution.
TCSPC data acquisition is often considered a somewhat slow process due to the high number
of photons that have to be collected per pixel for reliable data analysis. Our rapidFLIM approach
exploits recent hardware developments such as TCSPC modules and hybrid photomultiplier detector assemblies with ultra short dead times, enabling significantly higher detection count rates.
Thus, much better photon statistics can be achieved in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical
resolution. These shorter acquisition times allow imaging with several FLIM images per second
for monitoring FRET dynamics in transient molecular interactions as well as fast moving species
(e.g., vesicles) in living samples.
[1] M. Wahl, F. Koberling, M. Patting, H. Rahn, R. Erdmann, “Time-Resolved Confocal Fluorescence Imaging and Spectrocopy
System with Single Molecule Sensitivity and Sub-Micrometer Resolution”, Curr. Pharm. Biotechnol., 5, 299-308 (2004).
[2] B. Krämer, V. Buschmann, U. Ortmann, F. Koberling, M. Wahl, M. Patting, et al., “Advanced FRET and FCS measurements
with laser scanning microscopes based on time- resolved techniques”, Proc. SPIE, 6860, 68601D (2008).
58
Uwe Schröer
LaVision BioTec GmbH
Wednesday 10:35
Clearing and light sheet imaging of murine tissue and organs
In 1903 Siedentopf and Zsigmondy developed the light sheet technology. This first light sheet
microscope was used for imaging small particles in solutions. It was a long time before any
further significant developments took place. The concept of orthogonal, planar illumination was
mainly used for flow cytometry. It took until 1993 for the concept of light sheet microscopy to be
taken up again by Voie. He made use of the light sheet technology known as orthogonal-plane
fluorescence optical sectioning (OPFOS) for analyzing cochlea. In 2004, the light sheet concept
was again reported in an article by Stelzer. In 2007, Dodt reported the advantages of light sheet
technology for characterizing large clarified samples. This time the light sheet technology was
combined with large sample-clearing, as described by Spalteholz in 1914. Two main clearing
techniques have been developed since then. In case of organic solvent clearing, dehydration is
followed by merging the remaining refractive indices. On the other hand aqueous buffer clearing
protocols based on hydrogel embedding, hyperhydration and immersion have been developed
in parallel. Today several projects based on both techniques, clearing and light sheet imaging,
had been successfully published. An overview of the more common clearing protocols will be
given here by presenting some of these projects.
60
Anne Grünewald
Luxembourg Centre for Systems Biomedicine
Wednesday 11:30
Mitochondrial-nuclear interplay: Exploring the cause of respiratory chain deficiency in
single Parkinson’s disease neurons
We applied a quantitative immunofluorescence assay to postmortem midbrain sections of idiopathic Parkinson’s disease (IPD) patients and controls to (i) characterize the extend of respiratory chain defects in IPD and to (ii) explore the cellular pathways underlying this dysfunction in
single nigral dopaminergic neurons. In addition, individual neurons were isolated by means of
laser-capture microdissection to assess the integrity of the mitochondrial genome with a triplex
real-time PCR assay. In combination, these approaches revealed that the respiratory chain deficiency profile in IPD encompasses complex I as well as complex II. However, while the complex
I deficit results from impaired TFAM-mediated mtDNA transcription and replication, the lack of
complex II is more likely due to compromised nuclear transcription activation. These findings
implicate mitochondrial-nuclear signaling in the pathogenesis of IPD.
62
Berta Cillero-Pastor
Maastricht University, Netherlands
Wednesday 12:00
Unraveling the osteoarthritis disease with imaging mass spectrometry
Osteoarthritis (OA) is the most prevalent form of arthritis. OA affects 80% of the population over
the age of 65. It is a complex pathology because diverse factors interact causing the process
of deterioration of the cartilage. The current view is that OA is a group of diseases that can be
differentiated based on the risk factors and on the pathophysiological mechanisms underlying
the joint damage. However, current trials with potential therapies cannot induce the formation of
new cartilage. In this sense, mesenchymal stem cells (MSC) are an interesting alternative for cellbased therapy of cartilage defects due to their capacity to differentiate towards chondrocytes in
a process called chondrogenesis. Recently, the metabolism of lipids has been associated with
the modulation of this process and also with the development of pathologies related to cartilage
degeneration. Information about the distribution and modulation of lipids during chondrogenesis
could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers, could be highly valuable to improve MSC-based cartilage therapies. Analysis
of MSCs micromasses at days 2 and 14 of chondrogenesis by matrix-assisted laser desorption
ionization-mass spectrometry imaging (MALDI-MSI) led to the identification of 20 different lipid
species, including fatty acids (FA), sphingolipids (SLs) and phospholipids (PLs). In addition to
this, preliminary work of our group has classified OA and healthy groups based on the peptidomic and lipidomic profiles of human cartilage and synovium. Application of MSI in the field of drug
delivery also provides insight in the relation between tissue distribution, activity in target tissue
and possible local side effects in other tissues, e.g. osteoporotic bone changes, small molecule and tissue characteristics determining penetrance. In summary, we propose an innovative
workflow for the study of OA combining high spatial resolution and high molecular sensitivity and
specificity.
64
Rainer Pepperkok
EMBL Heidelberg, Germany
Wednesday 12:30
High Throughput Microscopy for Systems Biology
We have developed a microscopy platform automating and integrating all steps in microscope-based experiments. The technology also allows automated performance of complex
imaging protocols such as FRAP or FCCS experiments at high throughput or the extensive multiplexing of immunocytochemistry in fixed cells.
Application of this technology to several genome-scale siRNA screens addressing basic questions of membrane traffic and organelle integrity reveal unexpected relationships between secretory pathway function and genes involved in general metabolic integrity or signal transduction
pathways. This provides the basis for an integrative understanding of the global cellular organization and the regulation of the secretory pathway and organelle biogenesis.
We have also applied the technology to investigate the genetics of cholesterol homeostasis and
the processing, transport and function of disease relevant ion channels CFTR or ENac, which
play key roles in cystic fibrosis.
Further information:
• Almaca et al., Cell.154:1390-400.
• Bartz et al., Celll Metab., 10:63-75.
• Neumann et al., Nature 464: 721-727.
• Conrad et al., Nat. Methods, 8:246-249.
• Wachsmuth et al., Nat Biotechnol. 33:384-389.
• Simpson et al., Nat. Cell Biol., 14:764-774
66
Heinrich Leonhardt
Ludwig Maximilian University Munich, Germany
Thursday 09:00
Visualization and Manipulation of the Invisible
Fluorescence light microscopy allows multicolor visualization of cellular components with high
specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution and the lack of specific detection tools. To circumvent these limitations, we applied three-dimensional structured illumination microscopy (3D-SIM, Science, 320, 1332-6) and high-throughput STED microscopy in combination with automated image analysis. For detection of cellular
structures, we have generated fluorescent, antigen-binding proteins, termed chromobodies, by
combining epitope-recognizing fragments with fluorescent proteins (Nature Methods, 3, 8879). These chromobodies can be expressed in living cells and used to target or trace epitopes
in subcellular compartments providing an optical readout for novel high content analyses and
functional studies (Nature Struct. Mol. Biol., 17, 133-139). These antigen-binding fragments can
also be recombinantly produced, chemically functionalized and directly used for super-resolution
microscopy (Science, 331, 1616-20). To study the dynamics of genome organization we have
repurposed prokaryotic DNA binding proteins (TALEs and CRISPR/Cas) for the detection of specific DNA sequences in living cells (NAR 42, e38 and Nucleus, 5, 163-172). This combination of
detection tools and microcopy techniques provides new insights into the structure and function
of mammalian cells.
68
Corinne Lorenzo
ITAV Toulouse, France
Thursday 09:35
Spatio-temporal dynamics of the bystander effects on cellular and tissular levels in an
integrated model: a study with light sheet microscopy
In radiobiology, the effects of ionizing radiations were for a long time attributed to the targeted
induction of damage on the cellular components (DNA, organites, membranes) either directly
or indirectly by the production of free radicals within the irradiated cells. Until recently, we supposed that these effects were limited to cells directly crossed by ionizing radiations. Currently,
there are numerous facts that call into question the classic paradigm of the irradiation resulting
from targeted effects. We know now that the irradiation can also lead to “non-targeted” and
delayed effects which include genomic instability, low-dose hypersensitivity and bystanders
effects. Better understanding the processes leading to the induction and the distribution of the
bystanders’ effects remains a major importance for the scientific community. In the case of a
non-uniform exposure such as those arising naturally, or still in a therapeutic context such as the
radiotherapy, the biological consequences of these effects have to be controlled. Consequently,
it becomes necessary to determine exactly how the integration of signals at the cellular scale
influence these proximity effects within tissues and/or human organs and how they are implemented temporarily and spatially.
The aim of this project is to study the spatiotemporal dynamics of appearance and integration
of radio induced damages in particular in the non-exposed areas, in an integrated biological
model closer to the in vivo tissular reality. For that we wish to observe the impact of various types
of spatio-temporarily targeted damages (double and simple DNA stranded breaks, DSB and
SSB respectively) on the cellular pesponse outside the targeted areas. Unlike all the studies
carried out based on endpoint-experiments, the answer monitoring will be made in a dynamic
way with the use of innovative biological and technological tools (engineering of the tissue mimics, optogenetic, ionization of materials by optical breakdown, light sheet microscopy). We shall
characterize and compare the distribution of the bystanders effects from a qualitative but also
quantitative point of view according to the targeted zone, to the type and to the amplitude of the
generated damage. We shall set up original tools of processing and analyses of images to extract quantitative parameters of the bystander effects distribution (trajectory, speed, acceleration)
in numerous experimental conditions.
70
David Wiles
arivis AG
Thursday 10:10
Introduction to arivis Vision4D
Leveraging new technologies for fast data access and large data handling in imaging. Introduction
to the visualisation and analysis capabilities of arivis Vision4D.
72
Daniel Reisen
Bitplane AG
Thursday 10:35
Imaris - The Ideal Solution to Interactively Analyze Microscopy Images
Bitplane is the world’s leading interactive microscopy image analysis software company and was
founded in 1992 in Switzerland. Through their constant innovation and a clear focus on 3D and
4D image visualization analysis, Bitplane actively shapes the way scientists process multi-dimensional microscopic images.
Imaris includes a set of key tools, which cater for the needs of researchers in live cell imaging, in
particular developmental biology, but also in neurobiology where it enables efficient 3D tracing in
large images and complicated neural networks. The available tools in Imaris include:
•
•
•
•
•
•
•
•
•
visualization of terabyte multi-dimensional data sets;
detection, tracking and analysis of cells and organelles;
tracking of cell division, lineage analysis;
rotational and translational drift correction;
angle measurements;
Neuron and spine analysis;
Colocalization studies;
advanced interactive plotting for results exploration
and comparison between samples;
a wide range of plugins (Matlab XTensions).
The presentation given by Dr. Daniel Reisen will bring an
overview of the latest introduced features in the Imaris software.
74
Sebastian Munck
VIB Leuven, Belgium
Thursday 11:30
Analyzing plasma membrane distribution patterns by an inhomogeneity-based method
Laura Paparellia,b,c, Nikky Corthouta,c, Benjamin Paviea,c, Devin L. Wakefieldd, Ragna Sannerudb,c,
Tijana Jovanovic-Talismand, Wim Annaertb,c, Sebastian Muncka,c
VIB Bio Imaging Core, Herestraat 49, Box 602, 3000 Leuven
Laboratory of Membrane Trafficking; Department of Human Genetics, KU Leuven, Herestraat 49, Box 602, 3000 Leuven
VIB Center for the Biology of Disease, Department of Human Genetics, KU Leuven, Herestraat 49, Box 602, 3000 Leuven
d
Dept. of Molecular Medicine, Beckman Research Institute of the City of Hope Comprehensive Cancer Center, Duarte, California
a
b
c
Unraveling how proteins and lipids distribute on the cell surface is important to elucidate their
functions and interactions. Therefore, we developed a method to quantify such alterations that,
unlike many current tools, is compatible with diverse types of cellular organization, including
polarity. The analysis involves three main steps. The first step consists of dividing the image
of the protein of interest in polygons through a process called tessellation. The second step is
the analysis of the histogram of the tile areas. As the shape of the histogram changes with the
organization pattern, it can be used to describe the distribution of a given protein or lipid. This
analysis scheme allows to use the intensity information per tile and thus can be used to (partially)
correct for unresolvable objects per tile. Therefore our method can be applied to analyze images
stemming from different imaging modalities such as Widefield -, Structured Illumination -, and
Single Molecule –Microscopy. Finally, in the third step the deviation from a random distribution
is used to describe the inhomogeneity of a spatial pattern. Hence, our tool can be employed to
screen for changes in membrane constituents in a straightforward manner.
76
Christel Genoud
FMI Basel, Switzerland
Thursday 12:05
Correlative techniques for acquisition and analysis of large datasets with serial-block face
scanning electron microscopy following light microscopy image acquisition
3D imaging of tissue ultrastructure by serial block face scanning electron microscopy (SBEM)
is an approach to reconstruct large neuronal circuits in order to analyze their computational
functions as well as to localize molecules in cells in culture. In order to correlate light microscopy
signals with ultrastructure to reconstruct for example synaptic connectivity among neurons in
the zebrafish olfactory system or to identify the cell compartment in which a protein of interest is
located, we have developed tools and refined various steps in the imaging pipeline for SBEM.
First, we optimized protocols to acquire stacks of EM images with high signal-to-noise ratio. Second, we developed software workflows for image registration. Third, software tools have been
used to identify structures in stacks of EM images that were visualized by fluorescence microscopy in the same samples before fixation.
Applications of these methods include the reconstruction of all neurons in the olfactory bulb of a
zebrafish larva or identification of protein localization in cells.
In summary, these methods have substantially increased the speed and quality of our SBEM
approaches and are likely to be useful for a wide spectrum of applications in which acquisition
of large datasets are necessary and the correlation with fluorescent data acquired in vivo are
required.
78
Jens Rietdorf
ACQUIFER AG
Thursday 12:40
Re-thinking data flow at service facilities in times of terabyte-scale research projects
intermediate storage, network bandwidth, de-central versus centralized processing options, data
transfer, remote access, virtual machines, remote viewing, modularity and scalability, integration
into pre-existing network architectures, compatibility of open source and popular commercial
software solutions, data deconvolution and data compression, intermediate versus long term
storage, data integrity, risk management and software maintenance.
To address these issues Acquifer provides the highly modular solution HIVE, a computer architecture achieving an optimal cost-performance ratio for image based applications. Featureing
a CPU with up to 2x 22 cores, 512 GB EEC RAM, CUDA GPU with up to 24 GB RAM; 1,4GB/s
read&write speed (RAID6) which easily achieves simultaneous streaming from two 4MPx 16bit
scientific cameras. Network speed of >2,3GByte/s reading and >2,0 GByte/s writing, which
removes any network bottleneck. Storage is scalable up to the PetaByte range. Global remote
access provided by my.acquifer.net, Acquifer Remote Desktop LoQin technology gives personalized access on a project, directory and individual file level. HIVE has been tested and
approved to execute commonly used software packages like Huygens Professional (SVI), arivis
Vision 4D (arivis AG), Amira (FEI), Fiji, Imaris (Bitplane), Matlab (Mathworks), NIS (Nikon), LAS X
(Leica), ZEN (Zeiss), KNIME; HIVE can host an OMERO Image Data Server (Glencoe Software,
OME) in a Hyper-V virtual machine.
With respect to high content screening applications, Acquifer provides a second product, the
imaging machine; key feature here is the unique fixed stage architecture, which ideally supports
work with sensitive organoids, embryos and model organisms like Zebrafish. Integrated complete
workflows greatly profit from the combined use of HIVE.
Further up-front information about Acquifer’s data-solutions and high content screening solutions
is available at https://www.acquifer.de/data-solutions/ and https://www.acquifer.de/screening/.
80
Lars Hufnagel
EMBL Heidelberg, Germany
Thursday 14:00
Bioimaging across scales with light-sheet microscopy
Selective-plane illumination microscopy has proven to be a powerful imaging technique due to
its unsurpassed acquisition speed and gentle optical sectioning. We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. However, even in the case of multi-view imaging techniques that illuminate and
image the sample from multiple directions, light scattering inside tissues often severely impairs
image contrast. Here we combine multi-view light-sheet imaging with electronic confocal slit
detection (eCSD) on modern sCMOS sensors. In addition to improved imaging contrast, eCSD
doubles the acquisition speed in multi-view setups with two opposing illumination directions as
it allows for simultaneous dual-sided illumination without the otherwise inherent loss in image
quality. This eliminates the need for specimen-specific data fusion algorithms and thus greatly
reduces image post-processing, eases data handling and storage. We will demonstrate application of light sheet imaging for probing large scale tissue interaction and embryonic development
in fly and early mouse. Furthermore, we will present optical manipulation tools for tissue ablation,
cauterization and opto-genetic manipulations.
82
Konstantinos Kampas
Democritus University of Thrace, Greece
Thursday 14:30
NETs as a common pathogenetic mechanism - The paradigm of autoinflammation in FMF
Although neutrophil extracellular traps (NETs) were initially described as an antimicrobial mechanism of neutrophils, recent studies demonstrate their involvement in a plethora of pathological
conditions. Clinical and experimental data indicate that NET release constitutes a common
mechanism, which is involved in various manifestations of non-infectious diseases. Even though
the backbone of NETs is similar, there are differences in their protein load in different diseases,
which represent alterations in neutrophil protein expression in distinct disorder-specific microenvironments.
In the case of autoinflammation and specifically the prototypic IL-1β-dependent disease of Familial Mediterranean fever (FMF), our group demonstrated that IL-1β during attacks is expressed
on neutrophil extracellular traps (NETs). Moreover, stress-related protein REDD1 is significantly
over-expressed during these attacks. Neutrophils from FMF patients during remission are resistant to autophagy- mediated NET release, which can be overcome via REDD1 induction.
Stress-related mediators (e.g. epinephrine) lower this threshold leading to autophagy-driven
NET release, while the synchronous inflammatory environment of FMF attack leads to intracellular production of IL-1β and its release through NETs. REDD1 in autolysosomes co-localizes
with pyrin and NALP3. Mutated pyrin prohibits this co- localization, leading to higher IL-1β levels
on NETs. These findings propose a link between stress and initiation of inflammatory attacks in
FMF. REDD1 emerges as an upstream to pyrin regulator of neutrophil function, is involved in NET
release, regulation of IL-1β and, may constitute an important piece in the IL-1β-mediated inflammation puzzle.
84
Saskia Suijerbuijk
Hubrecht Institute Utrecht, Netherlands
Friday 09:00
High-resolution intravital imaging of cancer plasticity
Saskia J.E. Suijkerbuijk and Jacco van Rheenen
Although histological techniques have provided important information on epithelial stem cells and
cancer, they draw static images of dynamic processes. To study dynamic processes, we have
developed various imaging windows to image intestinal, liver and breast tissue, and visualize the
behavior of individual cells at subcellular resolution for several weeks with two-photon intravital
microscropy (IVM). In this talk I will discuss how we have used these techniques to establish
the short-term dynamics of intestinal stem cells and mammary cancer stem cells. Our IVM experiments illustrate that cellular properties and fate of cells are highly dynamic and change over
time. For example, we show in healthy and tumorigenic tissues, that cells can acquire and lose
stem cell properties, illustrating that stemness is a state as opposed to an intrinsic property of
a cell. Moreover, we show that mammary tumor cells that are surrounded by T cells acquire
migration properties. An additional aspect that complicates tumor heterogeneity is that cells may
exchange active biomolecules through the release and uptake of extracellular vesicles (EVs).
Our data shows, in living mice, that malignant tumor cells, through transfer of EVs, enhance the
migratory behavior and metastatic capacity of more benign cells. Taken together, these data
exemplify that tumor heterogeneity and tumor microenvironment are far more complex than currently anticipated, which has profound consequences for our ideas on the mechanisms of tumor
progression and for designing optimal treatment strategies.
86
Jean-Marie Vanderwinden
ULB Brussels, Belgium
Friday 09:35
Sample clearing, a clear(er) view in fluorescence microscopy deep imaging
Statement of need:
Tissue clearing methods have been mushrooming in recent years. Getting a clear view proves
arduous as there is no “one size fits all” solution and protocols keep evolving rapidly.
Topic and content:
Tissue clearing methods have been developed to mitigate the signal degradation that occurs in
fluorescence microscopy while imaging “deep” into biological material. All tissue clearing methods basically tackle, on one hand, the scattering of light in (fixed) biological tissues, and, on the
other hand, the matching of refractive index of the sample, embedding medium and immersion
medium.
I’ll present a grassroots overview of tissue clearing methods currently available, relying on an
extensive literature survey, multiple encounters and personal hands-on experience at an academic light microscopy facility. I’ll briefly discuss the rationale underlying the clearing process
of biological material. Next, I’ll exemplify mainstream tissue clearing techniques, using various
imaging modalities on diverse biological samples. Finally, I’ll discuss the current challenges and
perspectives of these fascinating techniques.
Expected outcome:
Attendees will gain a clear(er) view of the possibilities and challenges of the tissue clearing methods current available.
Attendees will be able to dig deeper on their own to find out which clearing method is best suited
to address their respective biological questions.
Supporting information:
Many excellent original papers and reviews dealing with tissue clearing have been published in recent years - and for sure many
more will appear in the future.
As a priming on tissue clearing methods, I recommend the following outstanding reviews:
• Richardson D.S. & Lichtman J.W. (2015) “Clarifying Tissue Clearing.” Cell 162(2): 246-257.
• Susaki, E. A. and H. R. Ueda (2016). “Whole-body and Whole-Organ Clearing and Imaging
Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals.”
Cell Chem Biol 23(1): 137-157.
• Tainaka, K., et al. (2016). “Chemical Principles in Tissue Clearing and Staining Protocols for
Whole-Body Cell Profiling.” Annu Rev Cell Dev Biol 32: 713-741.
Selected weblinks for detailed protocols & tips:
• CLARITY & PACT/PARS:http://clarityresourcecenter.org
http://www.chunglabresources.com/clarity
• CUBIC: http://cubic.riken.jp/
• DISCO: http://idisco.info/
http://www.erturk-lab.com/index.php/3disco-clearing
88
90
Practicals
abstracts
91
Practical sessions take place in parallel. Each workshop room hosts
one or two collaborating companies and groups of four participants
follow a rotation schedule and thus visit every practical session.
For group assignments, please refer to separate leaflet.
92
Andor Technologies
Room 4.410
A New Imaging Platform from Andor
Keywords: Confocal, spinning disk, TIRF, deconvolution, live cell, fixed sample, software, 3D
capture, 3D visualisation.
Microscopy is now a routine part of many research projects, fundamental to understanding biological processes or targets like protein localisation, dynamics and function, capturing detail
not only at the sub-cellular scale, but also multi-cellular or whole organism. In order to study a
wide variety of biological mechanisms using multi-scale model systems (e.g. single cell to whole
drosophila embryo), you typically need to capture images using more than one imaging system
according to the biological question you are asking.
Andor has designed and manufactured a new microscopy platform which in a single device
includes high-speed confocal, widefield-deconvolution, and simultaneous multi-colour TIRF imaging. This novel solution is driven by new and dedicated software to facilitate the multi-modal
imaging through a user friendly interface. The imaging workflow covers capture to real-time
multi-dimensional rendering and deconvolution, designed to then compliment Imaris for analysis.
During the workshop we will present our new imaging platform and its many benefits including:
1. Improving imaging quality through our patented Borealis illumination technology, delivering better signal to noise performance, illumination throughput and uniformity, and extended
spectral range.
2. Significantly increasing the speed of confocal image capture by at least 10-fold compared
to traditional point scanning technology, and so delivering faster 3D volume data for
a. Studying high-speed multi-dimensional cellular dynamics
b. High-throughput 3D volume capture and rendering.
3. Engaging additional modes of imaging on a single sample or experiment to reveal more
from one investigation.
a. widefield-deconvolution for high- sensitivity photo-sensitive imaging (e.g. yeast & Dictyosteliida), and
b. TIRF imaging for cell membrane related physiology (e.g. receptor localisation and
exocytosis).
4. Reducing phototoxicity and/or photobleaching for prolonged live cell imaging and deeper
imaging before losing signal
5. Controlling sample illumination and detection parameters as additional creative tools for
deeper investigations such as single molecule and localisation studies.
93
Argolight
Room 4.200
Quality control of a confocal fluorescence microscope
This workshop aims to present the assessment of the performances a confocal fluorescence
microscope with Argolight solutions (Argo-HM hardware and Daybook software). In particular,
different aspects of the microscope will be evaluated: illumination and collection homogeneity,
distortion of the field of view, spatial colocalization, lateral resolving power, stages repositioning
accuracy, intensity and spectral responses of the system, etc. The demonstration aims to show
how the quality control of the instrument can be simple and fast with Argolight solutions.
Confocal.nl
Room 4.200
Re-scan Confocal Microscopy (RCM) for improved resolution and sensitivity. A new microscope developed by researchers for researchers.
The RCM (Re-scan Confocal Microscope) is a standard confocal microscope extended with a rescan detection unit. With a simple optical trick a lateral resolution of 170nm can be achieved for
any pinhole diameter. The pinhole is only needed for Z-sectioning. Since RCM has also a strongly improved sensitivity (4x better signal-to-noise ratio) here is no need for high laser power, being
friendlier for cells. RCM can work in multi-colour mode for different colour combinations. It is our
mission to go further by extending the spectral range into the IR for deep tissue imaging and the
use of NIR probes. Many more biological applications are on the horizon.
In the workshop we will explain how the system works and how it easily can be tuned for specific
biological application. We will also show that with this RCM confocal microscope, Confocal.nl
introduces a new, affordable confocal system that can be plugged in-between your camera and
microscope.
RCM is a technology that has been developed by Giulia De Luca and Ronald Breedijk in the
group of Erik Manders at the University of Amsterdam. Confocal.nl is a spin-off company from the
University of Amsterdam that introduces RCM to the market.
94
Hamamatsu Photonics
Room 4.020
Cameras and detectors used in modern microscopy applications
During this workshop, we will discuss the current lineup of Hamamatsu’s lineup of high-end research cameras and detectors and have a closer look at actual hardware implementations in a
selective plane illumination microscope (in collaboration with LUXENDO).
LaVision BioTec
Room 4.190
LaVision BioTec UltraMicroscope II:
The Light Sheet Microscope for CLARITY, CUBIC, iDISCO and ECi
Keywords: light sheet microscope, clearing
Today we present the 2nd generation of our light sheet microscope which has been inspired by
our user’s feedback. This new UltraMicroscope II utilizes 6 thin light sheets to excite samples
while the fluorescence light is detected with a sCMOS equipped macroscope that is mounted
perpendicular to the plane illumination. Some of the most prevalent clearing protocols like CUBIC
[1], 3DISCO [2] or iDISCO [3] have been
developed using the UltraMicroscope. It
is the only light sheet microscope which
can image samples ranging in size from
over 1 cm to few µm in aqueous buffers
also as in organic solvents. The 10x zoom
optic allows fast switching in between
smallest to highest resolution without
changing objective lenses. Different
working distances from 4 mm to 10 mm
are available. The UltraMicroscope II is
used by facilities, institutes and pharmaceutical companies because they prefer
a system which is flexible, robust and
easy to operate.
Mouse Brain (CLARITY), Maximum Intensity Projection, 3 x 3 tiled imaging, courtesy of: Deisseroth Lab, Stanford University
[1] Cell. 2014 Nov 6;159(4):911-24 Whole-body imaging with single-cell resolution by tissue decolorization. Tainaka K, Kubota SI,
Suyama TQ, Susaki EA, Perrin D, Ukai-Tadenuma M, Ukai H, Ueda HR.
[2] Nature protocols. 2012 Oct Three-dimensional imaging of solvent-cleared organs using 3DISCO Ali Ertürk, Klaus Becker, Nina
Jährling, Christoph P Mauch, Caroline D Hojer, Jackson G Egen, Farida Hellal, Frank Bradke, Morgan Sheng & Hans-Ulrich Dodt
[3] Cell. 2014 Nov 6;159(4):896-910 iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.
Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M.
95
Leica Microsystems
Room 4.180
Beyond the diffraction limit: Leica STED Nanoscopy
Nanoscopy has revolutionized the study of subcellular architecture and dynamics and is on its
way to becoming the new gold standard in fluorescence imaging (Nobel Prize for Chemistry
2014).
The fully integrated STED (STimulated Emission Depletion) system Leica TCS SP8 STED meets
the requirements of daily research and provide fast, intuitive, and purely optical access to structural details far beyond the diffraction limit.
Resolution becomes tunable in x, y and z, in multicolor, on living or fixed samples.
LUXENDO
Room 4.020
High speed volume imaging with the MuVi-SPIM: fluorescence light sheet microscopy with
multiple views
Light sheet microscopy has become the state of the art methodology to address a wide variety
of biological questions. Key features of this technique are the extremely minimized photo toxicity,
the high speed image acquisition, and the large imaging depth. This allows for imaging delicate
samples in a volumetric manner over long time. Fast cellular processes and interactions can be
observed in a global context of an organ or organoid.
In this workshop we will give an introduction to the MuVi-SPIM (Multiple View – Selective Plane
Illumination Microscope) from LUXENDO, and then guide the participants through the necessary
steps to run high speed light sheet microscopy. Participants will observe a drosophila embryo
during its development, but also are invited to bring their own samples. They will learn how to
mount the sample, set up an experiment, acquire a timelapse movie, process (registration and
fusion) and visualize the acquired data.
An entire world of new applications is about to be discovered, come and explore with us!
Drosophila embryo during development
96
Nikon Instruments
Room 4.030
Widefield imaging of the future: less is more
Nikon is presenting a workshop on widefield imaging with emphasis on the multi-functionality of
the new inverted microscope Ti2. The Ti2 will be equipped in fluorescence and brightfield mode.
See how you can tune this microscope into multiple systems dedicated to live cell timelapse imaging, high content multiwell plate screening and slide scanning. Key to this is speed, accuracy
of focus and smart software. The concept of triggering will be explained, as will be the hardware
focus control (Perfect Focus System) and the impact of the newly designed tube lens for massive
field of view matching full frame sensor camera’s. A significant bigger field of view reduces the
number of images required, reduces stitching artefacts and gives more insight into biological context. An intuitive software interface with real-time analysis possibilities is key in designing these
applications. The concept of JOBS building blocks will be shown. Entire JOBS protocols will be
run leading to the mentioned widefield microscopy imaging applications.
Olympus Microscopy
Room 4.010
FLUOVIEW FV3000 - The new confocal laser scanning microscope
Enhance the quality of your live cell imaging with a new confocal laser scanning microscope
(cLSM) system allowing you to work with highest speed, outstanding sensitivity and a great combination of macro- and micro-confocal imaging capabilities.
With the FLUOVIEW FV3000 series, Olympus introduced a new cLSM which enables you to highspeed imaging with 438 fps to capture rapid in vivo responses, and offers new levels of total system transmission efficiency with the TruSpectral detection concept. The fully spectral FV3000 has
improved overall sensitivity and signal-to-noise ratio for excellent multi-color confocal imaging.
Get more details at resolutions down to 120 nm with the latest addition to the Olympus
FLUOVIEW range of laser scanning microscopes. Widen up your possibilities with the new TruSpectral detection concept based on patented Volume Phase Hologram (VPH) transmission enabling you to select the detection wavelength of each individual channel to 1 nm precision.
Olympus invites you to experience the FLUOVIEW FV3000 cLSM system in this workshop.
97
Zeiss Microscopy
Room 4.300
Axio Zoom.V16- Fluorescence Stereo Zoom Microscope for Large Fields
Combined with ApoTome.2 for Optical Sectioning
Axio Zoom.V16 combines a 16x zoom with a high numerical aperture of NA 0.25, moving to the
forefront of all known stereo and zoom microscopes. It achieves a very high aperture in the medium zoom range already: you get superior brightness in large object fields. With Axio Zoom.V16,
the fluorescence zoom microscope for large samples you view complete model organisms in
fluorescence contrast.
Optimized Zoom for Your Applications
The eZoom of Axio Zoom.V16 works with a motorized iris diaphragm coupled to the zoom. Simply
select the best mode for your purpose:
• Brightness mode: Observe fluorescence images over the complete zoom range
with highest possible brightness.
• Eyepiece mode: This is ideal if you work mainly with ocular observation using
conventional illumination. You zoom from large object fields with maximum depth
of field to high magnifications with maximum resolution.
• Camera mode: Axio Zoom.V16 adapts to the performance of your camera. You
get an optimal relation between resolution and depth of field across the whole zoom
range.
Intelligent Transmitted Light Over the Whole Zoom Range
In addition to brightfield, darkfield and oblique illumination, you get an increased contrast brightfield at the touch of a button. With the Best Mode button, the stereo zoom microscope Axio
Zoom.V16 determines the actual optical state and optimizes transmitted light automatically. Use
the Adjust control to fine-tune Best Mode more precisely to your application. Then simply save
your setting and reload it for your next experiment - once again, at the touch of a button.
ApoTome.2 – Create Optical Sections of Your Fluorescent Samples
With structured illumination, you know that only the focal plane appears in your image: ApoTome.2 recognizes the magnification and moves the appropriate grid into the beampath. The
system then calculates your optical section from three images with different grid positions. It’s a
totally reliable way to prevent scattered out-of-focus light, even in your thicker specimens. You
get images with high contrast in the best possible resolution. ApoTome.2 is the perfect option for
your stereo zoom microscope to create simply brilliant optical sections.
98
Data analysis
abstracts
99
Data analysis sessions take place in parallel. Each analysis room hosts
a team of software experts and groups of ten participants follow a rotation schedule and thus visit each of the three analysis sessions.
For group assignments, please refer to separate leaflet.
100
Sebastian Munck – VIB Leuven
Room 4.510
ImageJ/FIJI
In recent years, imaging and image processing has become more and more
important for life scientists. In addition, dealing with scientific images has
evolved from showing examples images towards quantitative biology. This
course is aiming at the starter level of image processing and will give an overview of commonly available tools and will introduce the usage of ImageJ/FIJI.
The course consists of lectures, demos and practical exercises.
Daniel Reisen – Bitplane
Room 4.410
Imaris
During the Imaris workshop participants will learn the workflow to load and
visualize (large) microscopy image data. Focus will be put into the latest features on how to trace neuron structure using the new patented Torch™ tool
in Filament Tracer. The new membrane based cell boundary detection from
the Cell module will as well be addressed to segment 3D membrane strained
cells.
David Wiles – arivis
Room 4.300
Vision4D
Introduction to very large image handling and processing in arivis Vision4D
software. Learn how to import images, adjust and display them in the most effective fashion and export high quality movies and snapshots for publication.
Introduction to 4D image analysis.
101
102
Participants
contact details
103
Registered participants
—A—
Al Heib, Razan
University of Luxembourg
Esch-sur-Alzette
Luxembourg
—B—
Becker, Björn
Saarland Univeristy
Saarbrücken
Germany
[email protected]
Berenguer, Clara
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—F—
Folz-Donahue, Kat
Max Planck Institute for Biology of Ageing
Cologne
Germany
[email protected]
—G—
Galtsidis, Sotirios
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Grzyb, Kamil
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—H—
Heurtaux, Tony
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
104
Howard-Till, Rachel
University of Vienna
Vienna
Austria
[email protected]
—I—
Illig, Christin
Ludwig Maximilian University
Munich
Germany
[email protected]
—J—
Jarazo, Javier
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—K—
Kleine-Borgmann, Felix
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Koeglsberger, Sandra
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Koncina, Eric
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Kondratyeva, Olga
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Krishtal, Jekaterina
Tallinn University of Tech-
nology
Tallinn
Estonia
[email protected]
—L—
Larsen, Simone
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Lucumi Moreno, Edinson
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—M—
Martins, Teresa
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Massouh, Amid
Instituto Gulbenkian de
Ciência
Oeiras
Portugal
[email protected]
Meier, Roger
ETH Zurich
Zurich
Switzerland
[email protected]
Moein, Mahsa
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Moes, Michele
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—O—
Ouzren, Nassima
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—S—
Sandberg, Elin
Royal Institute of Technology
Stockholm
Sweden
[email protected]
Schuster, Anne
Luxembourg Institute of
Health
Luxembourg City
Luxembourg
[email protected]
Schwirz, Jonas
Institute of Molecular Biology
gGmbH
Mainz
Germany
[email protected]
Siavash, Mansouri
Saarland University Medical
Center
Homburg
Germany
[email protected]
Sousa, Carole
Luxembourg Institute of
Health
Luxembourg City
Luxembourg
[email protected]
Stojevski, Dunja
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Sullivan, Adrienne
Imperial College
London
United Kingdom
adrienne.sullivan8@gmail.
com
—T—
Tosheva, Zornirza
University of Luxembourg
Luxembourg City
Luxembourg
[email protected]
—W—
Wasner, Kobi
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
Academic speakers
—C—
Cillero-Pastor, Berta
Maastricht University
Maastricht
Netherlands
[email protected]
—E—
Egner, Alexander
Laser-Laboratorium Göttingen
e.V.
Göttingen
Germany
[email protected]
—G—
Genoud Christel
Friedrich Miescher Institute
for Biomedical Research
Basel
Switzerland
[email protected]
Grünewald, Anne
University of Luxembourg
Esch-sur-Alzette
Luxembourg
[email protected]
—H—
Heintzmann, Rainer
Leibniz Institute of Photonic
Technology
Jena
Germany
rainer.heintzmann@uni-jena.
de
Hendrix, Jelle
University of Hasselt
Diepenbeek
Belgium
[email protected]
—K—
Kampas, Konstantinos
Democritus University of
Thrace
Alexandroupolis
Greece
[email protected]
Kuchenov, Dmytri
European Molecular Biology
Laboratory
Heidelberg
Germany
[email protected]
—L—
Leonhardt, Heinrich
Ludwig Maximilian University
Munich
Germany
[email protected]
105
Lorenzo, Corinne
Institut des Technologies
Avancées en sciences du
Vivant
Toulouse
France
[email protected]
—M—
Munch, Sebastian
Vlaams Instituut voor Biotechnologie
Leuven
Belgium
sebastian.munck@kuleuven.
vib.be
Manders, Erik
University of Amsterdam
Amsterdam
Netherlands
[email protected]
—P—
Pavie, Benjamin
Vlaams Instituut voor Biotech-
nologie
Leuven
Belgium
benjamin.pavie@kuleuven.
vib.be
Pepperkok, Rainer
European Molecular Biology
Laboratory
Heidelberg
Germany
[email protected]
Vanderwinden, Jean-Marie
Université Libre de Bruxelles
Brussels
Belgium
[email protected]
—W—
—S—
Widengren, Jerker
Royal Institute of Technology
Stockholm
Sweden
[email protected]
Stelzer, Ernst
Goethe University Frankfurt
Frankfurt am Main
Germany
[email protected]
Wittbrodt, Jochen
Heidelberg University
Heidelberg
Germany
[email protected]
Suijerbuijk, Saskia
Hubrecht Institute
Utrecht
Netherlands
[email protected]
—V—
—Z—
Zimmer, Christophe
Institut Pasteur
Paris
France
[email protected]
Company representatives
—B—
Baert, Philippe
Nikon Belux
Brussels
Belgium
[email protected]
Braquenier, Jean-Baptiste
Nikon Belux
Brussels
Belgium
jean-baptiste.braquenier@
nikon.com
—C—
Combettes, Bruno
Andor Technology Ltd
Belfast
106
United Kingdom
[email protected]
Coppieters, Jean-François
Carl Zeiss NV
Zaventem
Belgium
jean-francois.coppieters@
zeiss.com
—D—
Dierickx, Olivier
Carl Zeiss NV
Zaventem
Belgium
[email protected]
Dos Santos, Christophe
arivis AG
Rostock
Germany
christophe.dossantos@arivis.
com
Drent, Peter
Confocal.nl BV
Amsterdam
Netherlands
[email protected]
—E—
Eich, Florian
Olympus Europa SE & Co. KG
Hamburg
Germany
florian.eich@olympus-europa.
com
—F—
Fontaine, Frédéric
Olympus Belgium NV
Antwerpen
Netherlands
[email protected]
—G—
Gabler, Thomas
LASOS Lasertechnik GmbH
Jena
Germany
[email protected]
Golfis, Georgia
Bitplane AG
Zurich
Switzerland
[email protected]
—K—
Kaakour, Ziad
Olympus France SAS
Rungis
France
[email protected] —L—
Laigle, Clément
Leica Microsystems SAS
Nanterre
France
[email protected]
Löschinger, Monika
LUXENDO GmbH
Heidelberg
Germany
[email protected]
Lotter, Andreas
Leica Microsystems GmbH
Wetzlar
Germany
[email protected]
—M—
Mayer, Jürgen
LUXENDO GmbH
Heidelberg
Germany
[email protected]
—O—
Orthaus-Mueller, Sandra
PicoQuant GmbH
Berlin
Germany
[email protected]
—P—
Pfuhl, Andreas
LUXENDO GmbH
Heidelberg
Germany
[email protected]
Pirson, Emmanuel
Hamamatsu / Axisparc Technology
Mont-Saint-Guibert
Belgium
[email protected]
—R—
Rabis, Claudia
LASOS Lasertechnik GmbH
Jena
Germany
[email protected]
Rapp, Gert
Rapp OptoElectronic GmbH
Hamburg
Germany
[email protected]
Reisen, Daniel
Bitplane AG
Zurich
Switzerland
[email protected]
Pforzheim
Germany
[email protected]
Royon, Arnaud
Argolight SA
Pessac
France
[email protected]
—S—
Schröer, Uwe
LaVision BioTec GmbH
Bielefeld
Germany
[email protected]
Sommerauer, Michael
AHF analysentechnik AG
Tübingen
Germany
[email protected]
—T—
Tudor, Cicerone
Carl Zeiss NV
Zaventem
Belgium
[email protected]
—W—
Weiß, Alexander
Leica Microsystems GmbH
Wetzlar
Germany
[email protected]
Wiles, David
arivis AG
Rostock
Germany
[email protected]
Rietdorf, Jens
ACQUIFER Digital Biomedical
Imaging Systems AG
107
108
Notes
109
Participating companies