waves, acousto and light, tissues optics, interoperability, image processing
http://www.leica-microsystems.com/website/download.nsf?opendatabase&language=english
Leica AOBS SP2 confocal microscopy
http://www.aecom.yu.edu/aif/instructions/aobs/index.htm
The Leica confocal is one of a few laser scanning confocal microscopes in the AIF. The Leica may be programmed to scan many different wavelength ranges. The Leica AOBS uses variable spectral detection instead of traditional emission filters. There are a few laser lines for excitation and the dyes are limited by these. Any dye that is excited at one of these following wavelengths can be used: 405; 458; 476; 488; 514; 561; and 633 nm. According to the sales literature, the lasers are: diode 20 mW 405 nm; Ar 100 mW 457 nm, 488 nm, 514 nm; diode 10mW 561 nm; HeNe 10 mW 633 nm.
Objectives:
63X N.A. 1.4-0.60 Oil lBL HCX PL APO
40X N.A. 1.25-0.75 Oil CS HCX PL APO
20X N.A. 0.70 1mm corr lBL HC PL APO
Automated stage for tiling XY, XYZ or XYT volumes with 166.6 mm of travel in Z.
Here’s a picture of the new system as installed at the AIF on November 6, 2002.
Contents:
Leica Application Notes:
Sample method section for a paper:
Images were collected with a Leica TCS SP2 AOBS confocal microscope (Mannheim, Germany) with 25X and 60X oil immersion optics. Laser lines at 488nm and 543nm for excitation of Cy2 and Cy3 were provided by an Ar laser and a HeNe laser. Detection ranges were set to eliminate crosstalk between fluorophores.
Selected Bibliography
http://www-lsp.ujf-grenoble.fr/Collaborations-et-contrats
Contrat Plan état-Région 2007-2014 » Imageries biomédicales », volet « imagerie par optique non-linéaire ».
I) Pourquoi une plateforme ? Le Laboratoire dispose actuellement de 2 chaînes laser femtoseconde Sa :Ti. (laser femtoseconde et sa pompe) L’une (n°1, sous la responsabilité de J.C. Vial, DR CNRS) est immobilisée sur la plateforme « microscopie intravitale ». Elle est utilisée à de manière intensive pour des expériences sur le petit animal en collaboration avec des biologistes, dans le cadre du nouvel Institut des Neurosciences de Grenoble. L’autre (n°2, sous la responsabilité de P. Baldeck, DR CNRS) est utilisée pour des études de caractérisation spectroscopique de molécules aux propriétés photochimiques remarquables (collaboration avec plusieurs groupes de chimistes) en liaison avec la mise au point de nouveaux marqueurs fluorescents, et plus récemment de fabrication de nanostructures métalliques et polymériques.
En parallèle avec leur utilisation principale, ces installations ont permis régulièrement ces dernières années la réalisation d’expériences de faisabilité de plusieurs projets de recherche de différentes équipes du Laboratoire et de leurs collaborateurs extérieurs (ce qui d’ailleurs a conduit certains d’entre eux à l’acquisition ultérieure de leur propre système).
De fait il est banal de dire que les propriétés des lasers à impulsions courtes en font un outil devenu un outil incontournable pour de nombreuses applications. Ces installations sont désormais (déjà !) vieillissantes et le système n°2 n’est pas compatible avec l’adaptation d’un système de « cavity dumping ».
Une troisième chaîne est en cours d’assemblage : un oscillateur laser Sa:Ti a été acheté en 2006 sur fonds propres. Le modèle choisi (Coherent Mira 900) est susceptible d’accueillir le « cavity dumping » indispensable pour les applications nécessitant les fortes puissances crêtes (effet Kerr et génération du continuum par exemple). Elle ne dispose pas pour l’instant de laser de pompe, nous en proposons l’achat dans la présente demande de financement mi-lourd.
Nous pensons en effet qu’il est essentiel de disposer d’une nouvelle plateforme complète, mise en place autour d’un club d’utilisateurs bénéficiant d’un parc d’outils et assurant un partage de compétences techniques pour
développer de nouveaux projets de recherche dont certains sont brièvement décrits ci-dessous
préparer la jouvence de nos installations plus anciennes, disposer d’équipements complémentaires (« cavity dumping », système de compression d’impulsion, système d’allongement de durée d’impulsion).
pouvoir continuer à mettre à disposition de l’ensemble des chercheurs du Laboratoire ou de leurs collaborateurs une installation modernisée afin de réaliser avec un délai d’attente réduit des expériences de faisabilité, et pouvoir continuer à développer leur programme de recherche.
Cette plateforme sera couplée à plusieurs microscopes, tant pour le développement de méthodes d’imagerie non conventionnelle, que pour les applications en nanosciences ou nanobiologie. II) Projets scientifiques en cours ou futurs qui bénéficieront de la plateforme a) Microscopie optique non-linéaire pour l’investigation du vivant Intervenants : J.C. Vial et J. Douady, collaboration avec la ligne médicale de l’ESRF, le Grenoble institut des Neurosciences. A la suite de la démonstration de l’efficacité des méthodes non-linéaire classiques (2-photon et SHG) pour la microscopie intravitale [1] au sein du laboratoire, la plateforme servira à la mise en oeuvre de nouvelles méthodes d’optique non linéaire pour la microscopie du vivant, particulièrement l’effet Kerr, la génération de 3ieme harmonique et l’émission Raman cohérente.
b) Changements de phase du quartz dans la région d’opalescence par génération de second harmonique Responsable Gérard Dolino. La grande stabilité des impulsions femtosecondes issues du laser TiSa, en mode bloqué, est parfaitement adaptée à la génération de second harmonique, au sein du Quartz [2] pour sonder les changements de phase. c) Détection d’états transitoires dans les cristaux moléculaires Responsable Dominique Block. Le laser TiSa dans son mode à extraction de cavité (cavity dumping) peut conduire à la génération d’un continuum spectral utilisable dans la détection des états transitoires des cristaux moléculaires [3]. d) Spectroscopie des gaz dans les cavités à haute fréquence Responsable : Daniele Romanini. Les nombreux modes spectraux bloqués au sein des lasers TiSa femtosecondes peuvent être injectés simultanément dans des cavités à haute finesse, être doublés en fréquence et conduire à une spectroscopie de haute sensibilité [4]. e) Femtotechnologie des matériaux Intervenants : Patrice Baldeck et Olivier Stephan. Les impulsions laser femtoseconde peuvent créer de la photochimie et photopolymérisation par transition à 2 photons produisant des structures nanométriques à 3 dimensions [5] au sein de matériaux inorganiques, organiques et biologiques. f) Spectroscopie par corrélation de Fluorescence (FCS)[6] Responsable : Antoine Delon. Les transitions à 2 photons, par excitation femtoseconde, définissent efficacement des zones tri dimensionnelles favorable à la FCS . III) Description du matériel rassemblé sur la plateforme La plate-forme « Femto » possède déjà un laser femtoseconde (Achat en 2006) de type TiSa mais doit s’équiper d’un laser de pompe efficace et s’associer un environnement qui le rend utilisable sur des expériences complexes . Elle accueillera le matériel suivant, pour lequel un nécessaire multifinancement est espéré.
1) Un laser de pompe Type Millénia ou Verdi, Puissance 10W afin de pouvoir pomper 2 TiSa 60 k€ HT Objet de la présente demande 2) Laser Sa:Ti Coherent Mira 900 49k€ HT (déjà acheté sur fonds propres Labo) 2) Le doubleur et tripleur de fréquence 14 k€ HT 3) Le passage en mode picoseconde 18 k€ HT 4) Un microscope à balayage pour la mise en oeuvre de l’imagerie Kerr 35 k€ HT 5 ) Un cavity dumping 43 k€ HT 6) Un microscope à balayage pour le développement des nouvelles microscopies non-linéaire (biphoton, second harmonique, troisième harmonique, CARS) 214 k€ HT (En commun avec le CPER « imagerie biomédicale »)
IV) Plan de financement de la plateforme
60 Keuros Mi-lourd CNRS Demandé 35 Keuros Id-Nano Obtenu 49 Keuros Financement interne Obtenu 75 Keuros Financement interne + BQR Demandé 214 Keuros CPER Imagerie bio-médicale Demandé.
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– Jean-Claude Vial, physicist, DR CNRS (Laboratory of Physical Spectrometry: “SPECTRO”, UMR UJF-CNRS 5588)
– Jonathan A. Coles, physiologiste, DR CNRS (Inserm-UJF, GIN: Funtional Neuroimaging)
– Boudewijn Van der Sanden, biologist for applications in neuro-oncology, CR1, (Inserm –UJF, GIN: Medical applications of synchrotron radiation)
Within the GIN: UJF Engineer (“Ingénieur d’études”) on transfer
Within SPECTRO: Julien Douady, Maître de Conférences, Joseph Fourier University (recruited in 2006)
A “club biphoton” has been created, it groups researchers from the various teams of the GIN. It will help in the scientific direction of the facility for neuroimaging by non-linear optics.
Why a non-linear optics facility in the GIN?
The major themes of research in the GIN concern the investigation on the nervous system in conditions such as Parkinson’s disease, epilepsies and pain, the microvasculature, particularly in brain tumors and stroke and pathological re-organization of neurons and astrocytes. These projects are mainly carried out on animal models, and involve imaging brain structures at micron scales.
The GIN has strong competences in many in vivo nuclear magnetic resonance imaging (MRI) techniques. These techniques have the advantage of being non-invasive but do not have the micron (or less) spatial resolution that is needed to image blood microvessels, cells and synapses. There are fewer functional contrast agents and markers for MRI than the increasing number of available fluorescent indicators. Therefore, in experiments on animal models, MRI is routinely complemented by histological and quantitative micromorphometric studies on fixed tissues. The GIN Teams can also image changes in intracellular free calcium concentration in the superficial layers of brain slices. Further identified needs within the GIN include: in vivo imaging of the state of cerebral vasculature in brain pathologies such as tumours or after radiation damage; rapid, high-resolution imaging of electrical activity in brain slices characteristic of pathologies such as Parkinson’s disease or epilepsy; and rapid techniques for quantitative micromorphometry. The non-linear optical techniques we have acquired, developed or are currently exploring, can meet these needs.
Non-linear optical phenomena occur when the incident light has a very high intensity. In practice, the only commercially available light source suitable for non-linear optics in neuroscience is the titanium-sapphire infrared femtosecond laser. The femtosecond laser delivers a very high peak power with an acceptable average power by producing very short pulses (< 10-13 sec). The wavelength is usually adjusted within a 700 – 1000 nm range. In neuroscience, the laser is usually the source for a two-photon microscope, in which fluorophores are excited by the simultaneous absorption of two photons. This microscope allows the imaging of fluorophores with lateral resolution of about 500 nm and an axial resolution of about 3 micron down to depths of about 600 micron in turbid materials such as brain tissue. Two-photon microscopy has been used for in vivo studies of the cortex of mice and rats through a cranial window and has allowed, for example imaging of the cortical vasculature, intracellular calcium dynamics and long-term changes in the morphology of dendritic spines.
In 2001, we set up a two-photon microscope in the Laboratoire de Spectrométrie Physique (SPECTRO) on the campus of the Université Joseph Fourier (UJF). It is routinely used for imaging the microvasculature in the cortex of the anaesthetized mouse after intravenous infusion of fluorescent molecules. A 3D image can be acquired in under less than one minute and has typically an area of 512 x 512 pixels in the horizontal plane (a pixel being 0.5 x 0.5 micron) and a depth of 200 pixels of between 1 and 3 microns.
We have developed new quantitative methods for measuring brain hemodynamics. The simultaneous use of two fluorescent probes, a large one that remains intravascular and a small one that escapes from leaky microvessels, has led to the development of a novel method of quantifying extravasation (Vérant et al 2007). These techniques have been used to characterize the damage to normal brain tissue by experimental X-ray tumour treatments being tested at the Medical Beam Line of the European Synchrotron Research Facility in Grenoble (Serduc et al 2006, Vérant et al 2007).
Non-linear phenomena can also be used for Second Harmonic Generation (SHG). Incident light on arrays of orientated molecules gives rise to emission at half the wavelength of the incident light. This second harmonic light is produced from ordered endogenous molecules such as collagen. Of more interest to neuroscience, exogenous lipophilic fluorescent molecules that insert themselves in neuronal membranes give rise to SHG that is sensitive to the membrane potential. Two laboratories have reported that, with a suitably engineered probe molecule, the SHG is considerably more sensitive to changes in membrane potential than the traditional one photon fluorescent signal (Pons et al. 2003; Millard et al. 2004) and SHG has been used to record potentials in proximal dendrites (Dombeck et al. 2005 ) and dendritic spines (Nuriya et al., 2006). Within the “Interface Physique-Science du Vivant” program of the 12th Contrat de Plan Etat-Région (CPER) a grant was obtained in 2003 for developing SHG for measurement of membrane potential with a view to imaging functional neuronal networks. A group within the Chemistry Laboratory of the Ecole Supérieure de Lyon (Chantal Andraud) is collaborating in this project in the development of new probe molecules. A dedicated SHG set-up is now available at SPECTRO (which shares the existing beam from the Ti-sapphire femtosecond laser), a PhD student (Davy Cottet) is now engaged on this subject and a grant from the “RTRA-nanosciences” has been obtained (coordinator : Julien Douady).
The experience of non-linear optical techniques applied to neuroscience that has been acquired at the SPECTRO site will make it possible to provide a facility in the new building of the Grenoble Institute of Neuroscience that will be readily useable by neuroscientists. At the same time, following the model of the successful NMR imaging platform, new techniques and applications will continue to de developed.
Dombeck DA, Sacconi L, Blanchard-Desce M, Webb WW (2005) Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. J Neurophysiol 94:3628-3636
Millard AC, Jin L, Wei MD, Wuskell JP, Lewis A, Loew LM (2004) Sensitivity of second harmonic generation from styryl dyes to transmembrane potential. Biophys J 86:1169-1176.
Nuriya, M., Jiang, J., Nemet, B., Eisenthal, K. B. and Yuste, R. (2006). Imaging membrane potential in dendritic spines. Proc Natl Acad Sci U S A 103, 786-790.
Pons T, Moreaux L, Mongin O, Blanchard-Desce M, Mertz J (2003) Mechanisms of membrane potential sensing with second-harmonic generation microscopy. J Biomed Opt 8:428-431.
R. Serduc, P. Vérant, J. C. Vial, R. Farion, L. Rocas, C. Rémy, T. Fadlallah, E. Brauer, A. Bravin, J. Laissue, H. Blattmann and B. van-der-Sanden, « In vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature, » International Journal of Radiation Oncology Biology Physics 64, 1519-1527 (2006).
Premises: A large room with air-conditioning
Two-photon microscope adapted for in vivo imaging of mouse cortex
Infrared Ti-sapphire femtosecond laser (Spectra Physics) with pump laser and associated diagnostic measurement instruments
Confocal microscope scanning head (Bio-Rad)
Upright microscope with epi-illumination, video camera and motorized objective
Non-descanned port with two photomultipliers
Imaging software
Large motorized microscope stage
Stereotaxic frames for mice and rats
Set-up for microdissection under volatile anaesthetics
Large range of optical filters, microscope objectives and other optical elements.
Set-up for imaging by Second Harmonic Generation (SHG)
Upright microscope
Photomultipliers,
Scanning mirrors with drivers
Equipment for patch-clamping cells: micromanipulator, amplifier, software.
Experimental control and image analysis
4 PCs
Home made software for interfacing and image processing
Labview 7
Platform activities
To provide state-of-the-art two-photon microscopy for the Neuroscience, and to explore and develop new optical imaging techniques relevant to research topics of the GIN.
Scientific report
Techniques developed and used
– Measurement of blood volume and of the permeability of the blood-brain barrier to dyes in the cortex of anaesthetized mice (thesis Pascale Vérant).
– Use of multiple intravascular dyes to characterise damage to the blood-brain barrier and to cells in the parenchyma caused by synchrotron X-rays (Serduc et al, 2006) along protocols in experimental radiotherapy: micro-beams radiotherapy (cf. Serduc thesis) and photoactivation (cf. Clément Ricard thesis)
– Imaging of unfixed tissue to a depth greater than that accessible with a conventional confocal mono-photon microscope.
– Combined imaging of tissue using SHG and two-photon excited fluorescence (Clément et al., 2007)
Articles in peer-reviewed journals:
J. Coste, J. C. Vial, G. Faury, A. Deronzier, Y. Usson, M. Robert Nicoud and J. Verdetti, « NO synthesis, unlike respiration, influences intracellular oxygen tension, » Biochem Biophys Res Commun 290, 97-104 (2002).
R. Serduc, P. Vérant, J. C. Vial, R. Farion, L. Rocas, C. Rémy, T. Fadlallah, E. Brauer, A. Bravin, J. Laissue, H. Blattmann and B. van-der-Sanden, « In vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature, » International Journal of Radiation Oncology Biology Physics 64, 1519-1527 (2006).
P. Vérant, R. Serduc, B. van-der-Sanden, C. Remy and J. C. Vial, « A direct method for measuring mouse capillary cortical blood volume using multiphoton laser scanning microscopy, » Journal of Cerebral Blood Flow&Metabolism 27, 1072-1081 (2007).
A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. van der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. Lilian, P. Baldeck and J. F. Nicoud, « Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation., » Microscopy Research and Technique 70, 880-885 (2007).
P. Vérant, R. Serduc, B. van-der-Sanden, C. Remy, C. Ricard, J. A. Coles and J. C. Vial, « A subtraction method for intravital two-photon microscopy: intraparenchymal imaging and quantification of extravasation in mouse brain cortex., » Journal Of Biomedical Optics In press (2007).
C. Ricard, J. C. Vial, J. Douady and B. van-der-Sanden, « In Vivo Imaging of Elastic Fibers using Sulforhodamine B, » Journal Of Biomedical Optics In press (2007).
Book chapter:
C. Ricard, J. A. Coles, R. Serduc, B. van der Sanden, P. Vérant and J. C. Vial, in New Encyclpedia of Neurosciences, Third edition., edited by G. Adelman and B. Smith (Elsevier, 2007).
Theses:
P. Vérant, Thesis in Physical sciences, University Joseph Fourier, Grenoble, France, 2006.
Imagerie intravitale par microscopie biphotonique : application à l’étude des effets de la radiothérapie synchrotron par microfaisceaux sur la microvascularisation corticale de la souris.
R. Serduc, Thesis in Biological science, University Joseph Fourier, Grenoble, France, 2006. Effets de la radiothérapie par microfaisceaux synchrotron sur la microvascularisation cérébrale saine et tumorale chez la souris.
Congress Proceedings:
Vérant P, Serduc R, Coles JA, Farion R, Rémy C, Van der Sanden B, Vial JC (2004) A method for measuring cerebral blood volume of mouse using multiphoton laser scanning microscopy. Proc SPIE 5463:1-12.
Vérant P, Serduc R, Coles JA, Farion R, Rémy C, Van der Sanden B, Vial JC. (2005) An intravital two photon microscopy method for imaging mouse brain tissues. In: Proceedings of Imaging for Medical and Life Sciences (IMVIE2), Ilkirch, France, March 1-3, 2005.
P. Vérant, J. C. Vial, R. Serduc, C. Ricard, C. J., C. Rémy, B. van-der-Sanden, E. Brauer and A. Bravin, presented at the Biomedical Topical Meeting (BIO) Fort Lauderdale, Florida March 19, 2006 Poster Session II (ME), 2006 published in “applied-optics OSA series”.
Vérant P, Serduc R, Coles JA, Farion R, Rémy C, Van der Sanden B, Vial JC (2004) A method for measuring cerebral blood volume of mouse using multiphoton laser scanning microscopy. Photonics Europe, Strasbourg, 26 –30 avril.
Vérant P, Serduc R, Coles JA, Farion R, Rémy, C,Van der Sanden B, Vial JC. (2005), In vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature In : Progress in biomedical Optics in Imaging, Confocal, Multiphoton, Nonlinear Microscopy Imaging II. Munich 12-16 juin 2005.
Vérant P, Serduc R, Coles JA, Farion R, Rémy C, Van der Sanden B, Vial JC. (2005) An intravital two photon microscopy method for imaging mouse brain tissues. In: IMVIE2, Ilkirch, France, March 1-3, 2005.
Vial JC, Vérant P, Serduc R, Coles JA, Farion R, Rémy C, Van der Sanden B (2007) A subtraction method for intravitaltwo-photon microscopy : intraparenchymal imaging and quantification of extravasation in mouse brain cortex. 10th International conference : « Signal transduction in the blood-brain barriers » Potsdam September 13-16 2007.
Rémy C, Serduc R,Vérant P, Vial JC, Farion R, Brauer E, Bravin A, Laissue J, Segebarth C, van der Sanden B, 2005. Effect of microbeam radiation exposure to the microvasculature of healthy mouse brain, European Association For NeuroOncology (EANO), 5-8 May, Edinburgh.
Serduc R, Farion R, Rémy C, Vérant P, Vial JC, Brauer E, Laissue J , Blattmann H, Bravin A, van der Sanden B, 2005, In vivo two photon microscopic study of short term effects of microbeam radiation therapy on the microvasculature in healthy mouse brain, European Conferences on Biomedical Optics (ECBO), (OSA meetings), 12-16 June, Munich.
Pascale Vérant, Jean Claude Vial, Raphaël Serduc, Clement Ricard, Jonathan Coles, Chantal Rémy, Elke Brauer, Alberto Bravin, Boudewijn van der Sanden. A differential in vivo 2 photon microscopy method for quantitative imaging of dye extravasation in mouse cortex, Biomedical Optical Topics meeting, OSA, Fort Lauderdale, Floride, march 2006.
Clément Ricard, Pascale Verant, Raphaël Serduc, Jean-Claude Vial, Régine Farion, Chantal Remy, Christoph Segebarth & Boudewijn van der Sanden. Multiphoton Microscopy as a Powefull Tool to Investigate the Healthy and Diseased Brain. European Bioalpine Covention, Grenoble, October 2006.
Olivier HUGON, Eric LACOT, Irina Alexandra PAUN, Boudewijn VAN DER SANDEN. Laser optical feedback microscopy, European Bioalpine Covention, Grenoble, October 2006.
Clement Ricard, Jean-Claude Vial, Sonia Teypaz, Jerome Gastaldo, Manuel Fernandez, François Estève, Christoph Segebarth & Boudewijn van der Sanden. Short-Term Effects of a 15Gy – 79keV Synchrotron Tomographic Irradiation on Healthy Mice Brain Microvasculature. ICRR San Francisco, July 2007
Boudewijn van der Sanden, Clément Ricard, Raphaël Serduc, Jean-Claude Vial, Julien Douady,Pascale Vérant, Elke Brauer-Krisch, Alberto Bravin, Jerôme Gastaldo, Manuel Fernandez, Hélène Elleaume, Herwig Requardt, Hans Blattmann, Jean Laissue, François Estève. In Vivo Two-Photon Microscopy of Cerebral Vascular Damage after Synchrotron Irradiation Protocols. Medical applications of Synchrotron Radiation MASR 2007, 25 – 29 august 2007, Saskatoon, Canada.
Mathieu Maurin, Patrice L. Baldeck, Ali Hayek, Frederic Bolze, Jean-François Nicoud, Jean-Claude Vial, Boudewijn P. J.van der Sanden, Multifunctional dye for two-photon microscopy, MRI and photon activation therapy using synchrotron irradiation, BIOS 2008, SPIE Photonic West, communication orale, paper 6867-4.
Clement Ricard, Jean-Claude Vial, Julien Douady, Boudewijn P. J.van der Sanden, Imaging elastic and collagen fibers with sulforhodamine B and second-harmonic generation, BIOS 2008, SPIE Photonic West, communication, paper 6867-23.
Coles JA, Van der Sanden B, Vial JC, Gadelle A, Leprêtre JC. (2003) Synthèse et utilisation de nouveaux marqueuers bifonctionnels détectables en microscopie biphotonique et en IRM pour l’étude des pathologies de la perfusion sanguine dans le cerveau. In: Réunion bilan du programme interdiscipolinaire CNRS-CEA- Inserm « Imagerie du petit animal », Marseille, décembre 2003.
Serduc R, Vérant P, Farion R, Vial JC, Rémy C, Brauer E, Bravin A, Van der Sanden B (2004) Microscopie à 2 photons du cerveau de souris en cours de traitement radiothérapeutique par mirco-faisceaux X. 5ème Colloque National de Diagnostic et Imagerie Optique en Médecine/Biologie, Paris (France)
Vial, Jean-Claude; Vérant, Pascale; Serduc, Raphaël; Rémy, Chantal; van der Sanden, Boudewijn P J, 2003, Imagerie par microscopie à 2 photons du cerveau de la souris. Mise au point de la méthode, des marqueurs et des applications médicales, Atelier Imagerie Optique in vivo du Petit Animal, La Tronche (France)
Vérant, Pascale; Serduc, Raphaël; Coles, Jonathan A; Farion, Régine; Rémy, Chantal; van der Sanden, Boudewijn P J; Vial, Jean-Claude, 2004, Détermination du volume capillaire cérébral des souris par microscopie à 2 photons, 5ème Colloque National de Diagnostic et Imagerie Optique en Médecine/Biologie, Paris (France)
Serduc, Raphaël; Vérant, Pascale; Vial, Jean-Claude; Farion, Régine; Rémy, Chantal; Brauer, Elke; Bravin, Alberto; Laissue, Jean A; Blattmann, Hans; Segebarth, Christoph; van der Sanden, Boudewijn P J, 2004, Anatomical and physiological changes of the microvasculature in healthy mouse brain tissue after microbeam radiation exposure, Réunion d’automne de la Société Circulation et Métabolisme Cérébral, Paris
Posters:
Coles JA, Fernandes LAL, Foucher A, Martiel JL, Vial JC (2005) Using fluorescence recovery after photobleaching (FRAP) to study glucose uptake by rat vagus nerve. Society for Neuroscience. Washington, DC 12-16 November.
Clément Ricard, Jean-Claude Vial, Julien Douady, Christoph Segebarth & Boudewijn van der Sanden. MICROSCOPIE NON LINEAIRE : DE L’HISTOLOGIE CLASSIQUE À L’HISTOLOGIE INTRAVITALE, Application au marquage des fibres élastiques in vivo. OPT-DIAG, Paris, 15/16 mai 2007.
Julien Douady, Davy Cottet, Clément Ricard, Boudewijn van der Sanden & Jean-Claude Vial. IMAGERIE DE SECOND HARMONIQUE : EN AVANT TOUTE ! OPT-DIAG, Paris, 15/16 mai 2007
Clément Ricard, Pascale Verant, Raphaël Serduc, Jean-Claude Vial, Christoph Segebarth & Boudewijn van der Sanden. Microscopie Biphotonique Intravitale et Neurosciences, Applications à l’Etude des Effets de la Photoactivation Therapy par Irradiation Synchrotron, journéé SFO, Paris, 30 Novembre 2006.
Publications for the general public
– Le journal du CNRS Juillet-Août 2005, rubrique « La vie des labos » article « Zoom dans la tête des souris ». P. Vérant et al. http://www2.cnrs.fr/presse/journal/2330.htm/
– web site : http://www-lsp.ujf-grenoble.fr/-Microscopies-intravitales-
– Le journal PAPYRUS de l’université J. Fourier : « Microscopie Intravitale »
Thesis in physics: Pascale Vérant, 2006 (Dir. Jean Claude Vial and B. van der Sanden).
Two theses in biology: 1) Raphaël Serduc 2006 (Dir. Chantal Rémy and B. van der Sanden),
2) Clément Ricard 2008 (Dir. B. van der Sanden et Christoph Segebarth)
Placements: Chemistry – characterisation of new fluorescent probe molecules
Electronics – new microscope/computer interface
Optics – new scanning method for microscopy
Material sciences – New chromophors for intravital staining
Biology – Optical microscopy by Second Harmonic Generation of biological tissues
Biology – Electrical excitation of neurons
Practical labs for physics students: 3 sessions.
INSERM formation on non linear optics for microscopy.
Coles JA, Vial JC Mise en place d’un microscope biphotonique pour l’imagerie du cerveau de la souris. Contrat de Plan Etat-Région « Nouvelles Approches physques des Sciences du Vivant » 2000-2001 : 173 k€.
Vial JC, Coles JA Programme interdisciplinaire CNRS- Inserm -CEA » Imagerie du Petit Animal » – « Synthèse et utilisation de nouveaux marqueurs bi fonctionnels détectables en microscopie biphotonoqiue et en IRM pour l’étude des pathologies de la perfusion sanguine dans le cerveau » Nov 2002 : 36 k€
Coles JA, Vial JC. Extension d’un microscope biphotonique : la détection simultanée de fluorescence et du second harmonique. Application à l’imagerie de l’activité neuronale électrique dans le cortex cérébral de l’embryon de la souris. – CPER 2003 : 32 k€
Vial JC, van der Sanden B. – Imagerie de l’angiogénèse tumorale : de la microscopie à deux photons à l’imagerie par résonance magnétique.- CPER. 2003-2004 : 25 k€
Vial JC Soutien SPM-CNRS, équipement mi-lourd 2003 : 30 k€
Vial JC Programme « Nanobio pour la physique 2004» : 31 k€
Vial JC Développement de l’imagerie par effet Kerr, Programme de l’institut des nanosciences de Grenoble- IDNANO 2005 : 35 k€
Vial JC Elaboration de nanocristaux moléculaires fluorescents pour l’imagerie du cortex cérébral de souris par luminescence excitée à deux photons, Programme de la région Rhône Alpes CIBLE 2007 (coordinateur A. Ibanez) : 10 k€
Douady J Imagerie par génération de seconde harmonique des potentiels dendritiques. RTRA 2007 (financement pas encore communiqué).
Collaborations (Outside GIN).
European Synchrotron Research Facility (ESRF), medical line ID17, Alberto Bravin and Elke Brauer
Laboratoire de Chimie, ENS Lyon (Chantal Andraud).
Laboratoire de Cristallographie (CNRS Grenoble, A.Ibanez).
Laboratoire de Chimie LEOPR (Organic electrochemistry and redox photochemistry laboratory (CNRS-UJF, Grenoble, A.Deronzier).
Institut de Physique et Chimie de Strasbourg (IPCMS) de STRASBOURG (UMR 7504, J.F. Nicoud)
Institut Néel, Grenoble (Catherine Villard)
CEA Grenoble, Jacques Baudier))
The facility needs to spread its activities in register with the needs of other Teams in the GIN (besides Teams 5 and 6, which are already involved in the current activities), whose scientific projects may benefit from analyses via non linear optics.
In collaboration with physicists and clinicians, the ten research teams of the GIN will investigate brain pathologies such as cerebral tumors, epilepsies and neurodegenerative diseases using both animal models and in vitro preparations. To this end, the optical techniques based on non linear optics open new prospects of addressing biological questions otherwise inaccessible. These techniques now allow analyses from the cellular level up to in vivo investigations on intact animals (intravital microscopy).
Some promising projects are listed in the following:
-a) Neuronal networks responsible for movement (Mireille Albrieux, team #10). High frequency electrical stimulation of the subthalamic nucleus is a very effective functional treatment of Parkinson’s disease. This project will identify the neural circuits that, within the basal ganglia, are involved in the pathological neuronal activity. Two-photon and intravital microscopy will help to investigate neuronal and glial cell networks involved in disturbances of basal ganglia.
-b) X synchrotron radiotherapy.(Boudewijn van der Sanden, team #6) Two protocols of experimental X-synchrotron radiotherapy: microbeam radiotherapy (MRT) and radiotherapy by photo-activation of heavy ions, such as platinum (pat-plat) will be studied in nude mouse glioma models and in healthy brain by intravital two-photon microscopy.
-c) The role of late endosomes in synaptic plasticity (Yves Goldberg, team #2). The aim is to study the impact of a mutation of Alix or CHMP2B on the morphological in situ development of dendrite spines in the cortex of KO mice. The outline of the spines will be made visible by the expression of a fluorescent protein. The use of a two-photon microscope is mandatory for such investigations.
-d) Measurement of neuronal electrical activity by second harmonic generation(SHG)(Jonathan Coles, team #5). Analysis of electrical activity in neural networks by fluorescent probes responding to membrane potential will be useful to projects (a) and (e). We will develop use of SHG, the technique that is currently the most promising, and requires a powerful femtosecond laser.
-e) Cortical generator of absence epilepsy seizures (Antoine Depaulis, team #9) .The project concerns the characterization of the cortical circuits responsible for absence seizures in a genetic rat model and their development during brain maturation. Two-photon microscopy will help in understanding the role of astrocytes during cortical maturation and the involvement of glia-neuron relationships in adults.
-f) The role of cancer stem cells in the genesis of cerebral tumors and their radiosensitivity (Jacques Baudier, CEA). Recent investigations have shown the existence of cancer stem cells endowed with the capacity to initiate and maintain cerebral tumors. The goal of the research is to: i) characterize the interactions between these cancer stem cells and the vasculature via two-photon microscopy during the genesis of glioblastomas, and ii) analyze the sensitivity of these cancer stem cells to the pat-plat protocol (see project b).
The facility will not contribute directly to the general overhead costs of the GIN. The platform will receive basic services (electricity, heating, cleaning, etc) but will not be further subsidized by the GIN. The platform will be financed by (1) grants (2) contributions from Teams using the platform.
At the GIN
A two-photon microscope will be set-up with the following equipment:
Ti-sapphire femtosecond laser 150 k€
Anti-vibration table, various optical components 35 k€
Ready to use confocal microscope adapted for two photon and SHG microscopy 450 k€
Microsurgical equipment 100 k€
Total 735 k€
Technological developments
They will be done at SPECTRO where the environment is favorable.
– New non linear phenomena, promising for intravital microscopy, such as the third harmonic generation and the Kerr effect will be investigated. They need a large spectral range based on a powerful TiSa laser associated with a parametric oscillator and a cavity dumping.
450 k€
– A fast scanning unit 65 k€
Total 515 k€
Running costs
The principle running costs are incurred during collaborative biological projects, notably the purchase of animals and fluorescent probes. These costs will be covered by the collaborating biological Teams. In addition, there is the cost of maintaining the lasers and optical systems (20 k€), and the purchase of dyes for development purposes (10 k€). These costs will be covered by the program grants of the teams involved.
http://www-lsp.ujf-grenoble.fr/-Microscopies-intravitales
Nous cherchons à surpasser les inconvénients de la vidéomicroscopie classique. en utilisant l’optique non linéaire en microscopie à balayage : la microscopie biphotonique et la microscopie par génération de second harmonique.
Présentation générale : un diaporama de l’activité
A la différence de l’imagerie par résonance magnétique (IRM) et de la Tomographie par émission de positron (PET), les techniques optiques sont plus rarement applicables directement chez l’homme.
La plateforme de développement de microscopie intravitale, En SPECTRO
Bientôt en SPECTRO La plateforme FEMTO en SPECTRO
Bientôt au GIN, une plateforme ouverte de microscopie intravitale
Une info sur les manifestations en relation avec la microscopie intravitale
Les Conférences, cours, séminaires en relation avec la microscopie intravitale. Les propositions de stage…etc
La microscopie à balayage de la luminescence excitée à 2 photons de colorant injecté dans le circuit sanguin (de la souris) autorise une tomographie optique à grande profondeur (jusqu’a 0.6 mm).
Sous-rubriques :
Stereology (from Greek stereos = solid) was originally defined as `the spatial interpretation of sections’. It is an interdisciplinary field that is largely concerned with the three-dimensional interpretation of planar sections of materials or tissues. It provides practical techniques for extracting quantitative information about a three-dimensional material from measurements made on two-dimensional planar sections of the material. See the Examples below. Stereology is an important and efficient tool in many applications of microscopy (such as petrography, materials science, and biosciences including histology, bone and neuroanatomy). Stereology is a developing science with many important innovations being developed mainly in Europe. New innovations such as the proportionator continue to make important improvements in the efficiency of stereological procedures.
In addition to two-dimensional plane sections, stereology also applies to three-dimensional slabs (e.g. 3D microscope images), one-dimensional probes (e.g. needle biopsy), projected images, and other kinds of `sampling’. It is especially useful when the sample has a lower spatial dimension than the original material. Hence, stereology is often defined as the science of estimating higher dimensional information from lower dimensional samples.
Stereology is based on fundamental principles of geometry (e.g. Cavalieri’s principle) and statistics (mainly survey sampling inference). It is a completely different approach from computed tomography.
Classical applications of stereology include:
* calculating the volume fraction of quartz in a rock by measuring the area fraction of quartz on a typical polished plane section of rock (« Delesse principle »);
* calculating the surface area of pores per unit volume in a ceramic, by measuring the length of profiles of pore boundary per unit area on a typical plane section of the ceramic (multiplied by 4 / π);
* calculating the total length of ca
Stereology is not tomography
Stereology is a completely different enterprise from computed tomography or for DOT. A computed tomography algorithm effectively reconstructs the complete internal three-dimensional geometry of an object, given a complete set of all plane sections through it (or equivalent X-ray data). On the contrary, stereological techniques require only a few `representative’ plane sections, and statistically extrapolate from them to the three-dimensional material.
Stereology exploits the fact that some 3-D quantities can be determined without 3-D reconstruction: for example, the 3-D volume of any object can be determined from the 2-D areas of its plane sections, without reconstructing the object. (This means that stereology only works for certain quantities like volume, and not for other quantities).
biomedical optics 
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