The book abstracts - Université Paris Saclay

Transcription

4 - 5 - 6 NOVEMBER 2015
AUDITORIUM INSTITUT CURIE
CENTRE UNIVERSITAIRE ORSAY, BÂT. 111
PhysChemCell 2015
PHYSICAL CHEMISTRY OF THE CELL:
INNOVATIVE BIOIMAGING
Cell signalling and trafficking
New cellular reporters
Innovative structural and chemical imaging
Multimodal imaging
In vivo & clinical applications
Plenary speakers
Crédits photos : CLUPS, IBAIC, ISMO, LCP
Philippe Bastiaens (Dortmund)
Stefan Jakobs (Göttingen)
Kai Johnsson (Lausanne)
Abraham Koster (Leiden)
Nicolas Beziere (Munich)
REGISTRATION AND INFORMATION
http://www.cpps.u-psud.fr
2
4 - 5 - 6 NOVEMBER 2015
AUDITORIUM INSTITUT CURIE
CENTRE UNIVERSITAIRE ORSAY, BÂT. 111
OBJECTIVES
Cell imaging and microscopy play a prominent role in biological and medical research and have considerably progressed in the past fifteen years. Optical imaging
techniques track the spatio-temporal transformations, interactions and dynamics
of defined biomolecules in their functional context. A variety of chemical mapping
methods give access to the biodistribution of cell metabolites as well as of exogenous
drugs, nanocarriers or trace elements. Label-free or fast cryogenic methods better
preserve living cell and tissue integrity, to analyze their subcellular as well as multicellular organization. Correlative approaches overlay various spectral or chemical
information into high-content images. Most of these techniques now operate at or
close to the molecular scale, i.e., at nanometric resolution.
From the single cell to the living animal, these advances will ultimately give access
to a detailed, in situ physical and chemical analysis of biological functions, thereby
addressing the challenge of biological complexity and integration. The intimate
knowledge of biological processes thus gained at the molecular level brings in turn
essential information for understanding and modeling diseases, opening the way to
innovative therapeutic strategies.
The conference will bring together an interdisciplinary community to discuss the
latest advances in the fields of analytical cell imaging. Conference topics will cover
cell signaling and trafficking studies, the development of new organic, inorganic or
genetically encoded probes, innovative imaging contrasts and instruments, and will
explore their potential for in vivo and biomedical applications.
3
ORGANIZING COMMITTEE
Larbi Amazit
Céline Boutin
Cédric Bouzigues
Catherine Chapon
Ariane Deniset
Sandrine Lécart
Amélie Leforestier
Florence Mahuteau
Anne Mantel
Rachel Méallet-Renault
Fabienne Mérola
Valérie Nicolas
Oliver Nüsse
Robert Pansu
Ève Ranvier
Matthieu Réfrégiers
Béatrice Satiat-Jeunemaître
Marie-Noëlle Soler
Marie-Paule Teulade-Fichou
François Treussart
Boris Vauzeilles
ACKNOWLEDGMENTS
4
SUMMARY
TIME TABLEP. 6
WEDNESDAY 4 NOVEMBER
SESSION 1: CELL SIGNALLING AND TRAFFICKING
SESSION 2: INNOVATIVE STRUCTURAL AND CHEMICAL IMAGING
P. 9
P. 19
THURSDAY 5 NOVEMBER
SESSION 1: NEW CELLULAR REPORTERSP. 27
SESSION 2: MULTIMODAL IMAGINGP. 35
FRIDAY 6 NOVEMBER
SESSION 1: IN VIVO AND CLINICAL APPLICATIONS
P. 43
POSTERS ABSTRACTSP. 51
5
TIME TABLE
Wednesday 4 November
8:30 - welcoming session
8:45 - Patricio Leboeuf, Deputy Director for Research, FCS Campus Paris Saclay
Session 1 - 9:00-13:00 - Cell signalling and trafficking
Chairs : Oliver Nüsse, Béatrice Satiat-Jeunemaître
The interdependence of vesicular membrane dynamics and signal processing
Philippe Bastiaens, Dortmund
Spatio-temporal coordination between DNA replication and mitotic commitment
Olivier Gavet, Villejuif
Oxidative stress signaling during pathogen phagocytosis
Sophie Dupré, Orsay
11:00 - coffee-break
Following the autophagosome formation: a live cell imaging challenge
Romain le Bars, Gif
Oxidant signalling in cells revealed by single rare-earth based nanoparticle imaging
Cédric Bouzigues, Palaiseau
Industrial session
Exploring life science with HORIBA Scientific
Chiraz Frydman, Palaiseau
Click4Tag markets new tools for labelling membranes of living microorganisms
Sam Dukan, Marseille
13:00 - lunch
Session 2 - 14:30-18:30 - Innovative structural and chemical imaging
Chairs : Marie Erard, François Treussart
Live-cell RESOLFT nanoscopy
Stefan Jakobs, Göttingen 3D superlocalization microscopy thanks to supercritical angle fluorescence
Sandrine Lévêque-Fort, Orsay
Label-free deep UV imaging at SOLEIL synchrotron
Frédéric Jamme, Saint Aubin
Biological applications of lipid imaging with cluster-TOF-SIMS and MALDI-TOF mass spectrometry
Alain Brunelle, Gif
How to investigate bio-molecules in microorganisms by infrared nanoscopy technique
Alexandre Dazzi, Orsay
Multidisciplinary analysis of the cell membranes electropermeabilization
Lluis Mir, Villejuif
Thursday 5 November
Session 1 - 9:00-13:00 - New cellular reporters
Chairs : Anton Granzhan, Robert Pansu
Synthetic and semisynthetic probes for live-cell imaging
Kai Johnsson, Lausanne 6
Metabolic reporters of live bacteria
Boris Vauzeilles, Gif
Switchable probes for labelling nucleic acids in live cells
Marie-Paule Teulade-Fichou, Orsay
Scanning transmission electron microscopy cathodoluminescence
Mathieu Kociak, Orsay
Engineering cyan fluorescent proteins with ultimate performances for live cell biosensors
Hélène Pasquier, Orsay
Laser-polarized xenon for the study of biological cells
Céline Boutin, Saclay
13:00 - lunch
Session 2 - 14:30-18:30 - Multimodal imaging
Chairs : Sandrine Lévêque-Fort, Rachel Méallet-Renault
Institutional session
Jean-Pierre Mahy, Paris-Saclay Chemistry Department work group
Nadine Peyriéras, France-BioImaging
Sandrine Lévêque-Fort, GDR 2588 Microscopie et Imagerie du Vivant (MIV)
Zooming in on cells and macromolecules with correlative light-electron electron microscopy
Abraham Koster, Leiden
Chemical imaging tools for intracellular tracking of nanoparticles
Sergio Marco, Orsay
Soft X-ray microscopy with synchrotron radiation
Bertrand Cinquin, Saint-Aubin
In situ quantitation of collagen fibrils diameter using absolute measurements of SHG signals
Marie-Claire Schanne-Klein, Palaiseau
Diffusion, localization and bioavailability of clinically-used antibiotics in staphylococcus aureus biofilms
Karine Steenkeste, Orsay
Friday 6 November
Session - 9:00-13:00 - In vivo and clinical applications
Chairs : Emmanuel Beaurepaire, Matthieu Réfrégiers
Optoacoustic Imaging: light and sound for in vivo biomedical imaging
Nicolas Bézière, Munich
Liver graft quality control by infrared spectroscopy
François Le Naour, Villejuif
In vivo imaging techniques to explore the immune system in non-human primates
Catherine Chapon, Fontenay-aux-Roses
Optical imaging for biodistribution studies in preclinical research
Frédéric Ducongé, Fontenay-aux-Roses
In vivo imaging of signaling pathways activation in drosophila’s mushroom bodies neurons
Nicolas Gervasi, Paris
Thermal effects of high power density light stimulation for optogenetics control of deep brain structures
Frédéric Pain, Orsay
Closing remarks
7
8
SESSION 1: CELL SIGNALLING AND TRAFFICKING
SPEAKERS :
Philippe Bastiaens, Dortmund
Olivier Gavet, Villejuif
Sophie Dupré, Orsay
Romain Le Bars, Gif-sur-Yvette
Cédric Bouzigues, Palaiseau
INDUSTRIAL PRESENTATION
HORIBA Scientific
Click4Tag
CHAIRMANS:
Olivier Nüsse and Béatrice Satiat-Jeunemaître
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N-STORM 4.0
Nikon’s
Next Generation
N-STORM
Super-Resolution
Microscope System
High Power objectives optimized
for N-STORM Imaging
Newly developed illumination magnifying lens
for high quality Super-Resolution images
Newly developed optics for expanded flexibility in the
imaging field of view
Improved laser excitation design and a sCMOS camera
to increase acquisition rates from minutes to seconds
www.nikoninstruments.eu
10
The interdependence of vesicular membrane dynamics and signal processing
Philippe Bastiaens
Systemic Cell Biology, Max Planck Institute for Molecular Physiology,, Dortmund, Germany
[email protected]
Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables diverse, context-dependent
responses to extracellular signals but comes at the price of autonomous, ligand-independent
activation. Reactions in and on membranes play an important role in extracellular information
processing by cells. The local concentration of signaling proteins is maintained by membrane
dynamics to tightly control the qualitative response properties of signaling systems. In order to
illuminate the relevance of this spatial dimension in signaling, I will describe how vesicular membrane
dynamics control the autocatalytic activity of receptor tyrosine kinases such as EGFR and EphA2.
Spontaneous RTK activation is suppressed by vesicular recycling and dephosphorylation by protein
tyrosine phosphatases (PTPs) at the pericentriolar recycling endosome. This spatial segregation of
catalytically superior PTPs from RTKs is essential to preserve ligand responsiveness of receptors at
the plasma membrane. Ligand-induced clustering, on the other hand, promotes phosphorylation of cCbl docking sites and ubiquitination of the receptors, thereby redirecting them to the late
endosome/lysosome. This switch from cyclic to unidirectional receptor trafficking thereby converts a
continuous suppressive safeguard mechanism into a finite ligand-responsive signaling mode.
11
Spatio-‐temporal coordination between DNA replication and mitotic commitment Olivier GAVET1, 2 1
2
Sorbonne Universités, UPMC Paris VI, UFR927, F-‐75005, Paris, France Present address: Institut Gustave Roussy, UMR 8200 CNRS, PR2, 114 rue Edouard Vaillant, F-‐94805 Villejuif Cedex, France Abstract Eucaryotic cell proliferation requires the orderly progression through two major events of the cell cycle, DNA replication and Mitosis, to ensure the stable inheritance of the genetic material to the cell progeny. How this tight coordination is achieved in space and time remains poorly elucidated. We investigated underlying regulatory network by the development of FRET-‐based kinase biosensors and real time live single cell assays. We previously determined that activation of the master mitotic driver, CyclinB1-‐Cdk1, is rapidly initiated in late G2 cells and immediately triggers prophase onset. Here, we investigated upstream regulatory mechanisms focusing on the involvement of Polo-‐like kinase 1 (Plk1). We found that activation of Plk1 pool reproducibly precedes Cyclin-‐B1-‐Cdk1 one by a few minutes during G2 phase progression and that its activity is critical for mitotic commitment. Plk1 activation relies on upstream Aurora-‐A kinase and appears further related to interphasic CyclinA2-‐Cdk1&2 activity, a main regulator of DNA replication. Indeed, we found that stimulating CyclinA2-‐Cdk activity from late S triggered a premature activation of Plk1 preceding unscheduled entry into mitosis. Consistently, using Knock-‐In CyclinA2-‐Venus human cells, we observed that nuclear CyclinA2 translocates to the cytoplasm during G2 phase progression and progressively interacts with Plk1. Therefore, our results uncover a molecular link between DNA replication and activation of the Mitotic Entry Network. 12
Oxidative stress signaling during pathogen phagocytosis
Dupré S., Song Z.M., Bouchab L., Hudik E., Le Bars R., Nusse O.
Université Paris Sud, LCP, UMR8000/ INSERM UMR1174, 91405 Orsay
[email protected]
Neutrophils are white blood cells that engulf pathogens at the site of infection.
This process named phagocytosis drives the formation of a closed compartment inside
the cell: the phagosome. In the phagosome, reactive oxygen species (ROS) are
produced. These ROS, produced by the NADPH oxidase (NOX2), are crucial for
pathogen killing. The NADPH oxidase is activated when the cytosolic subunits of
NOX2 (p67, p47, p40, Rac) assemble with the membrane subunits (gp91 and p22) at
the phagosomal membrane. P67 triggers the electron flow from NADPH to O2.-.
We stilO GRQ¶W NQRZ ZKLFK IDFWRUV UHJXODWH D VXVWDLQHG 526 SURGXFWLRQ DW WKH
phagosome and maintain the key cytosolic subunit p67 at that phagosomal membrane.
Some recent data indicate that phosphoinositol-3phosphate (PI3P) activate ROS
production in human neutrophils via its binding to p401.
In this study, we wanted to understand the role of PI3P in the regulation of
phagosomal ROS production. In a first approach we used a pharmacologic inhibitor of
PI3P formation, wortmannin, to decrease phagosomal PI3P. Using spinning disk
confocal video microscopy and a fluorescent protein-tagged PI3P probe we observed
the disappearance of PI3P within 3-4 minutes after wortmannin addition. The addition
of wortmannin also dramatically decreased the time of presence of tagged p40 and
p67 at the phagosome, which both left the phagosome at the same time. We then
conducted a genetic approach to manipulate the PI3P content of the phagosome.
Using a siRNA approach we were able to increase the level of PI3P at the phagosome
in space and time. Thanks to the ROS biosensor DCFH2-yeast2, we observed that the
increase of PI3P at the phagosome triggered increased ROS production inside the
phagosome and an extended accumulation of p67 at the phagosome. We also
overexpressed the PI 3-phosphatase MTM1 at the phagosomal membrane with the
FRB-FKBP dimerization system. This overexpression induced the disappearance of
PI3P from the phagosome and a strong decrease in ROS production.
In conclusion, PI3P regulates ROS production in the phagosome. Our data suggest
that PI3P could regulate the termination of the oxidative burst and the disassembly of
NADPH oxidase complex at the phagosome.
1. Matute JD et al, 2009. ³A new genetic subgroup of chronic granulomatous disease with autosomal
recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity´. Blood,
114(15):3309-15
2. Tlili A, Dupré-Crochet S, Erard M, Nüsse O.,2011. ³Kinetic analysis of phagosomal production of
UHDFWLYHR[\JHQVSHFLHV´ Free Radicals in Biology and Medecine, 50(3):438-47
13
Following the autophagosome formation: a live imaging challenge
Romain Le Bars1, Jessica Marion1,2, Claire Boulogne1, Béatrice Satiat-Jeunemaitre1,2, and
Michele W. Bianchi2.
1
CNRS, I2BC, Pôle d’Imagerie et de Biologie Cellulaire IMAGERIE-GIF, 91198 Gif sur Yvette, France.
2
CNRS, I2BC, Dynamique de la Compartimentation cellulaire dans les cellules de plantes
supérieures, 91198 Gif sur Yvette, France.
[email protected]
Macroautophagy (here referred to as autophagy) is a bulk degradation mechanism that sequesters
cytosolic compounds inside a double membrane bound vesicle termed the autophagosome, which is
then delivered to the vacuole for breakdown and turnover. Genetic studies in yeast identified genes
involved in the different steps of autophagosome biogenesis. Subsequently, homologs of several of
the ATG proteins have been identified in plants. These findings allowed a better understanding of the
roles of autophagy such as: nutrient recycling, cell detoxification, organelles turnover, remobilization
during leaf senescence
However the early steps of autophagosome formation are still poorly
understood and the origin of the membrane is still a matter of debate.
Autophagy is a very dynamic process within plant cells and also highly sensitive to any kind of
environmental stress. A precise and accurate study of this process required the use and development
of multiple tools coupled with live imaging technics.
The use of such an up to date microscopic setup on Arabidopsis roots, allowed us to show that ATG5,
a protein involved in the early steps of autophagosome formation, labels a subdomain of the
autophagosome membrane. The ATG5 domain marks the growing autophagosome and gave us the
opportunity to closely follow their formation in vivo. The interactions between autophagosomes
markers (ATG5, ATG8) and markers for endomembrane compartments were also monitored,
revealing a close link with the cortical ER network. This observation was also confirmed by correlative
microscopy. Our findings will contribute to the understanding of autophagosome formation in plants.
References
Le Bars et al. (2014) ATG5 defines a phagophore domain connected to the endoplasmic reticulum
during autophagosome formation in plants Nat Commun 5, 4121.
Le Bars et al. (2014) Folding into an autophagosome: ATG5 sheds light on how plants do it.
Autophagy 10, 52-54.
14
Oxidant Signaling in Cells Revealed by Single Rare-Earth Based Nanoparticle Imaging
Cedric Bouzigues, Mouna Abdesselem, Thanh-Liêm Nguyên, Rivo Ramodiharilafy, PierreLouis Tharaux and Antigoni Alexandrou
Laboratoire Optique et Biosciences
Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay Palaiseau France
[email protected]
For many cell functions, spatio-temporal organization of signaling pathways is important for the cell
response regulation. Reactive oxygen species (ROS) are common second messengers involved in the
control of numerous normal and pathological cell responses. ROS homeostasis is thus essential for
the cell physiology and their local concentration is thus tightly regulated. However, the dynamics of
ROS production and organization is so far mostly unknown, due to the lack of efficient probes.
3+
By imaging single Eu -doped nanoparticles, we quantitatively probed the intracellular ROS response
with temporal and spatial resolution. We thus measured the absolute ROS concentration in vascular
cells and revealed specific temporal patterns of ROS production under different types of stimulation
(PDGF and ET-1). We furthermore quantitatively revealed mechanisms of transactivation of the EGFR
pathway, which notably control the dynamics of the cell response. These mechanisms of temporal
control are impaired in tumoral cells, which may be of great pathophysiological relevance for the
metastatic transition. By using a microfluidic system, we furthermore apply spatially controlled
stimulations and displayed the maintenance of asymmetric ROS concentration in the cell under a
migration inducing PDGF gradient. This likely relies on a balance between ROS diffusion and
degradation, whose molecular basis is still to be investigated.
Altogether, our results reveal the regulation mechanisms of the ROS organization in the cell, which
illustrates how the spatio-temporal control of transduction pathways is crucial for the buildup of the cell
response. We now propose to extend these methods, by using different types of nanoparticles in a
ratiomectric approach, to detect ROS at high temporal resolution (!2Hz) in order to elucidate the
molecular mechanisms responsible for the observed patterns at the cellular scale.
Left: HeLa cells in a microfluidic device creating a permanent PDGF gradient. Right: YVO4:Eu
nanoparticles in HeLa cells submitted to a PDGF gradient.
15
Exploring Life Science with HORIBA Scientific Mme Chiraz Frydman Horiba Scientific, Avenue de la Vauve, Passage Jobin Yvon, 91120 Palaiseau, France HORIBA Scientific is a worldwide leader in development and production of analytical measurement equipment for research, analysis for laboratories, and quality control. We offer instrumentation for Fluorescence-‐ and Raman-‐Spectroscopy, Ellipsometry and other methods measuring thin films In addition measurement techniques like Particle analysis, methods for the determination of water quality as well as X-‐Ray fluorescence and SPRi (surface plasmon resonance imaging) belong to our expertise. The better part of these instruments can be used not only for macroscopic analysis, but also for micro-‐measuring techniques -‐ often in a non-‐destructive manner, which is a major advantage over many other analytical methods. 16
Click4Tag markets new tools for labeling membranes of living microorganisms
Audrey Dumont, Emilie Fugier and Sam Dukan
CLICK4TAG, 163 avenue de Luminy, Zone Luminy Biotech, Case 922, 13288 Marseille cedex 09.
[email protected]
Click4Tag created in December 2014, develops innovative solutions for detection, enumeration, concentration
and/or rapid identification of culturable microorganisms using a real technological breakthrough exclusively
licensed at the end of 2014.
The technology is based on specific functionalization of culturable microorganisms via natural assimilation and
integration of a "hook" (probe) into their membrane. This anchorage point permits to associate conventional
labels by "Click" reaction, to (i) a fluorescent label to detect, count and/or identify culturable microorganisms of
interest, (ii) magnetic beads in order to concentrate/isolate culturable microorganism of interest.
Figure 1: The concept of Click4Tag technology
Nowadays, Click4Tag commercialized molecules allowing bacterial membranes functionalization for industrial
and academic research. Right now we commercialized L. pneumophila lipopolysaccharides labeling and in few
month a new tools that will label membrane from all kind of microorganisms.
References
- Fugier E., Dumont A., Malleron A., Poquet E., Mas Pons J., Baron A., Vauzeilles B. and Dukan S. (2015)
Rapid and specific enrichment of culturable Gram negative bacteria using non-lethal copper-free click chemistry
coupled with magnetic beads separation. Plos One 10(6) :e0127700.
- Pons J. M., Dumont A., Sautejeau G., Fugier E., Baron A., Dukan S. and Vauzeilles B. (2014) Identification of
Living Legionella pneumophila Using Species-Specific Metabolic Lipopolysaccharide Labeling. Angew. Chem.
Int. Ed. 53, 1275-1278.
- Dumont A., Malleron A., Awwad M., Dukan S. and Vauzeilles, B. (2012) Click labeling of bacterial
membranes via metabolic modification of the LPS inner-core. Angew. Chem. Int. Ed., 51, 3143-3146.
17
18
SESSION 2: INNOVATIVE STRUCTURAL AND CHEMICAL IMAGING
SPEAKERS:
Stefan Jakobs, Göttingen
Sandrine Lévêque-Fort, Orsay
Frédéric Jamme, Saint Aubin
Alain Brunelle, Gif-sur-Yvette
Alexandre Dazzi, Orsay
Lluis Mir, Villejuif
CHAIRMANS:
Marie Erard and François Treussart
19
THE FIRST MULTI-PROTEINS
PRINTING PLATFORM
PRIMO is an optical unit docked
to an inverted microscope (Nikon Ti)
for printing several proteins on cell
culture slides.
3
Based on the combination of a UV
illumination system and a patented
chemistry (PLPP) which catalyzes
the proteins patterning.
2
Fibroblasts arranged
in ALVÉOLE pattern
Projected pattern
on culture slide
PRIMO BRINGS YOU NEVER SEEN CAPABILITIES
Multiple proteins printing (>3)
v
v
Resolution <1 micrometer over the entire field of view
v
Quantitative printing (255 grayscale gradient)
v
Fast printing (20s for a 4mm2 pattern)
Connection of PRIMO
to the microscope
UV
400 µm
3 color fluorescence
microscopy image.
This complex micro pattern
combines three different
fluorescent proteins (GFP,
mCherry and NeutravidinAto647) which have been
printed sequentially at the
same location
PRIMO SOFTWARE KEY FEATURES
v Automatic calibration
v
Multipatterning
v
Oversized patterns management
v
Successive patterns alignment
MICROSCOPE
NIKON ECLIPSE Ti-E
1
20
ALVÉOLE - 68 Bd de Port-Royal, 75005 PARIS - FRANCE - ALVEOLELAB.COM
Image file
sent to PRIMO
Live-cell RESOLFT nanoscopy
Stefan Jakobs
Max Planck Institute for Biophysical Chemistry, Dept. of NanoBiophotonics
& University Medical Center of Göttingen, Dept. of Neurology
Am Fassberg 11
37077 Goettingen, Germany
www.RSFP.de
E-mail: [email protected]
All super-resolution microscopy (nanoscopy) concepts that fundamentally overcome the
diffraction barrier in far-field optical microscopy utilise fluorophore transitions between two states,
typically a fluorescent on- and a non-fluorescent off-state. In RESOLFT (reversible saturable optical
fluorescence transition) and the related STED (stimulated emission depletion) microscopy concepts, a
doughnut or a pattern is scanned across the sample, determining at any point in time the nanosized
coordinate range where the fluorophores are in the on-state.
RESOLFT microscopy utilizes reversibly switchable fluorescent proteins (RSFPs) that can be
repeatedly photoswitched between fluorescent and non-fluorescent states by irradiation with distinct
light wavelengths to overcome the diffraction barrier in live cell imaging. Previous RSFPs allowed only
a limited number of switching cycles before photodestruction. We have generated a number of new
and improved RSFPs with different photophysical properties; they exhibit strong resistance against
photobleaching and switching fatigue, and thus allow dual-color as well as massively parallelized
RESOLFT microscopy. Recent progress in the development of new RSFPs as well as their application
in live-cell RESOLFT nanoscopy will be discussed.
References
Chmyrov, A.,Keller, J.,Grotjohann, T.,Ratz, M.,d'Este, E.,Jakobs, S.,Eggeling, C. and Hell, S.W.
(2013). Nanoscopy with more than 100,000 'doughnuts'. Nature Methods 10 (8): 737-740
Grotjohann, T., Testa, I., Reuss, M., Brakemann, T., Eggeling, C, Hell, S.W., and Jakobs, S. (2012).
rsEGFP2 enables fast RESOLFT nanoscopy of living cells. eLife, DOI:10.7554/eLife.00248
Grotjohann, T., Testa, I., Leutenegger, M., Bock, H., Urban, N.T., Lavoie-Cardinal, F., Willig, K.I.,
Eggeling, C., Jakobs*, S., and Hell*, S.W., (2011). Diffraction-unlimited all-optical imaging and writing
with a photochromic GFP. Nature, 478(7368):204-8
Brakemann, T., Stiel, A.C., Weber, G., Andresen, M., Testa, I., Grotjohann, T., Leutenegger, M.,
Plessmann, U., Urlaub, H., Eggeling, C., Wahl, M.C., Hell, S.W., and Jakobs, S. (2011). A reversibly
photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nature
Biotech, 29(10):942-7.
21
3D superlocalization microscopy thanks to Supercritical Angle Fluorescence
N. Bourg1,2, G. Dupuis2, S. Lécart2, E. Fort3, S. Lévêque-Fort1
1
Institut des Sciences Moléculaires d’Orsay (ISMO), CNRS, Univ. Paris-Sud, Université Paris-Saclay,
F91405 Orsay, France
2
Centre Laser de l’Université Paris Sud (CLUPS/LUMAT), Univ. Paris-Sud, CNRS, IOGS, Université
Paris-Saclay, F-91405 Orsay, France
3
Institut Langevin, EPSCI ParisTech, CNRS, PSL Research University, F-75005 Paris, France
[email protected]
Point Spread Function (PSF) engineering methods combined to Single Molecule Localization
Microscopy (SMLM) allow ones to retrieve 3D position of fluorescent molecules with nanometer
accuracy. Unfortunately, achieving high 3D localization precision is often at the cost of an increased
experimental complexity. In addition, all these strategies only provide the relative axial positions of the
fluorophores with respect to an arbitrary focal plane. Hence, 3D optical nanoscopy would strongly
benefit of a method that combines high nanometer axial precision, simplicity of implementation and
absolute axial positioning.
Here we propose to take advantage of the
forbidden light, also called SupercriticalAngle Fluorescence (SAF)[1,2], at the single
molecule level. Indeed, when a fluorophore is
located in the vicinity of the coverslip
interface, its near-field SAF component
become propagative and can be collected by
a high numerical aperture objective (Fig. 1).
In the objective back focal plan, the SAF
component appears as a ring beyond the
critical angle !c. For a fluorophore at the
interface, the number of photons in the SAF
SAF
represents 50% of all the photons
ring N
tot
SAF
As
N
decreases
collected
N .
exponentially with the dye depth distance
from the coverslip surface, absolute axial
Figure 1 Principle of the SAF emission
position of each fluorescent dye is retrieved
SAF
tot
versus N . In practice,
by comparing N
only the detection path of our SMLM setup is modified to insert a compact homemade dual view
SAF
and Ntot, and retrieve in live the absolute
module, which permits to simultaneously measured N
axial position of each fluorescent dye. This 3D absolute method, called “Direct Optical Nanoscopy with
Axially Localized Detection (DONALD)” gives an isotropic 3D localization precision of 20-nm within an
axial range of ~150 nm above the coverslip [3]. The localization performances of DONALD allows us
to observe the actin network with an axial resolution ~35 nm (Fig.2), and to super-resolve the 3D
hollowness of microtubules. Thanks to the absolute localization, 3D reconstruction of biological
structures such as adhesion complex composed of several proteins can be easily retrieved.
Figure 2 : COS7 cell with actin network labelled with Alexa 488, diffraction limited image on the left,
DONALD super-resolved image on the right.
References
[1] T. Ruckstuhl et al.,Forbidden light detection from single molecules, Analytical chemistry, 2000
[2] T. Barroca et al., Full-field Near-Field Optical Microscope for Cell Imaging, PRL, 2012
[3] N. Bourg et al., Direct Optical Nanoscopy with Axially Localized Detection, Nat. Photon. 2015.
22
Label-free deep UV Imaging at SOLEIL synchrotron
Frederic Jamme1, Alexandre Giuliani1,2, Frank Wien1 and Matthieu Réfrégiers1
1
Synchrotron SOLEIL, L’Orme des Merisiers, 91192 Gif sur Yvette, France
Cepia, Institut National de la Recherche Agronomique (INRA), BP 71627, 44316 Nantes, France
2
DISCO Beamline is a bending magnet beamilne at synchrotron SOLEIL covering the
unusual 1-21 eV energy range. It is composed of two branches and three
endstations, SRCD, Atmospheric pressure photoionisation and DUV imaging. I will
focus on the Imaging station.
Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities
in biology because, it does not need external specific probes or labeling, but instead
takes profit of the intrinsic fluorescence that arise from many biomolecules under
deep ultraviolet excitation. Indeed, observation of label free biomolecules or active
drugs ensures that the label will not modify the biolocalisation or any of its properties.
UV monophotonic excitation does present real spectral excitation, leading the way to
excitation imaging and a better selectivity of the chromophores. DUV excitation may
also be used to track exogenous drugs or toxic compounds that present different
spectral behaviour. Moreover, due to diffraction limit the lateral resolution is always
increased when looking in the UV range allowing nanometric spatial resolution3.
Examples in cell biology, enzymology and tissue diagnosis will be presented and
compared to multiphotonic excitation.
1. Giuliani, F. Jamme, V.Rouam, F. Wien, J.L. Giorgetta, B. Lagarde,O. Chubar,S. Bac, I. Yao,S. Rey,
C. Herbeaux, J.L Marlats, D. Zerbib, F. Polack and M. Réfrégiers, J. Synchrotron Rad. 2009, 16: 835841.
2. Jamme, F., Villette, S., Giuliani, A., Rouam, V., Wien, F., Lagarde, B., & Refregiers, M. Microscopy
and Microanalysis, 2010, 16(5): 507-514.
3. Jamme, Kascakova, S., Villette, S., Allouche, F., Pallu, S., Rouam, V., & Réfrégiers, M. Biology of
the Cell,, 2013,105(7), 277–288.
23
Biological applications of lipid imaging with cluster-TOF-SIMS and MALDI-TOF mass
spectrometry
Alain Brunelle
Institut de Chimie des Substances Naturelles, CNRS-ICSN UPR2301, Université Paris-Sud, Avenue
de la Terrasse, 91198 Gif-sur-Yvette, France
[email protected]
Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) using keV energy metal cluster beams
as primary ions is now recognized as a powerful method for in situ chemical, biological and medical
applications. It opens a new field of surface imaging, particularly for biological tissue sections.
Compared to the more established MALDI (Matrix Assisted Laser Desorption Ionisation) imaging
approach, TOF-SIMS provides the incomparable advantages of a routine sub-micrometre scale
resolution and of an easy sample preparation which does not require matrix coating of the surface.
However TOF-SIMS suffers from some limitations, such as the narrow mass range, the lack of
structural analysis of the species by tandem mass spectrometry, and the fact that mainly lipids are
preferentially released from the biological samples.
This lecture intends to show the wealth of powerful information that can be obtained from the chemical
analysis of biological surfaces, with several examples chosen among various applications such as the
localization of xenobiotics, natural substances, lipid markers from neurodegenerative diseases, and
even cultural heritage samples. The strengths and weaknesses of lipid imaging using TOF-SIMS and
MALDI-TOF will also be compared, showing the complementarity between the two methods, the
compatibility with histology, and very recent developments towards increases of sensitivity and
resolution.
Images recorded by mass spectrometry: (from left to right) swarming of a bacterium colony, drug in a
small area of eye, secondary metabolites in wood, lipids in mouse brain, lipid droplets in fat liver.
References
Touboul D, Laprévote O, Brunelle A (2011) Micrometric molecular histology of lipids by mass
spectrometry imaging Curr Opin Chem Biol 15, 725-732
Bich C, Touboul D, Brunelle A (2014) Cluster TOF-SIMS imaging as a tool for micrometric histology of
lipids in tissue Mass Spectrom Rev 33, 442-451
Bich C, Touboul D, Brunelle A (2015) Biomedical studies with TOF-SIMS imaging Biointerphases 10,
2329-2335
24
How to investigate bio-molecules in microorganisms by infrared nanoscopy technique
A. Dazzi, R. Rebois, J. Mathurin, D. Onidas, A. Deniset-Besseau
Laboratoire de Chimie Physique – Université Paris-Sud – 91405 - ORSAY
[email protected]
Abstract
AFM-IR (1) (coupling an Atomic Force Microscope with an IR laser) is a simple and efficient technique
to make nanoscale infrared spectroscopy. Using this technique, we were able to obtain exciting data
showing the detection of bio-molecules such as bio-polymer produced by Rhodobacter (2,3) and size
estimation of triacylglycerol (TAG) vesicles inside dried microorganisms like Streptomyces bacteria (4)
or microalgae.
The production of bio-plastic is still challenging because their properties are not always optimum for
industrial process as polymer from petrochemistry. (PHB) poly(3-hydroxybutyrate) has been receiving
more interest because of its thermo-plastic and mechanical properties similar to those of synthetic
polymer (isotactic polypropylene). However, the crystallinity of this polymer is often an issue for
specific applications. Using the nanoIR, we have estimated the amorphous and crystal phases of PHB
granules directly inside bacteria (Rhodobacter) and study the effect of solvent on these granules
(figure below).
Figure : (Left) Chemical mapping at 1736 cm-1 revealing vesicles (in violet). (Right) Spectra on a vesicles (in red)
and on a bacterium (in green). Black spectrum correspond to the FTIR spectrum of the full culture.
With the same idea to look for alternatives to petrochemical product, several oleaginous
microorganisms have been intensively explored to produce biodiesel. Numerous works focus on
microalgae and little on a peticular bacteral specie called Streptomyces. Thanks to nanoscale IR
spectroscopy, we were able to investigate the production of those biodiesel precursors (triacylglycerol
or TAG) at the subcellular level.
References
1. A.Dazzi et al, Opt. Lett. 30, Issue 18, 2388-2390 (2005).
2. C. Mayet et al Analyst 135, 2540-2545 (2010).
3. C. Mayet et al, Biothechnology advances, vol.31, issue 3, 369-374 (2013).
4. A.Deniset-Besseau, et al, J. Phys. Chem. Lett., 5 (4), pp 654–658 (2014).
25
Multidisciplinary approach to the molecular analysis of the cell membranes impermeability
rupture caused by electric pulses (electroporation)
L.M. Mir1, M. Breton1, A. Silve1,2, A. Azan1, I.Leray1,
M. Scherman3, M. Tarek4, B. Attal-Trétout3
1
Vectorology and Anticancer Therapies, UMR 8203, CNRS, Univ.Paris-Sud, Université Paris-Saclay,
Gustave Roussy, Villejuif.
2
present address: Institute for Pulsed Power and Microwave Technology, Karlsruhe Institute of
Technology, Karlsruhe, Germany.
3
Département de Mesures Physiques, ONERA, Palaiseau.
4
Structure et Réactivité de Systèmes Moléculaires Complexes, UMR 7565, CNRS, Université de
Lorraine, Nancy.
[email protected]
Cell electroporation, also termed cell electropermeabilization, can be achieved both in vitro
and in vivo, and is already used in medical treatments such as the antitumor electrochemotherapy. In
vitro, ultrashort electric pulses (for example with a 10 nanoseconds duration) can induce the same
biological effects as the classical electric pulses of 100 microseconds. Interestingly, the 10 ns pulses
allowed to experimentally validate the results of molecular dynamics simulations of the evolution of
membrane models exposed to these same electric pulses. We thus showed that electropores are
formed in the 5 nm-thick membrane during the pulses and that they close very rapidly after the end of
the pulse (M. Breton et al, JACS, 2012).
To explain the permeability of the cell membranes which is observed for minutes after the
pulse(s) delivery, we elaborated a new model to explain all the steps of the cell membranes
impermeability rupture induced by the electric pulses. The originality of the model is the introduction of
electrically-facilitated chemical reactions occurring in the pores of the membrane, during the pulses.
We have recently proved this model experimentally using chemical approaches, including mass
spectrometry (M. Breton et al., submitted).
In order to obtain dynamic information on these processes, we have developed a wide-field
Coherent Anti-Stockes Raman Scattering (CARS) microscope with a special illumination geometry that
minimizes the non-resonant contribution of the bulk water. We will conclude the presentation by
showing preliminary results on the changes of the interfacial water before and after the electric pulses
delivery.
References
M. Breton, L. Delemotte, A. Silve, L.M. Mir, M. Tarek. Transport of siRNA through Lipid Membranes
driven by Nanosecond Electric Pulses: an Experimental and Computational Study. J. Am. Chem. Soc.,
134, 13938-13941, 2012.
26
THURSDAY 5 NOVEMBER
SESSION 1: NEW CELLULAR REPORTERS
SPEAKERS:
Kai Johnsson, Lausanne
Boris Vauzeilles, Gif-sur-Yvette
Marie-Paule Teulade-Fichou, Orsay
Mathieu Kociak, Orsay
Hélène Pasquier, Orsay
Céline Boutin, Saclay
CHAIRMANS:
Anton Granzhan and Robert Pansu
27
Innovation in organic
synthesis
A partnership with Synth-Innove laboratories
will accelerate your move from in-vitro concept
to medical drug commercialization
François Scherninski
Research manager
Tél. : +33 (0)1 45 58 36 90
Mobile : 33 (0) 6 86 66 49 97
Fax : +33 (0)1 47 00 89 84
Email : [email protected]
33, Boulevard du Général28Martial Valin 75015 Paris
“Synthetic and Semisynthetic Probes for live-cell imaging”
Kai Johnsson
Institute of Chemical Sciences and Engineering; École Polytechnique Fédérale de Lausanne; CH-1015
Lausanne; Switzerland. [email protected]
In my presentation I will demonstrate how a combination of protein engineering and synthetic chemistry can be
exploited to generate fluorescent probes for live-cell imaging.
In the first part of my presentation I will introduce a highly permeable and biocompatible near-infrared
fluorophore that can be specifically coupled to intracellular proteins in live cells and tissues using different
labeling techniques. The fluorogenic character of the probe and its high brightness permit live-cell imaging
experiments without washing steps. The fluorophore is ideally suited for live-cell superresolution microscopy
approaches using stimulated emission depletion (STED) microscopy. The excellent spectroscopic properties of
the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.
In the second part of my talk, I will talk about our attempts to introduce a new class of fluorescent sensor
proteins that permit to visualize drug and metabolite concentrations in living cells. I will also discuss how these
sensor proteins can be utilized for point-of-care therapeutic drug monitoring.
References
1) “Fluorogenic probes for live-cell imaging of the cytoskeleton” Lukinavicius, G.; Reymond, L.; D'Este, E.;
Masharina, A.; Gottfert, F.; Ta, H.; Guther, A.; Fournier, M.; Rizzo, S.; Waldmann, H.; Blaukopf, C.;
Sommer, C.; Gerlich, D. W.; Arndt, H. D.; Hell, S. W.; Johnsson, K. Nature Methods 2014, 11, 731
2) “Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring” Griss, R.; Schena, A.;
Reymond, L.; Patiny, L.; Werner, D.; Tinberg, C. E.; Baker, D.; Johnsson, K. Nature Chem Biol 2014, 10,
598
3) “A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins” Lukinavicius, G.;
Umezawa, K.; Olivier, N.; Honigmann, A.; Yang, G.; Plass, T.; Mueller, V.; Reymond, L.; Correa, I. R., Jr.;
Luo, Z. G.; Schultz, C.; Lemke, E. A.; Heppenstall, P.; Eggeling, C.; Manley, S.; Johnsson, K. Nature
Chemistry 2013, 5, 132
29
Metabolic reporters of live bacteria
Boris Vauzeilles
Department of Chemical Biology, ICSN, CNRS UPR 2301, Université Paris-Saclay,
91198 Gif-sur-Yvette (France),
Synthesis of Bioactive Molecules and Macromolecules, ICMMO, CNRS UMR 8182, Univ. Paris-Sud,
Université Paris-Saclay, 91405 Orsay (France), and
Click4Tag, Zone Luminy Biotech, Case 922, 163 avenue de Luminy, 13009 Marseille (France)
[email protected]
Sudden outbreaks of new epidemics regularly warn us against the severe sanitary and economical
impact resistant bacterial infections could have on our industrialized and globalized societies. The
rapid identification of viable bacteria is therefore a fundamental challenge.
The outer membrane of Gram-negative bacteria is covered by a dense layer of Lipopolysaccharides
(LPS), which are considered to participate into cell integrity, as well as to the level of pathogenicity of a
given strain.
We have recently shown that, when they are metabolically active, Gram-negative bacteria can
specifically incorporate into their LPS a monosaccharide, which has been modified by the introduction
of an azido anchor.
This bioorthogonal chemical reporter can then be further exploited in the click-chemistry mediated
labeling of these bacteria. This overall procedure offers an efficient and rapid strategy to identify living,
or metabolically active, bacteria.
This communication will present our latest results in this field, including specific labeling of a serious
pathogen, Legionella pneumophila.
References
Dumont A, Malleron A, Awwad M, Dukan S, Vauzeilles B (2012) Click-mediated labeling of bacterial
membranes through metabolic modification of the lipopolysaccharide inner core Angew Chem Int Ed
Engl 51, 3143-3146
Mas Pons J, Dumont A, Sautejeau G, Fugier E, Baron A, Dukan S, Vauzeilles B (2014) Identification
of Living Legionella pneumophila Using Species-Specific Metabolic Lipopolysaccharide Labeling
Angew Chem Int Ed Engl 53, 1275-1278
Fugier E, Dumont A, Malleron A, Poquet E, Mas Pons J, Baron A, Vauzeilles B, Dukan S (2015) Rapid
and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click
Chemistry Coupled with Magnetic Beads Separation PLoS ONE 10(6): e0127700
30
Switchable probes for labelling nucleic acids in live cells: To be or not to be in the nucleus ?
Marie-Paule Teulade-Fichou
Laboratory Chemistry, Modelling and Imaging for Biology, CMRS UMR9187-Inserm U1196, Institut
Curie, Research Center of Orsay, University Paris-Sud, Bât 110, 91405 Orsay , France.
[email protected]
In view of the rapid developments of optical microscopies new fluorescent probes are constantly
needed in particular IR-NIR probes compatible with intravital investigations.
In this context we launched a program aimed at the design of new fluorophores excitable in the IR
range via a two-photon absorption (2PA) process. Based on the non-linear optical (NLO) properties of
push-pull triphenylamines (TP), we engineered this chemical family to make it compatible with
biological media and in particular cellular context. Several generations of 2 and 3 branched dyes
bearing cationic e-acceptors ( pyridinium, benzimidazolium) linked to the strong e-donor TP core via
1,2
These compounds were shown to combine strong 2PA
vinyl bonds has been synthesized.
absorption and high affinity for AT-rich DNA grooves. In addition, of utmost importance is the
switchable off/on fluorescence of the TP dyes that are virtually non-fluorescent in water and strongly
3
emissive when immobilized in DNA which results in a strong contrast when imaging (Fig. 1).
A
B
C
!
Figure 1: A) Model of TP-2Bzim after docking in the minor groove of AT-rich DNA. 2PA microscopy
imaging!of nuclear DNA in fixed HT29 cells at 1PM B) and 100nM dye C), Oexc: 800nm.!!
Finally we observed that the two-photon absorption cross-section of the dyes is dramatically enhanced
5
once bound to DNA (G=1080 GM vs G=110 GM for the free TP-2Bzim compound ). This is attributed
to a tight fit of the TP-2Bzim molecule inside the minor groove of the DNA matrix, which induces
geometrical rearrangements in the dye ground state and presumably in its excited state. This effect of
the DNA matrix on the nonlinear absorption of a bound dye is revealed for the first time and paves the
way for studying NLO processes in DNA. (WO 2008/055969, collaboration with. C Fiorini , F.Charra,
CEA-Saclay).
6
More recently it was shown that in live cells the TP dyes are trapped in mitochondria. Upon IR-light
7
excitation dye translocation to the nucleus is induced with concomitant induction of apoptosis. The TP
dyes thus represent promising drug prototype able to kill cancer cell upon IR light activation
(EP12306133.5.WO 09/13, collaboration with. P.Tauc, E. Deprez LBPA-IDA, Cachan).
.
!!!!!
References :
1- G. Bordeau et al. 2008, J. Am.Chem. Soc., 130, 16836.
2- R.Lartia et al. 2008, J.Org.Chem., 73,1732.
3- B. Dumat et al. 2012 Org.Biomol. Chem.,10, 6054.
5- B. Dumat et al. 2013 J.Amer.Chem.Soc.,135, 12697.
6- R.Chennoufi et al. 2015, Chem Comm, DOI: 10.1039/C5CC05970D;
7- R.Chenouffi et al. submitted.
Acknowledgements : We wish to thank ANR, CNRS and Institut Curie for fundings.
.
31
Scanning Transmission Electron Microscopy Cathodoluminescence: a
powerful tool for combined UltraStructure and luminescence microscopies
M. Kociak1, S. Nagarajan1, L. H. G. Tizei1, C-Y Fang2, J-R Bertrand3, C. Durieu4, E. Le Cam4,
H-C Chang2, M. de Frutos1, F. Treussart5
1
Laboratoire de Physique des Solides, CNRS UMR8502, Université Paris Sud,
Université Paris Saclay, 91405 Orsay, France
2
Institute of Atomic and Molecular Sciences, Academia Sinica,Taipei 106, Taiwan
3
Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, CNRS UMR 8203,
Université Paris Sud, Université Paris Saclay, 94805 Villejuif, France
4
Signalisations, Noyaux et Innovations en Cancérologie, CNRS UMR 8121,
Université Paris Sud and Institut Gustave Roussy, 94805 Villejuif, France
5
Laboratoire Aimé Cotton, CNRS UMR9188, Université Paris Sud, ENS Cachan,
Université Paris Saclay, 91405 Orsay, France
[email protected]
Techniques allowing to image cells are in constant evolution. Light microscopy (LM)
is particularly useful when it comes to study dynamics, while Electron Microscopy
(EM) gives access to structural details. The combination of both techniques, namely
Correlative Light Electron Microscopy (CLEM) has shown impressive success in the
past decade [1]. CLEM, however, is a demanding technique, as it requires several
handlings as well as a posteriori alignment of the EM and LM images.
Here we present a different approach, where luminescence signal and structural
information are generated by the same particle – the electron. Indeed, the
luminescence
created
by
an
electron
impinging
a
fluorophore
–
Cathodoluminescence, CL – can be tracked with sub-10 nm resolution [2] when
recorded in a Scanning Transmission Electron Microscope (STEM), where in addition
morphological images with sub-nanometer resolution can be acquired in parallel.
The demonstration of the ability of STEM-CL to correlate luminescence and ultrastructure imaging is given in the specific case of the uptake by human Ewing's
sarcoma cells in culture, of fluorescent nanodiamonds (NDs) coated with two different
cationic polymers (Polyethyleneimine (PEI) and Polyallylamine Hydrochloride (PAH)).
The specific advantage of the technique is illustrated with the use of two types of NDs
with a distinct spectral signature for each coating. Co-incubation of the cell with NDs
coated with both types of polycations resulted in changes of ND density in the
endosomal compartments [3]. Beyond this proof of principle, we believe that the
technique may be applied to other biological problems. The bottlenecks towards this
goal will be disserted, and we hope this workshop will be the place to discuss the way
to overcome them.
References
1. Caplan, J., et al., (2011) The power of correlative microscopy: multi-modal, multiscale, multi-dimensional. Current Opinion in Structural Biology, 21(5), 686.
2. Zagonel, L., et al. (2011) Nanometer Scale Spectral Imaging of Quantum Emitters
in Nanowires and Its Correlation to Their Atomically Resolved Structure, Nano
Letters, 11, 568.
3. Nagarajan, S., et al. (2015) Integrated Cathodoluminescence and Transmission
Electron Microscopy allows Simultaneous Ultrastructure and Spectral Imaging of
endocytosed Nanodiamonds, in preparation
32
Engineering cyan fluorescent proteins with ultimate performances
for live cell biosensors .
Hélène Pasquier, Asma Fredj, Yasmina Bousmah, Marie Erard, Fabienne Mérola
Laboratoire de Chimie Physique, UMR 8000, CNRS Université Paris Sud, Orsay,
France
[email protected]
Over the past fifteen years, genetically encoded reporters based on fluorescent proteins (FPs)
have contributed to major advances in biological imaging, from the dynamic study of biochemical cell
signaling to super-resolution optical nanoscopies. New FP variants are constantly discovered from
natural sources, and submitted to intensive genetic engineering based on large scale random
mutagenesis and directed evolution. However, in many cases, these newly developed FPs fail to
achieve all the performances and characteristics required for their bioimaging applications, while the
large scale remodeling strategies used have lost track of potentially crucial mutagenesis steps. The
design of highly optimized FP-based reporters, simultaneously displaying appropriate color, multimeric
state, chromophore maturation speed, brightness, photostability and environmental sensitivity will
require a better understanding of the structural and dynamic determinants of FP photophysics.
The recent development of new cyan fluorescent proteins (CFPs) like mCerulean3 (Markwardt
et al, 2011), mTurquoise2 (Goedhart et al, 2012), and Aquamarine (Erard et al, 2013) brings a different
view on these questions, as in this particular case, a step by step evaluation of critical mutations has
been performed within a family of spectrally identical and evolutionary close variants. These efforts
have led to CFPs with quantum yields close to unity, near single exponential emission decays, high
photostability and complete insensitivity to pH, making them ideal choices as robust energy transfer
donors in FRET and FLIM imaging applications. With the help of structural and theoretical
investigations, these works contribute to establish a rationale for the obtained improvements in the
context of FPs derived from hydrozoan species. In particular, we have shown that a proper amino-acid
choice at only two positions (148 and 65) is sufficient to completely transform the photophysics of CFPs
and achieve the above performances. These two positions critically control also the chromophore
photoisomerization kinetics, while other amino-acid locations can be targeted to accelerate
chromophore maturation and extend long term photostability.
Today, these results provide a useful toolbox for a rapid and reliable upgrade of the different
CFP donors carried by FRET biosensors. They also open new perspectives towards the de novo
engineering of new generations of FPs with ultimate performances by synthetic biology.
References
Markwardt ML, Kremers G-J, Kraft CA, Ray K, Cranfill PJC, Wilson KA, Day RN, Wachter RM, Davidson MW,
Rizzo MA (2011) An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible
Photoswitching. PLoS ONE 6:e17896.
Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TWJ,
Royant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature
Communications 3:751.
Erard M, Fredj A, Pasquier H, Betolngar D-B, Bousmah Y, Derrien V, Vincent P, Merola F (2013) Minimum set of
mutations needed to optimize cyan fluorescent proteins for live cell imaging. Molecular Biosystems 8:258.
33
Laser-Polarized Xenon for the Study of Biological Cells
Céline Boutin, Emilie Mari, Estelle Léonce, Guillaume Carret, Patrick Berthault
CEA Saclay, IRAMIS, NIMBE, UMR CEA/CNRS 3685, Laboratoire Structure et Dynamique par
Résonance Magnétique, 91191 Gif-sur-Yvette, France
[email protected]
Hyperpolarized xenon is a powerful sensor for magnetic resonance detection of molecular or
biological events. This is due to the huge signal obtained by the prior optical pumping step, to its
exogenous and gaseous nature, its solubility in biological media and finally its wide chemical shift
range. Using these properties, two approaches where the hyperpolarized noble gas is used alone as a
tracer or targeted to biological receptors via functionalized host systems are developed in the lab.
129
We have shown that, for various types of cells, either eukaryotic or prokaryotic, the Xe NMR
spectra contain a signal specific to the intracellular compartment, very distinct from the signal of xenon
in the external water pool.[1] (see figure 1) This signal depends on the cell type, the viability of the
cells, the presence of toxics, etc. We have applied this observation to the study of the multidrug
129
Xe NMR, it has been possible to distinguish
resistance phenotype on model cell lines. Using
resistant and sensitive cell lines.[2,3]
Figure 1. The simplicity of the approach is depicted here. In its original version hyperpolarized xenon
prepared in the batch mode by optical pumping is introduced in the NMR tube containing the cell
129
suspension under study. The one-scan Xe NMR spectrum displays two signals corresponding to
xenon in the cells and xenon in the medium.
129
Also, in collaboration with chemists (ENS Lyon and CEA/DSV), Xe NMR-based biosensors
consisting in xenon hosts bearing a biological ligand have been conceived and studied. They enabled
sensitive detection of different analytes or biological receptors. Some of these biosensors will be
presented.
References
129
[1] C. Boutin et al. (2011) Hyperpolarized Xe NMR signature of living biological cells NMR in Biomedicine 24
1264
[2] C. Boutin, P. Berthault (2012) Procédé de détermination de la résistance cellulaire aux médicaments France
Patent 12 52922
[3] P. Berthault, C. Boutin, (2015) Hyperpolarized Xenon-129 Magnetic Resonance: Concepts, Production,
Techniques and Applications RSC Book Chapter, New Developments in NMR no. 4, Edited by T. Meersmann and
E. Brunner
[4] P. Berthault et al. (2009) Biosensing using laser polarized xenon NMR/MRI Progress in NMR Spectroscopy
55 35
34
THURSDAY 5 NOVEMBER
INSTITUTIONAL PRESENTATION
Jean-Pierre Mahy, Paris-Saclay Chemistry Department work group
Nadine Peyriéras, France-BioImaging
Sandrine Lévêque-Fort, GDR 2588 Microscopie et Imagerie du Vivant (MIV)
SESSION 2: MULTIMODAL IMAGING
SPEAKERS:
Abraham Koster, Leiden
Sergio Marco, Orsay
Bertrand Cinquin, Saint-Aubin
Marie-Claire Schanne-Klein, Palaiseau
Karine Steenkeste, Orsay
CHAIRMANS:
Sandrine Lévêque-Fort and Rachel Méallet-Renault
35
FEMTONICS Ltd microscopes à 2 photons et imagerie de fluorescence
IDIL Fibres Optiques, le partenaire et distributeur en France
FEMTONICS Ltd est une spin off de l’IEM HAS Institut de médecine Expérimentale de l’Académie des
Sciences de Hongrie, créée en 2005. Le microscope multi-photon est basé sur un statif Olympus motorisé. En plus
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Femtonics s’appuie sur plusieurs technologies brevetées pour proposer des microscopes les plus rapides en imagerie
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§ (Photo-activation)
§ Kit d’epifluorescence
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Des références d’utilisateurs FEMTONICS en Europe, en Australie et aux Etats Unis sont disponibles.
IDIL Fibres Optiques cultive depuis vingt ans son expertise et son savoir-faire en ingénierie optoélectronique
fibrée. Notre équipe technique est composée essentiellement de collaborateurs possédant une formation d’un niveau
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Pour les microscopes biphotons, notre équipe sera capable de vous aider sur l’ensemble des éléments optiques du
système. Une de nos antennes se trouve à Orsay, non loin de votre site. Cette antenne est équipée de matériel pour
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également accès directement aux ingénieurs Femtonics qui ont développé et font la maintenance de la partie
informatique.
Dans le cas où nos solutions auraient un intérêt plus marqué pour vous, il est également possible de prévoir un
rendez-vous dédié par téléphone ou lors d’un rendez-vous de présentation d’un équipement d’un de nos utilisateurs
Français. Nous pouvons vous inviter chez FEMTONICS (Hongrie) à nos frais pour vous présenter nos solutions, faire
des démonstrations de mesures et discuter de vos applications et besoins spécifiques. Des expériences ciblées
peuvent être définies au préalable ensemble pour vous démontrer les performances et l’efficacité d’utilisation de nos
microscopes. www.idil.fr, http://femtonics.eu/ et [email protected] 02 96 05 40 20 [email protected] 04 50 28 34 81.
36
IDIL Fibres Optiques
21 rue Louis de Broglie F-22300 Lannion
Tel : 02 96 05 40 20 - Fax : 02 96 05 40 25
Email : [email protected] - Site : www.idil.fr Zooming in on cells and macromolecules with correlative light-electron electron microscopy
A.J. Koster
Department of Molecular Cell Biology
Leiden University Medical Centre
Leiden, The Netherlands.
[email protected]
In correlative light and electron microscopy (CLEM) imaging modalities are combined to study cellular
processes. Fluorescence light microscopy (FM) enables the imaging of dynamic events in relatively
large fields of view exploiting a wide range of available fluorescent markers, while electron
microscopy (EM) can reveal structural macromolecular arrangements in their cellular context in
1
relatively narrow fields of view at nm-scale resolution .
EM specimens prepared by conventional methods that incorporate chemical fixation and metal
2
staining steps can provide a wealth of information on the cellular architecture and processes . 3D
morphology of cell systems and tissue can be unravelled in sections of material several hundred nm
3
thick using electron tomography with transmission EM (TEM). 3D imaging of material 100’s µm in
3
size can be obtained with serial block face scanning electron microscopy .
At the molecular level however, the fidelity of interpretation of conventionally prepared specimens is
limited because of effects related to the fixation and staining. Imaging of cryo-immobilized frozenhydrated specimens excludes the use of stain and as such the molecular resolution is preserved.
Images of frozen hydrated specimens have an inherent low contrast and low signal to noise ratio
because of their electron dose sensitivity and the lack of heavy atoms. Fortunately, recent
technological improvements of image detectors and contrast-enhancing phase plates for TEM have
improved both the contrast and signal-to-noise ratio of cryo EM datasets.
4,5
For applications of EM, FM can be instrumental as a tool for selection of areas of interest . An
overview of CLEM developments will be given and workflows of different types of CLEM will be
5
illustrated by results obtained on a variety of biological systems, such as on fluorescently labelled
6
bacteria, virus-induced replication structures and blood-filtering structures in tissue .
References
1. Patwardhan et al. (2014) A 3D cellular context for the macromolecular world Nat Struct Mol Biol
21(10):841-5.
2. Barcena and Koster. (2009) Electron tomography in life scienc. Semin Cell Dev Biol 20(8):920-30.
3. Mourik et al. (2015) Towards the imaging of Weibel-Palade body biogenesis by Serial Block Face –
SEM J Microscopy 259(2):97-104.
4. Briegel et al. (2010) Correlated light and electron cryo-microscopy Methods Enzymol 481:317-4.
5. Faas et al. (2013) Localization of fluorescently labeled structures in frozen hydrated samples using
integrated light electron microscopy. J Struct Biol 181(3):283-90.
6. Mourik et al. (2015) Content delivery to newly forming Weibel-Palade bodies is facilitated by
multiple connections with the Golgi apparatus Blood 125(22):3509-16.
37
Chemical imaging by electron microscopy and secondary ion mass spectroscopy for
intracellular tracking of nanoparticles
Marco Sergio, Guerquin-Kern Jean Luc, Wu Ting-Di, Trepout Sylvain, Messaoudi Cédric
INSERM U1196/ Institut Curie/CNRS UMR 9187
Campus Universitaire d'Orsay, Rue Becquerel. Bat 110 91405 Orsay cedex FRANCE
[email protected]
The characteristics of condensed matter depend on its chemical composition and the understanding of
its properties frequently involves the study of the spatial distribution of its chemical elements. When
the results of this analysis are qualitative or quantitative chemical maps we talk about chemical
imaging. The progresses in biological sample preparation and the improvements in methods whose
usage is suitable for chemical imaging not only make this approach possible for the study of biological
samples, but also makes available the access to the three-dimensional (3D) distribution of chemical
elements. Thus chemical imaging offers nowadays large possibilities for a better understanding of
biological systems by allowing the identification of chemical components at tissue, cellular and
subcellular levels (Marco et al., 2014). Besides the biological questions which can be approached by
chemical imaging, nanoparticles characterization and their intracellular tracking is currently a major
topic because of their use in cosmetics and nanomedicine as well as their increasing presence in air
and water.
Since transmission electron microscopy (TEM) provides access to the 3D chemical maps by the use of
electron energy filtered (EFTEM) techniques (Messaoudi et al., 2013), having high spatial resolution
on biological samples, it is a method of choice to address the aforementioned questions. Moreover,
the use of nano-focused electron beam in scanning mode (STEM) provides access to atomic contrast
(Sousa et al., 2012), also call Z-contrast, also suitable for nano-particles characterization and tracking.
However, analytical TEM applied to biology is not a quantitative approach and it is limited by the
surface of the sample accessible to the analysis in a reasonable scale of time. Secondary ion mass
spectroscopy (nano-SIMS) imaging (Guerquin-Kern et al. 2005) offers a way to overcome this limits by
providing access to the isotopic quantification and the imaging of isotope labelled molecules (Bower et
al., 2009). Nevertheless, nano-SIMS imaging has lower spatial resolution than analytical TEM and
cannot be used to generate 3D chemical maps. Since TEM and nano-SIMS are complementary
approaches for chemical imaging, to study the same sample in a correlative approach can be the way
to overcome the limits of these two methods.
During this talk, the basis of analytical TEM, nano-SIMS and chemical correlative imaging will be
exposed and they will be illustrated by examples on nanoparticles, drugs and isotope labelled
molecules characterization and sub-cellular tracking.
References
x
x
x
x
x
Boxer SG, Kraft ML, Weber PK. (2009) Advances in imaging secondary ion mass spectrometry for
biological samples. Annu Rev Biophys. 38,53-74.
Guerquin-Kern JL, Wu TD, Quintana C, Croisy A. (2005) Progress in analytical imaging of the cell
by dynamic secondary ion mass spectrometry (SIMS microscopy). Biochim Biophys Acta.
1724,228-38.
Marco S. (2014) Towards Three-dimensional Chemical Imaging of Cells. J. Mol. Biol. & Mol.
Imaging. 1,2
Messaoudi C, Aschman N, Cunha M, Oikawa T Sorzano C.O.S, Marco S. (2013) EFTEM-TomoJ:
3D chemical mapping by EFTEM including SNR improvement by PCA and volume improvement
by noise suppression during the ART reconstruction process. Microsc. Microanal. 28:1-9.
Sousa AA, Leapman RD. (2012) Development and application of STEM for the biological
sciences. Ultramicroscopy. 123,38-49.
38
Soft X Ray Microscopy with synchrotron radiation
Bertrand Cinquin
Synchrotron Soleil, Orme des Merisiers, 92192 Gif sur Yvette
[email protected]
Utilizing soft x-ray in the water window energy range allow microscopic studies of hydrated biological
samples close to their near-native states since Oxygen atoms are more transparent than Carbon atoms.
Therefore, a natural good contrast may be obtained from carbonaceous species : different compaction of
DNA, rich lipid organelles such as mitochondria, lipid droplet!
However, living specimen are highly sensitive to Soft X-Rays and radiation damages are a primary concern.
3D observation with high resolution (with a voxel size of 125 Å3, i.e. 50 nm pixel side) need between one to
three hundred projections. Therefore, insuring the conservation of the structures of the sample along the
acquisition is mandatory. Presently, Cryo freezing and cryo environment is the best solution.
Furthermore, being a transmission technique, linear absorption coefficient can be retrieved allowing the
characterization of inner cellular features. Soft X rays microscopies offer an in between resolution compared
to electronic microscopy and conventional fluorescent techniques. However, super resolution techniques
such as SIM or SPIM can offer the same definition for a similar volume about 10 "m x 10 "m x 10 "m
where electronic microscopy is limited by the thickness of the sample (about 1 "m). More, being label-free,
other Soft X ray density probes (such as TiO2) could be used in combination with other fluorescent proteins.
A correlative work between cryofluorescence microscopy and Soft X ray microscopy will be presented.
Besides the soft x-ray microscopy technique2, I will talk about some interesting examples such as the inner
nucleus organisation along the cellular differentiation or the role of different proteins involved in the lipid
droplet autophagy in S. Cerevisiae as a result of using a soft X-ray microscope at the ALS on NCXT
beamline. I will conclude by presenting the principal challenges the technique need to address in order to
further explore the utility of such a powerful instrument within the biological community.
References
1. B Cinquin, R. Boudreau, M. Legros, C. Larabell (2013) 3D Organization of the Interphase Nucleus
using Soft X Ray Tomography. Biophysical Journal, 102, p478a
2. MA Le Gros, G McDermott, B Cinquin, E. Smith, M. Do, W. Chao, P Naulleau, C. Larabell (2014)
Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source. Journal synchrotron
radiation, 21, 1370-1377
39
In situ quantitation of collagen fibrils diameter using absolute measurements of SHG signals.
M.-C. Schanne-Klein1, S. Bancelin1, I. Gusachenko1, G. Latour1, L. Kowalczuk2, T. Coradin3 and C. Aimé3
1. Laboratoire d’Optique et Biosciences, École Polytechnique, CNRS, INSERM U1182, Université
Paris-Saclay, F-91 128 Palaiseau, France
2. Centre de Recherche des Cordeliers, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS
872, F-75 006 Paris, France
3. Laboratoire de Chimie de la Matière Condensée de Paris, Sorbonne Universités, UPMC Univ Paris
06, CNRS, Collège de France, F-75 231 Paris, France.
E-mail: [email protected]
Type I collagen is a major structural protein in mammals. This biopolymer is synthesized as a
triple helix, which self-assembles into fibrils (diameter: 10-300 nm) and further forms various 3D
organizations specific to each tissue. In recent years Second Harmonic Generation (SHG) microscopy
has emerged as a powerful technique for the in situ investigation of the fibrillar collagen structures in
matrices or tissues [1]. However, as an optical technique with typically 300 nm lateral resolution, SHG
microscopy cannot resolve most of the collagen fibrils. Moreover, in contrast to incoherent
fluorescence signals that scale linearly with the chromophore concentration, SHG is a coherent
multiphoton signal that scales quadratically with the density of collagen triple helices aligned in a
parallel way in the focal volume. Consequently, quantitative SHG measurements have been limited so
far to averaged phenomenological parameters [1].
In this study, we correlated SHG and
transmission electron microscopies to determine
the sensitivity of SHG microscopy and calibrate
SHG signals as a function of the diameter of the
collagen fibril [2]. To that end, we synthesized in
vitro isolated fibrils with various diameters and
successfully imaged the very same fibrils with both
techniques, down to 30 nm diameter (see figure 1).
We observed that SHG signals scale as the fourth
power of the fibril diameter, as expected from
analytical and numerical calculations. It validated
our quantitative bottom-up approach used to
calculate the non-linear response at the fibrillar
scale and demonstrated that the high sensitivity of
SHG microscopy originates from the parallel
alignment of triple helices within the fibrils and the
subsequent constructive interference of SHG
radiations. This calibration was then applied to
Fig. 1 : Correlative SHG-electron imaging of
intact rat corneas, where we successfully
isolated collagen fibrils.
recovered the diameter of hyperglycemia-induced
fibrils in the Descemet’s membrane without having
to resolve them [2,3].
Importantly, this calibration only applies to isolated fibrils. Nevertheless, complementary
techniques can probe the sub-micrometer structure of dense distributions of collagen fibrils. For
instance, we showed that the anisotropy ratio measured by polarization-resolved SHG microscopy
increases with the orientation disorder within the focal volume [4]. These results therefore represent a
major step towards quantitative SHG imaging of collagen biomaterials or tissues.
References
[1] M. Strupler et al (2008) Second Harmonic Microscopy to Quantify Renal Interstitial Fibrosis and
Arterial Remodeling, J. Biomed. Opt. 13, 054041
[2] S. Bancelin et al (2014), Determination of collagen fibril size via absolute measurements of secondharmonic generation signals, Nat Commun 5
[3] G. Latour et al (2012), Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The
Potential of Second Harmonic Generation Microscopy, PLos ONE 7, e48388
[4] I. Gusachenko et al (2012), Polarization-resolved second-harmonic microscopy in tendon upon
mechanical stretching, Biophys. J. 102, 2220-2229.
40
Diffusion, localization and bioavailability of clinically-used antibiotics in Staphylococcus
aureus biofilms: toward a better understanding of bacterial biofilms tolerance?
Karine Steenkeste, Rym Boudjemaa, Matthieu Revest, Cédric Jacqueline, Jocelyne Caillon,
Romain Briandet, Marie-Pierre Fontaine-Aupart
Institut des Sciences Moléculaires d’Orsay, Université Paris-Sud, Bât. 210, 91405 Orsay Cedex,
France
[email protected]
Staphylococcus aureus is one of the most frequent pathogens associated with biofilm-related
infections. Among clinically-used antibiotics for the treatment of S. aureus infections, very few enable
long-term successful treatments against biofilms. There is therefore a major need to gain a better
understanding of these biological structures tolerance towards antibiotics. Here, we developed an in
vitro model which holds particular relevance for assessing antibiotics activities using time-kill studies
but also for directly visualizing by fluorescence microscopy the interaction of fluorescently-labelled
antibiotics with S. aureus at different phases of growth.
MSSA and MRSA clinical and collection strains were grown in vitro at 37°C in TSB enriched with
proteins and calcium. Planktonic bacteria and 24h-biofilms grown in polystyrene microplates were
exposed to daptomycin 20µg/mL, vancomycin 40µg/mL or their combination with rifampicin 20µg/mL
during 72h. Antibiotics efficiency was screened by time-kill studies. Fluorescence confocal microscopy
was employed in addition to visualise biofilms three-dimensional structures and dead and live cells
distribution over time but also to probe antibiotics diffusion and availability inside biofilms using timelapse and FRAP modules.
Vancomycin and daptomycin alone were ineffective against biofilms, but an important part of viable
cells were not culturable. This subpopulation might be responsible for recurrent infections. Previously
vancomycin has been shown to diffuse through the whole biofilm structure, to interact with its target
but still remains inactive. Similarly, we confirmed that daptomycin penetrated, diffused and was
bioavailable inside biofilms. Unexpectedly, we showed that daptomycin could enter cell membranes
when bacteria were exponentially-growing. But as soon as bacteria secreted a matrix, daptomycin
mainly localized in the biofilm matrix, revealing a higher affinity for matrix components.
These in vitro biofilms constitute promising high throughput systems to visualize and quantify
antibiotics tolerance within biofilms and decipher the associated molecular mechanisms. They are
currently being compared with a mouse model of S. aureus infection on a medical implant to assess
their in vivo relevance.
References
Daddi Oubekka S, Briandet R, Fontaine-Aupart M-P, Steenkeste K. (2012) Correlative time-resolved
fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrob Agents
Chemother; 56, 3349–58.
41
42
FRIDAY 6 NOVEMBER
SESSION 1: IN VIVO AND CLINICAL APPLICATIONS
SPEAKERS:
Nicolas Bézière, Munich
François Le Naour, Villejuif
Catherine Chapon, Fontenay-aux-Roses
Frédéric Ducongé, Fontenay-aux-Roses
Nicolas Gervasi, Paris
Frédéric Pain, Orsay
CHAIRMANS:
Emmanuel Beaurepaire and Matthieu Réfrégiers
43
44
Optoacoustic Imaging: light and sound for in vivo biomedical imaging.
Nicolas Bézière, Vasilis Ntziachristos
Institute for Biological and Medical Imaging
Technische Universität München and Helmholtz Zentrum München
Ingolstädter Landstr.1
85764 Neuherberg, Germany
[email protected]
By relying on the conversion of light energy into ultrasound waves through thermo-elastic expansion,
resulting from light absorbance, optoacoustic imaging has effectively provided a new modality to
observe, time and quantify molecular events in preclinical and clinical settings. Its latest
implementations, built on Multispectral Optoacoustic Tomography (MSOT) approaches, have allowed
spectral identification of a variety of intrinsic (blood, melanin…) and extrinsic (optical reporters,
metallic nanoparticles, genetic tags) contrast sources(1), yielding unprecedented insight on physiology
and pharmacology(2).
Being able to follow simultaneously multiple biomarkers, MSOT provided a new perspective on cancer
imaging in particular, with accurate oxygenation status monitoring along with drug-delivery system
distribution tracking. Labeled liposomes specifically have highlighted the importance of nanoparticle
size in relation to tumor type for efficient drug delivery systems, a behavior that could also be seen
using gold nanoparticles, both paving the way for real-time theranostic applications.
Lastly, the emergence of suitable genetic tags for optoacoustic imaging, either photoabsorbing
proteins or through enzymatic activity has recently allowed for longitudinal studies of cell behavior at
high resolution and increased imaging depth. Application in different cancer models using either
genetically labeled cells or in vivo viral infection highlighted the potential of genetic labels in modern in
vivo biomedical imaging.
Through these specific applications, current state of the art in preclinical optoacoustic imaging will be
discussed, and built upon to provide a glimpse of the future of optoacoustic imaging, from bench to
bedside.
IndoCyanine Green labelled liposomes (LipoICG, left
panel). Transverse optoacoustic image of the
distribution of LipoICG (hot scale), overlayed on an
anatomic image (grey scale) of a mouse bearing a 4T1
tumor on its back 24h after LipoICG iv injection (right
panel).
References
1.
2.
Ntziachristos V & Razansky D (2010) Molecular imaging by means of multispectral optoacoustic
tomography (MSOT). Chemical reviews 110(5):2783-2794.
Taruttis A & Ntziachristos V (2015) Advances in real-time multispectral optoacoustic imaging and its
applications. Nature photonics 9(4):219-227.
45
Liver graft quality control by infrared spectroscopy
François Le Naour
INSERM, Unité 1193, Univ. Paris-Sud, hôpital Paul Brousse, F-94807 Villejuif, France
[email protected]
Liver transplantation constitutes a major treatment of patients with cirrhosis or hepatocellular
carcinoma. Due to the shortage of grafts, transplantation teams are using marginal grafts from
expanded criteria donors. These marginal grafts constitute an additional source of organs but also an
increased risk factor of primary non-function or dysfunction of the graft mainly due to their poor quality.
Steatosis is one of the most important factors affecting liver allograft function, mostly because of more
severe ischemia-reperfusion injury. Steatosis is characterized by the intra-cellular accumulation of
triacylglycerides (TG) resulting in the formation of lipid vesicles in the hepatocytes. The gold standard
to assess hepatic steatosis in liver grafts during the transplantation procedure is the histological
examination of frozen sections by a pathologist. The quantitative histological evaluation of steatosis is
based on the percentage of hepatocytes containing cytoplasm fat inclusions. The major issue is that
assessment of hepatic steatosis on histological sections is an imperfect and not reproducible method.
Furthermore, the histological estimation of steatosis is poorly correlated with the true lipid content of
the liver tissue. Thus, an alternative method to histopathology providing an objective assessment of
steatosis is an unmet need.
We addressed the potential of infrared (IR) microspectroscopy for grading steatosis on frozen
tissue sections. Infrared microspectroscopy is based on the determination of absorption of infrared
light due to resonance with vibrational motions of functional molecular groups. As such IR
microspectroscopy constitutes a valuable tool for biochemical investigations. The use of the bright
infrared source emitted by the synchrotron radiation (SR) allowed investigating the biochemical
composition at the cellular level. The variance in the huge number of spectra acquired was addressed
by principal component analysis (PCA). The study demonstrated that the progression of steatosis
corresponds not only to the accumulation of lipids but also to dramatic changes in the qualitative
composition of the tissue. Furthermore, the approach highlighted that dramatic biochemical changes
occur also in the non-steatotic part of the tissue despite normal histological aspect suggesting that the
whole tissue reflects the grade of steatosis. This latest observation opened the possibility to address
the grade of steatosis using a laboratory IR microscope. Thus, acquisition of IR spectra on unstained
frozen sections was achieved further using a commercially available laboratory IR microscope that
allows scanning a whole tissue section ranking between 1 to 5 mm! (up to 10 000 spectra with 50 x 50
2
"m aperture size). The quantification of the lipid content was addressed from the IR spectra by
-1
calculating the ratio of integrated intensity of bands attributed to lipids (2800-3000 cm ) related to
-1
proteins (Amide II: 1485-1595 cm ). This ratio allows normalizing the intensity variation of the bands
due to variations in thickness of the tissue section. For each patient, the average ratio lipids/proteins
measured by IR microspectroscopy was plotted as a function of the concentration of TG leading to
exhibit a marked linearity. A standard curve was further established to quantify the lipid content and
thus the level of steatosis. Finally, we took advantage of attenuated total reflection-Fourier transform
infrared (ATR-FTIR) spectroscopy that allows by a close contact with the sample the acquisition of an
2
average IR spectrum corresponding to a huge area of 2 x 2 mm in only 1 minute.
In conclusion, infrared microspectroscopy allows accurate quantification of lipid content on
tissue section. This rapid method can be easily used at the hospital for reliable assessment of graft
quality control in liver transplantation.
References
Peng C*, Chiappini F*, Ka#$áková S, Danulot M, Sandt C, Samuel D, Dumas P, Guettier C, Le Naour
F. Vibrational signatures to discriminate liver steatosis grades. Analyst. 2015, 140:1107-1118. (*)
Equal contribution.
Le Naour F, Gadea L, Danulot M, Yousef I, Vibert E, Wavelet M, Ka#$áková S, Castaing D, Samuel D,
Dumas P*, Guettier C*. Quantitative assessment of liver steatosis on tissue section using infrared
spectroscopy. Gastroenterology. 2015, 148:295-297. (*) Equal contribution.
46
In vivo imaging techniques to explore the immune system in non-human primates:
application to the study of the immune response to vaccination against HIV
1-2-3
1-2-3
1-2-3
4
3-5
Biliana Todorova
, Nina Salabert
, Frédéric Martinon
, Raphaël Boisgard , Gerard Zurawski , Sandra
3-5
1-2-3
1-2-3
6
3-5
3
, Antonio Cosma
, Thierry Kortulewski , Jacques Banchereau , Yves Levy ,
Zurawski , Nathalie Bosquet
1-2-3
1-2-3
and Catherine Chapon
Roger Le Grand
[email protected]
1Immunology of viral infections and autoimmune diseases, IDMIT Infrastructure, CEA –iMETI/Division of ImmunoVirology, Université Paris Sud, Inserm U1184, Fontenay-aux-Roses, France; 2- Université Paris-Sud, UMR E1, Orsay, France;
3- Vaccine research institute (VRI), Créteil, France; 4- CEA, Institute of Biomedical Imaging (I2BM), DSV/I2BM/SHFJ/INSERM
U1023, CEA, Orsay, France; 5- Baylor Institute for Immunology Research, Dallas, 75204 TX, USA; 6- CEA, Photonic
microscopy platform, Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), DSV, Fontenay aux Roses, France
Introduction: Non invasive and longitudinal imaging approaches are required to study the behavior of
antigen presenting cells (APCs) following immunization in order to better understand the mechanisms
leading to the induction of cellular and humoral immune responses after vaccination. Here we describe
in vivo fluorescence imaging approaches to track APCs and evaluate the local effects of an
intradermal (id) vaccination against HIV with different vaccine vectors (an anti-langerin-HIVgag fusion
protein in association with an adjuvant, the resiquimod (R-848); or a DNA plasmid combined with
electroporation (EP)).
Methods: Monoclonal fluorescent labelled anti-HLA-DR antibody (mAb) was injected id to specifically
target and track skin APCs in non-human primates (NHP). Two different vaccine vectors were injected
id to study the role of cutaneous APC at the site of vaccination. Anti-langerin-HIVgag labelled with
near infrared (NIR) fluorochrome (exc. 682nm; em. 710nm) was injected id, with or without R-848.
Moreover, animals were immunized with an id auxo-GTU-Luc-EGFP DNA plasmid injection associated
with noninvasive EP. Antigen expression was measured by in vivo bioluminescence, and by in vivo
fibered confocal fluorescence microscopy (Cellvizio® DualBand, Mauna Kea Technologies) from 24h
to 96h post-vaccination. Epidermal APCs were tracked with noninvasive in vivo fibered confocal
fluorescence microscopy for up to 96h post-vaccination. In addition, confocal fast laser scanning
microscopy was performed on whole skin biopsies to monitor the behavior of fluorescent-labelled
APCs in both dermis and epidermis. Three-dimensional image reconstructions and image analysis
(cell quantification and tracking) were performed using Volocity software (Perkin Elmer).
Results: APCs were visualized by in vivo fluorescence imaging after id injection of fluorescent
labelled anti-HLA-DR mAb for at least 48 hours. The NIR fluorescent-labelled anti-langerin-HIVgag
vaccine was visualized in the skin and in the draining lymph nodes by in vivo imaging for up to 48
hours post-injection. Confocal videomicroscopy confirmed that the anti-langerin-HIVgag specifically
targeted Langerhans cells (LCs). The number of APCs in the epidermis decreased significantly over
time, undoubtedly a consequence of migration out of the skin after vaccination with fusion protein and
also with DNA plasmid. Ex vivo monitoring confirmed the cell mobilization with a higher mobility and
displacement after DNA vaccination with EP compared to the condition without EP. Furthermore, after
EP, LCs interact with antigen producing expressing cells and leave the epidermis following directional
movement.
Conclusions: In vivo fluorescence imaging approaches allowed us to track the skin immune cells
noninvasively in their native environment, and to characterize early cellular local events postvaccination.
47
Optical imaging for biodistribution studies in preclinical research.
!
Ioanna Théodorou, Nam Nguyen Quang, Benoit Lelandais & Frédéric Ducongé
CEA, DSV, I!BM, MIRCen, CNRS UMR-9199, Université Paris Sud & Université Paris-Saclay, 18,
route du Panorama - Bât 61 p113 - BP6 - 92265 Fontenay aux roses, France.
[email protected]
Several types of compounds (antibodies, peptides, aptamers, nanoparticles") are developed
to specifically deliver contrast agents or drugs to pathologic cells. However their in vivo use will be
dependent of their ADME characteristics (Absorption, Distribution, Metabolism, and Excretion) which
remain difficult to predict. To address limitations of in vitro assays, in vivo imaging of vertebrates has
emerged as a powerful tool used in virtually all forms of modern biomedical research. Currently,
positron emission tomography (PET) and single-photon-emission computed tomography (SPECT)
represent well-validated imaging technologies that allow the quantification of radio-labeled probes
independently of their localization inside small animals and humans.
As an alternative, new imaging systems based on optics have been developed since the 90s
and have encountered huge success in preclinical research. Here, several optical imaging
technologies will be presented: planar fluorescence imaging, fluorescence diffuse optical tomography
(fDOT) and probe based confocal laser endomicroscopy (pCLE). The advantages and limitations of
these technologies as well as their complementarity with nuclear imaging will be discussed from
experiments
to
study
the
biodistribution
of
chemical
compounds(1),
biomolecules(2,3),
nanoparticles(4-6) and stem cells(7).
1.
2.
3.
4.
5.
6.
7.
Garofalakis, A., Dubois, A., Theze, B., Czarny, B., Tavitian, B. and Duconge, F. (2013) Fusion of [(18)F]FDG PET with
fluorescence diffuse optical tomography to improve validation of probes and tumor imaging. Mol Imaging Biol, 15,
316-325.
Garofalakis, A., Dubois, A., Kuhnast, B., Dupont, D.M., Janssens, I., Mackiewicz, N., Dolle, F., Tavitian, B. and
Duconge, F. (2010) In vivo validation of free-space fluorescence tomography using nuclear imaging. Opt Lett, 35,
3024-3026.
Cibiel, A., Nguyen Quang, N., Gombert, K., Thézé, B., Garofalakis, A. and Ducongé, F. (2014) From Ugly Duckling to
Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors. PLoS ONE, 9,
e87002.
Mackiewicz, N., Gravel, E., Garofalakis, A., Ogier, J., John, J., Dupont, D.M., Gombert, K., Tavitian, B., Doris, E. and
Duconge, F. (2011) Tumor-targeted polydiacetylene micelles for in vivo imaging and drug delivery. Small, 7, 27862792.
Gravel, E., Ogier, J., Arnauld, T., Mackiewicz, N., Duconge, F. and Doris, E. (2012) Drug delivery and imaging with
polydiacetylene micelles. Chemistry, 18, 400-408.
Gravel, E., Tanguy, C., Cassette, E., Pons, T., Knittel, F., Bernards, N., Garofalakis, A., Duconge, F., Dubertret, B.
and Doris, E. (2013) Compact tridentate ligands for enhanced aqueous stability of quantum dots and in vivo imaging.
Chemical Science, 4, 411-417.
Lewandowski, D., Barroca, V., Duconge, F., Bayer, J., Van Nhieu, J.T., Pestourie, C., Fouchet, P., Tavitian, B. and
Romeo, P.H. (2010) In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult
hematopoietic reconstitution. Blood, 115, 443-452.
48
In vivo imaging of signaling pathways activation in Drosophila’s Mushroom Bodies
neurons.
Nicolas Gervasi1 and Paul Tchénio2,3
1-INSERM/UPMC UMR-S 839 Institut du Fer à Moulin, Paris, France.
2-CNRS/Université Paris-Sud/ENS-Cachan Laboratoire Aimé Cotton, Orsay, France.
3-CNRS/ESPCI-ParisTech, PSL Research University Laboratoire Plasticité du Cerveau, Paris,
France.
Drosophila melanogaster can be classically conditioned both aversively and appetitively, by
association of an odorant with electric shock or sugar. The mushroom bodies (MBs) was the first brain
region to be shown to play a role in Drosophila olfactory learning. In adult Drosophila, the MBs consist
of approximately 2500 intrinsic neurons per brain hemisphere, named the Kenyon cells. Signaling
pathway activation from synapses to the nucleus are critical for this olfactory memory processing.
However, it remains unclear how the different components of this signaling pathways interact
dynamically in vivo and, in particular, in which MB neurons compartment they operate.
The brain of the fly is a very dense medium and the MBs spread across more than 200 micron
deep. We used and developed confocal, two-photon and Hilo microscopy for suitable and noninvasive fluorescence imaging with a high sensitivity and resolution of living animals. We performed
real-time measurements of Protein Kinase cAMP depend (PKA) activity using in vivo multiphoton
imaging coupled with the genetically encoded FRET probe. Our data show that MB dendrites and
axons integrate cAMP/PKA stimulation with different efficiencies and time courses. Furthermore, we
demonstrate subcellular compartimentation of PKA in axons. Using confocal spinning-disk and Hilo
microscopy with ArcLight voltage sensor, we accessed to the fast membrane potential modification of
the cell bodies of the Kenyons cells in response to aversive stimuli. Finally, I will present how spatial
mathematical modelling based on the kinetics data obtain by genetically encoded sensors can show
unexpected properties of signaling pathways dynamics in neurons.
Using advances in vivo microscopy coupled with specific neuronal expression of genetically
encoded sensor provide important information on neuronal integration. Modelling approaches can help
to decipher the complex signaling spatiotemporal dynamics process that take place during memory
formation.
49
Experimental assessment of thermal effects of high power density light stimulation for
optogenetics control of deep brain structures.
Pain F1, Sisniak I1, Chiang CC1,2, Martin C1, Palfi S3, Chaillet A4, Senova S3
1
Laboratoire Imagerie et Modélisation en Neurobiologie et Cancérologie, Université Paris sud,
Université Paris Sacaly, Bat 440, Orsay
2
3
4
National Tsing Hua University, Medical Imaging Physics Lab, Taïwan
Hôpital Henri Mondor, Service de Neurochirurgie, Créteil
Laboratoire L2S, Supélec, Université Paris Saclay
Contact : [email protected]
Optogenetics has become ubiquitous in fundamental neuroscience labs as a very powerful tool to unravel brain
networks connectivity and cellular mechanisms. Yet, its clinical translation requires a careful assessment of the
inocuity of repeated and sustained high power light stimulations. Objectives: We have studied in vivo in
anesthetized rats the potential damages and non-physiological effects produced by high power optical
neurostimulation in typical optogenetics experiments. 2D surface maps of light distribution and temperature
increase were recorded in wild type anesthetized rats brains during 90s light stimulation at 478nm (blue) and
638nm (red) with continuous or pulsed optical stimulations with corresponding power ranging from 100 up to
1200 mW/mm! at the output of an optical fiber. Post mortem maps were recorded in the same animals to assess
the cooling effect of blood flow. Post mortem histological analysis were carried out to assess whether high
power light stimulations had phototoxic effects or could trigger non physiological functional activation.
Temperature increase remains below physiological changes for stimulations up to 400mW/mm! at 40Hz.
Histology did not show significant irreversible modifications or damage to the tissues. The spatial profile of light
distribution and heat demonstrate as expected a rapid attenuation. On the basis of Monte Carlo the optimal
geometry for an optrode dedicated to optical stimulation of deep cortical tissues is proposed.
Keywords : optogenetics , red-shifted opsins, neuromodulation, nonhuman primate, heat map, light distribution.
Figure1 : Left : distribution of light in the living brain of a mouse. Right Corresponding thermal map.
References
Boyden, Edward S., Feng Zhang, Ernst Bamberg, Georg Nagel, et Karl Deisseroth. 2005. « Millisecond-Timescale, Genetically
Targeted Optical Control of Neural Activity. » Nature Neuroscience 8 (9): 1263̻68.
Dai, Ji, Ilker Ozden, Daniel I. Brooks, Fabien Wagner, Travis May, Naubahar S. Agha, Benjamin Brush, David Borton, Arto V.
Nurmikko, et David L. Sheinberg. 2015. « Modified Toolbox for Optogenetics in the Nonhuman Primate. »
Neurophotonics 2 (3): 031202.
Deng, Wei, Ewa M. Goldys, Melissa M. J. Farnham, et Paul M. Pilowsky. 2014. « Optogenetics, the Intersection between
Physics and Neuroscience: Light Stimulation of Neurons in Physiological Conditions. » American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology 307 (11): R1292̻1302.
Detorakis, Georgios Is, Antoine Chaillet, Stéphane Palfi, et Suhan Senova. 2015. « Closed-Loop Stimulation of a Delayed
Neural Fields Model of Parkinsonian STN-GPe Network: A Theoretical and Computational Study. » Frontiers in
Neuroscience 9: 237.
Vandecasteele, Marie, Yann-Suhan Senova, Stéphane Palfi, et Guillaume P. Dugué. 2015. « [Therapeutic potential of
optogenetic neuromodulation]. » Médecine Sciences: M/S 31 (4): 404̻16.
50
POSTERS ABSTRACTS
51
52
Rare-earth based nanoparticles for ROS detection and MRI: from cellular signaling to
inflammation imaging
Mouna Abdesselem1, Rivo Ramodiharilafy1, Pierre-Louis Tharaux2, Thierry Gacoin3, Cedric
Bouzigues1 and Antigoni Alexandrou1
1
LOB, INSERM U1182 - CNRS UMR7645, Ecole polytechnique, Palaiseau, France
2
PARCC, Hôpital Européen Georges Pompidou, Paris, France
3
PMC, UMR 7643, Ecole polytechnique, Palaiseau, France
[email protected]
Medical imaging, using multiple modalities such as MRI or fluorescence is a powerful tool for the
diagnosis and the treatment of complex pathologies. Multifunctional probes may be efficiently used to
generate simultaneously different signals and are thus crucial for reducing the toxicity and costs of
multimodal exams.
We developed rare-earth based nanoparticles (Gd0.6Eu0.4VO4) for optical microscopy and magnetic
resonance imaging (MRI). These particles combine paramagnetic properties with remarkable
luminescence properties: stable narrow emission, no blinking and reactive oxygen (ROS) detection. We
successfully used them for the quantitative and time-resolved (1 Hz) study of ROS-related intracellular
signaling pathways and contrast enhancing in small animal MRI.
We also demonstrated the feasibility of in vivo imaging with this multifunctional probe for the
characterization of tissue inflammation with two complementary approaches: (1) functional imaging
based on macrophages tracking by MRI in a renal inflammatory disease, and (2) optical molecular
imaging of ROS production during acute skin inflammation.
The possibility of collecting, at different scales, both molecular and functional information through a
single contrast agent (Gd0.6Eu0.4VO4 nanoparticles) opens perspectives for biomedical applications.
These nanoparticles are indeed promising candidates for the quantitative diagnosis and monitoring of
ROS related disease including inflammation associated pathologies, neurodegerations, immune
dysfunctions and cancer.
53
Developing QD-DNA bioconjugates for biological applications
a
b
b
a
c
Anusuya Banerjee , Chloé Grazon , Brice Nadal , Thomas Pons , Yamuna Krishnan and Benoit
a
Dubertret *
a
Laboratoire de Physique et d Etude des Matériaux, ESPCI ParisTech, CNRS UMR 8213, Université Pierre et
Marie Curie, 10 rue Vauquelin, 75005 Paris, France
b
Nexdot, 10 rue Vauquelin, 75005 Paris, France
c
929E, 57th St, E305, GCIS, Dept of Chemistry, University of Chicago, Chicago 60637, IL, USA
Email: [email protected]
Abstract:
Quantum Dots (QDs) have emerged as novel fluorescent probes for biomedical applications [1]. The
photophysical properties of QDs such as broad absorption, narrow emission spectrum, reduced
blinking, and enhanced photostability make them advantageous over organic fluorophores. However,
for some biological applications, QDs need to be first targeted to specific intracellular locations. It
parallel, DNA has been used as a versatile tool for cellular targeting and biosensing [2] and the
combination the photophysical properties of QDs and targettability of DNA has yielded fluorescent and
targetable nanosensors [3]. In this project, we present the synthesis of QD-DNA bioconjugates and
their applications in bioimaging.
We present a novel method to conjugate DNA to QDs solubilized in water using an amphiphilic copolymer. We quantify the DNA coupling efficiency, and show that the DNA coupled on the QD remains
available for hybridization. We have investigated and optimized the parameters that influence the
conjugation reaction and colloidal stability of QD-DNA conjugates.[4]* The coupling strategy we have
developed with the QDs has been successfully extended to gold nanoparticles. We also show that
using DNA hybridization, proteins can be bound to QDs with controlled stoichiometry. Our work
presents a general strategy for binding various biomolecules to different nanoparticles.
We will show few examples of these new QD-conjugates for targeted bioimaging in cells.
References:
[1] Medintz, I.L., Uyeda, H.T., Goldman, E.R. & Mattoussi, H. (2005). Quantum Dot bioconjugates for
imaging, labelling and sensing. Nat. Mater. 4, 435-446.
[2] Krishnan, Y., Bathe, M. (2012). Designer Nucleic Acids to probe and program the Cell. Trends in
Cell Biol. 22, 624-633. [3] Patolsky, F et al. (2003). Lighting-Up the Dynamics of Telomerization and
DNA Replication by CdSe ZnS Quantum Dots J. Am. Chem. Soc., 125, 13918 1391.
[4]* Banerjee, A., Grazon, C., Nadal, B., Pons, T., Krishnan, Y., and Dubertret, B. (2015). Fast, efficient
and stable conjugation of multiple DNA strands on colloidal quantum dots. Bioconjugate Chem. 26,
1582 1589.
*This work has been featured as the ACS Editors Choice Manuscript.
54
A fluorogen-based reporter for fluorescence imaging
Arnaud Gautier
École Normale Supérieure – PSL Research University, Department of Chemistry, UMR 8640
PASTEUR, 24 rue Lhomond, F-75005 Paris, France.
[email protected]
Selective labeling with fluorogenic probes represents a general strategy for highlighting proteins in
living systems. In this approach, a protein of interest (POI) is fused to a protein tag that binds a
fluorogenic ligand and activates its fluorescence. As the fluorogenic ligand is non-fluorescent by its
own and becomes strongly fluorescent only upon binding its target, unspecific fluorescence
background in cells remains minimal even in the presence of an excess of fluorogen, thus ensuring
high imaging contrast. Here, we present Y-FAST (Yellow Fluorescence-Activating and absorptionShifting Tag), a small protein tag enabling to fluorescently label proteins through the specific and
reversible binding of a cell-permeant and non-toxic fluorogenic ligand HMBR. High labeling selectivity
results from a unique fluorogen activation mechanism combining two spectroscopic changes: increase
of fluorescence quantum yield and absorption red-shift. Y-FAST was evolved from the 14-kDa
photoactive yellow protein (PYP) by directed evolution using yeast display technologies and high
throughput fluorescence activated cell sorting (FACS). Y-FAST is photostable, as bright as common
fluorescent proteins, and allows for imaging proteins in various subcellular locations and in a large
variety of systems, from mammalian cells (including neurons) to microorganisms and zebrafish. YFAST distinguishes itself from other labeling systems because the binding of the fluorogen is not only
instantaneous and specific but also highly dynamic: the exchange dynamics is characterized by a
binding/unbinding frequency of about 10 Hz enabling in particular (i) to rapidly switch the fluorescence
on and off at will by addition or withdrawal of the fluorogen, opening new perspectives for multiplexing
imaging, and (ii) to envision various applications for sub-diffraction-limit imaging using Single-molecule
Localization Microscopies (SLM) or Super-resolution Optical Fluctuation Imaging (SOFI).
References
Plamont, M.-A., Billon-Denis, E., Maurin, S., Gauron, C., Pimenta, F. M., Specht, C. G., Shi, J.,
Querard, J., Pan, B., Rossignol, J., Morellet, N., Volovitch, M., Lescop, E., Chen, Y., Triller, A., Vriz,
S., Le Saux, T., Jullien, L.* & Gautier, A.*. A small fluorescence-activating and absorption-shifting tag
for tunable protein imaging in vivo. PNAS in revision.
55
Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence
#
#
Slávka Ka!"áková1 , Cameron M. Kewish , Stéphan Rouzière, Françoise Schmitt, Rodolphe
Sobesky, Joël Poupon, Christophe Sandt, Bruno Francou, Andrea Somogyi, Didier Samuel,
Emmanuel Jacquemin, Anne Dubart-Kupperschmitt, Tuan Huy Nguyen, Dominique Bazin,
Jean-Charles Duclos-Vallée, Catherine Guettier, François Le Naour
#
These authors contributed equally to this work
Affiliation : Univ Paris-Sud XI/ INSERM Unité 1193, Villejuif, F-94800, France
Email : [email protected]
Wilson’s disease (WD) is a rare autosomal recessive disease due to mutations of the gene
encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of
symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and
the absence of reliable diagnostic test. We investigated the potential of X-ray fluorescence
(XRF) that allows quantitative analysis of multiple elements. XRF experiments were first
performed using synchrotron radiation leading to measure the distribution of copper, iron and
zinc on tissue sections at the cellular level. Investigations were further focused on formalinfixed paraffin embedded needle biopsies using a laboratory X-ray source. The intensity of
copper related to iron and zinc significantly discriminated WD from other genetic or chronic
liver diseases including cholestatic diseases with 97.6% specificity and 100% sensitivity. This
study established a definite diagnosis of Wilson’s disease based on XRF that can be easily
implemented at the hospital.
56
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57
Outsmarting Photobleaching Processes: The Case Study of Y-FAST, a
Fluorogenic Protein with a Chromophore with Fast Exchange Dynamics
Frederico M. Pimenta,a,b,c Thomas Le Saux a,b,c Ludovic Jullien a,b,c Arnaud Gautier a,b,c
École Normale Supérieure ± PSL Research University, Department of Chemistry, 24 rue
Lhomond, F-75005 Paris, France.
b Sorbonne Universités, UPMC Univ Paris 06, UMR 8640 PASTEUR, F-75005 Paris, France.
c CNRS, UMR 8640 PASTEUR, F-75005 Paris, France.
a
[email protected]
Overcoming photobleaching is arguably one of the remaining Holy Grails in Bioimaging.
In this combined photophysical and photochemical process, light absorption by a chromophore
leads to non-reversible loss of fluorescence through pathways which may radically differ
depending on the chromophore at hand. This creates limitations not only in long-term imaging
but, more importantly, in the accurate quantification of cellular processes over time.1 The
development of genetically-encoded fluorogenic approaches comes with the promise of greatly
improving the photostability of genetically-encoded fluorescent proteins )3¶V. The former are
based on the semi-rational design of a protein-chromophore duo that, upon interaction,
enhance and/or alter the emission and absorption properties of the fluorophore.2 Unlike typical
)3¶V, the (externally or endogenously-supplied) chromophore is usually in surplus amount with
respect to its protein counterpart, indicating that chromophore recycling in the protein binding
pocket could prevent photobleaching. However, studies on the photostability and
photochemical processes that occur in these fluorogenic systems lags behind their current
development.
To understand if fluorogenic systems can be used to reduce photobleaching in
fluorescence imaging we made use of the genetically-encoded system YFAST (YellowFluorescence Activating-Shifting Tag), which presents fast dynamics of binding and unbinding
of its exogenous chromophore, HMBR (4-hydroxy-3-methylbenzylidene-rhodanine).3 Through
a series of photophysical and photochemical studies, which can be extended to other
fluorogenic systems, we demonstrate that YFAST exchange dynamics reduce photobleaching
to a certain extent. Loss of fluorescence is still observed upon prolonged irradiation, most likely
through an electron-driven photooxidation pathway here explored. This study thus provides an
experimental starting point for fluorogenic approaches to be probed and improved upon based
on the mechanistic information obtained.
References
1
Remington, S. James. (2006) Fluorescent proteins: maturation, photochemistry and
photophysics, Current Opinion in Structural Biology, 16 (6), 714-721
2
Bruchez, Marcel P. (2015) Dark dyes-bright complexes: fluorogenic protein labelling, Current
Opinion in Structural Biology, 27, 18-23
3
Plamont et al. (2015) A small fluorescence-activating and absorption-shifting tag for tunable
protein imaging in vivo, under revision in Proceedings of the National Academy of Sciences
58
Expression-Enhanced Fluorescent Proteins Based on Enhanced Green
Fluorescent Protein for Super-resolution Microscopy
Duwé S1, De Zitter E1, Gielen V1, Moeyaert B1, Vandenberg W1, Grotjohann T2, Clays K,
Jakobs S2, Van Meervelt L1, Dedecker P1
1
2
Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium
Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany
3
University of Goettingen Medical School , Robert-Koch-Str. 40, 37075 Goettingen, Germany.
[email protected]
"Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced
fluorescence imaging. However, only a limited number of such labels is available, and many display
reduced biological performance compared to more classical variants. We present the development of
robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens,
that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold
higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP.
This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but
rather due to enhanced expression levels that allow many more probe molecules to be functional at
any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how
these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and
RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of
a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"conformation we were able to confirm the presence of a cis-trans isomerization and provide further
insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically
encoded "smart fluorophores" can be readily optimized for biological performance and provides a
practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.
References
S. Duwé et al, ACS Nano 2015: 10.1021/acsnano.5b04129.
59
Imaging Drosophila brain by combining cryo-soft X-ray microscopy of
thick vitreous sections and cryo-electron microscopy of ultrathin vitreous
sections
Amélie Leforestiera, Pierre Levitzb, Thomas Preatc, Peter Guttmannd, Laurent J. Michotb, Paul
Tchénioc,e
a
Laboratoire de Physique des Solides, CNRS, Université Paris-Sud, Universiét Paris Saclay, 91405
Orsay Cedex, France
b
PHENIX Laboratory, UMR 8234, CNRS-Université Pierre et Marie Curie, 75252 Paris, France
c
Genes and Dynamics of Memory Systems, Brain Plasticity Unit, CNRS, ESPCI-ParisTech, PSL
Research University, Paris, France
d
Helmholtz-Zentrum Berlin, Institute for Soft Matter and Functional Materials, Albert Einstein Str 15,
D-12489 Berlin, Germany
e
Laboratoire Aimé Cotton, CNRS, Université Paris-Sud, Universiét Paris Saclay, 91405 Orsay, France
[email protected]
Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron
microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at
lower résolution [1, 2] . We describe how the method, so far restricted to isolated small cells
or cell monolayers, can be extended to large cells and tissue. We image the synapses of the
Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of
thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show
how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 µm, by
tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted
on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of
synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution
better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel
at the nanometre level of resolution by cryo-electron microscopy [3].
References
[1] C.A. Larabell, K.A. Nugent (2010) Imaging cellular architecture with X-rays. Curr. Opin. Struct. Biol., 20
(5) pp. 623–631
[2] W.G. Müller, J.B. Heymann, K. Nagashima, P. Guttmann, S. Werner, et al. (2012) Towards an atlas of
mammalian cell ultrastructure by cryo soft X-ray tomography. J. Struct. Biol., 177, pp. 179–192 .
[3] A. Leforestier, P. Levitz , T. Preat , P. Guttmann , LJ. Michot, P. Tchénio (2014) Imaging Drosophila brain
by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin
vitreous sections. J Struct Biol. 188(2):177-82
60
"CPBM imaging center: confocal, time-resolved and 3D super-resolution fluorescence
microscopy setups”
1: S. Lécart, G. Dupuis
2: N. Bourg, C. Cabriel, I. Coto Hernandez, S. Sivankutty2, SY. Bak, S. Lévêque-Fort,
3: E. Fort
1. CPBM/CLUPS/LUMAT, Orsay; 2. Biophotonics group from ISMO, Orsay; 3. Institut Langevin,
ESPCI ParisTech, Paris
[email protected]
Abstract :
The biomedical photonic center is an open imaging facility of the Paris-South University. We work
together with different development teams (especially the biophotonics group from ISMO, Orsay and
the Institut Langevin from ESPCI, Paris) to develop innovative highly-resolved (in space and time)
fluorescence microscopes. At the CPBM, users can access these new microscopes and imaging
processes and benefit from the presence of a L2 culture laboratory to prepare their sample.
Our recent instrumental and methodological developments have been focused on circumventing the
diffraction limit in 3D to image nanometric cell’s structure, for precise colocalisation and understanding
protein association for exemple.
We developed different strategies to enhance lateral and/or axial resolution.
- An original approach based on the selective detection of supercritical angle fluorescence (SAF) will be
presented. While keeping a classical epifluorescence excitation, we will present how the SAF detection
module allows a simultaneous detection of membrane and inner part of the cells, with a high axial
discrimination.
- We will focus on our new super-resolved microscope: the direct optical nanoscopy with axially localized
detection (DONALD) which combines conventional dSTORM with the detection of a fluorescent probe's
evanescent light—specifically, its supercritical-angle fluorescence emission. Using this method, we were
able to demonstrate isotropic super-resolution imaging of actin and microtubules in mammalian cells.
- Finally, the poster will describe our running STED microscope design to be user friendly with a time
gated detection and also a better axial resolution using SAF technic.
61
Quantitative analysis of the assembly of the phagocyte NADPH oxidase in live cells using a
FLIM-FRET approach
C.S.Ziegler, L.Bouchab, S. Dupré, Y. Bousmah, F. Mérola, O. Nüsse, M. Erard,
Laboratoire de Chimie Physique, UMR8000, Université Paris Sud
[email protected]
The phagocyte NADPH oxidase (NOX2) is generating superoxide anions, which are precursors for
other reactive oxygen species. NOX2 is a major enzyme of the innate immune response. Accordingly,
dysfunctions of the NOX2 are associated with a plethora of diseases and thus detailed knowledge
about its regulation is needed. This oxidase is composed of five subunits, the membrane-bound
gp91phox and p22phox and the cytosolic p47phox, p67phox, and p40phox. The cytosolic subunits are
assumed to be in a ternary complex which translocates together with the small GTPase Rac to the
membranous subunits during activation.[1]
Our aim is to reveal interactions of the NOX2 subunits in living cells using Förster Resonance Energy
Transfer (FRET) between fluorescent protein-tagged subunits. In our study, we took advantage of the
improved fluorescent properties of the new cyan fluorescent donor, Aquamarine, which allow a
quantitative detection of FRET by fluorescence lifetime imaging microscopy (FLIM).[2]
We observed heterodimeric interactions between all cytosolic subunits. Our data allow for the first time
an estimation of the fraction of bound subunits and the apparent affinity of these interactions inside
living cells. The molecular FRET efficiency allows as well a rough approximation of the distances
between the binding partners which give new ideas of the spatial formation of the complex in live cell.
B
A
Figure 1 A. Design of the FRET experiment to probe the interaction between p67phox and p47phox.
B. FRET efficiency (Eapp [%]) and fraction of bound proteins (β) for p67phox and p47phox heterodimer.
References
[1] Dupré-Crochet S; Erard M, Nüsse O (2013) ROS production in phagocytes, why, when and where?
Journal of Leukocyte Biology 94:657-670
[2] Erard M, Fredj A, Pasquier H, Betolngar BD, Bousmah Y, Derrien V, Vincent P, Merola F (2013)
Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging Molecular
Biosystems 9:258-267
62
Fluorescent LbL Self-assembled Films for Bioimaging Applications
Yayang Tian1, 2, Rachel Méallet-Renault2, Gilles Clavier1
1
PPSM, CNRS UMR 8531, ENS-Cachan, Cachan, 94235, France
2
ISMO, UMR 8214, Université Paris-Sud, Orsay, 91405, France
[email protected]
[email protected]
[email protected]
Abstract: Two oppositely charged BODIPY-based fluorescence polymer chains were alternatively
deposited on the substrate by means of electrostatic attraction. The numbers and top layer of
fluorescent Layer-by-Layer (LbL) self-assembled films were easy to control by changing the deposition
times. In addition, the photophysical and surface properties of LbL films were characterized by
steady-state UV-vis and fluorescence spectroscopy, AFM, confocal intensity and lifetime microscopy
imaging. After a certain time of growing E. coli bacteria on FPC LbL film surface, fluorescence imaging
shown that most of bacteria became fluorescent, while few of them were not fluorescent. Such surfaces
possess a potential application in bioimaging. Indeed it seems that E. coli bacteria “eat” or digest the
polymer chains from the LbL architecture.
63
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catalogue anglais basse def janv 013

transmission 20-2015 june 19th highlights

Review - H

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