Date : 23/03/2011

Laboratory
Wave front engineering microscopy group, Neurophysiology and new microscopy laboratory
UMR8154
CNRS, INSERM, University Paris Descartes
45, rue des Saints Pères
75006 Paris
Website : http://www.biomedicale.univ-paris5.fr/neurophysiologie/Groups/emilianigroup.php
Main discipline : Physics
Lab director : Serge Charpak

PhD Supervisor
Valentina Emiliani
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : + 33 142864253

Subjects
1.: optogenetics
2.: voltage sensitive dyes
3.: brain connectivity

Tools and methodologies
1.: two photon excitation
2.: holographic light patterning
3.: temporal focusing

Summary of lab's interests

The research interest of the Emiliani's team focuses on the development of advanced optical methods to mimic and image the complexity of signal transmission in the brain. In particular, the group is focalized on a specific class of techniques where light patterning is engineered via phase modulation of the wave-fronts. The team activity is organized in three research lines: Spatiotemporal control of neuronal activity by patterned excitation; Super resolution and scanning less microscopy; Microendoscopy for awake animals. The group is one of the pioneers in the use of wave front engineering for high resolution photoactivation of optogenetics molecules (Lutz et al. Nature Methods 2008; Papagiakoumou et al. Nature Methods 2010). This new approach is currently used in a series of collaborative projects including the functional mapping of neuronal ion channels, the analysis of Zebrafish swim circuit, the investigation of short term memory, the study of calcium dynamics in respiratory-related Neurons.

Summary of project

The combination of optical methods with targeted expression of protein-based probes enables the analysis of well-defined neuronal population within intact neuronal circuits and systems. Existing optogenetic tools such as channelrhodopsin and halorhodopsin already provide a well-established control component for optogenetic excitation and inhibition, while genetically-encoded voltage sensitive reporters (VSFP) provide the recording component. Being genetically encoded, VSFPs offer several advantages over other used approaches to monitoring neuronal activity. They can essentially be 'programmed' for selective expression within specific subtypes of neurons or particular regions of the brain, and could be used to chart long-range neural circuits extending over considerable distances. Given the high degree of spatial and temporal resolution displayed by the VSFPs they will prove a useful tool for researchers hoping to understand how patterns of neuronal activity correlate with behavior or physiological changes in the living brain. To date, VSFP imaging has been performed only with single photon (1P) whole field excitation. Whole field excitation is preferable considering that high photon rates are required at the detection side in order to achieve a reasonable signal-to-noise ratio (S/NR) at high temporal resolution. Two-photon excitation (2PE) improves upon 1PE in terms of tissue penetration, phototoxicity and axial resolution. However the limited number of chromophores excitable within a conventional 2PE volume is too small to permit a good S/NR. 2P scanning approaches permit extending the excitation area and therefore increasing the number of collecting photons, however due to the relatively low photo-cycle of VSFP, this approach has limited temporal resolution (given by the scanning and the residence time). In our group, we have recently developed a number of scan-less 1P and 2P techniques for the generation of broad axially confined excitation area based on the a combination of the technique of temporal focusing (TF) for the control of the axial resolution with the principle of digital holography or of generalized phase contrast for the control of lateral light patterning [1-4]. We applied this last approach to activate ChR2 in cultured cells and brain slices enabling for the fist time, 2P excitation of multiple neurons or multiple neuronal compartments separately or together [4]. In this project we propose to use of 2P TF-GPC for the excitation of VSFPs and (for comparison) classical voltage sensitive dyes (VSD). This approach will permit simultaneous excitation of VSFP over larger volumes without deterioration of temporal and axial resolution. The system will be first validated in primary neuronal cultures. Cultured neurons will be transfected with plasmids encoding VSFPs as described in [5]. Once the optimal parameter will be found (excitation wavelength, power and excitation spot size) experiments will be repeated in brain slices. Gene transfer in intact tissue will be done by either by in utero electroporation [5], viral delivery or using transgenic mice that express VSFPs. These experiments will be done in close collaboration with Thomas Knopfel RIKEN Brain Science Institute, Japan. For applications in brain slices, a further challenge needs to be faced which is to image functional responses from 3D processes. It is know that neuronal processes are rarely confined to a single plane; following signal propagation along 3D processes will therefore require the use of axial scanning with inherent loss in temporal resolution. Here we propose to do it with the technique of remote focusing [6] recently implemented in our group to perform scan-less functional imaging along tilted planes [7]. This research project will give to the candidate the opportunity to acquiring complementary skills ranging from non linear optics to neurophysiology.

1. C. Lutz, et al. Nat Methods 5, 821 (2008)
2. M. Zahid, et al. PLoS One 5, e9431 (2010)
3. E. Papagiakoumou, et al. Optics Express 16, 22039 (2008)
4. E. Papagiakoumou, et al. Nat Methods 7, 848 (2010)
5. W. Akemann et al. Nat Methods 7, 643 (2010)
6. E. J. Botcherby et al.Optics Letters 32, 2007 (2007)
7. F. Anselmi et al.V. Emiliani, in preparation (2011)