Date : 10/4/2010

Laboratory

Laboratory of Neurophysiology and new microscopy
Inserm U603
45 rue des Saints-Pères 75006 paris
Director : Serge Charpak

PhD Supervisor

Serge Charpak
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : +33 142864148

Subjects / Tools-Methodologies

1 : Development of a new microscope/two photon imaging, optics
2 : Neuronal network activity/imaging calcium and spikes
3 : Olfactory bulb/odor

Summary of lab's interests

The research objective of the team is to investigate several aspects of glomerular neurophysiology in the rodent, combining electrophysiological recording and two-photon imaging, both in vivo and in vitro. We investigate functional hyperaemia in the olfactory bulb by addressing the differential roles of neurones, astrocytes and pericytes during odor. Because we are convinced that blood flow regulation cannot be understood without determining precisely which neuronal networks are mobilised, we also study intrinsic and synaptic properties of glomerular neurones. Finally, and it is the topic of the present project, we maintain an activity in technological developments, in particular by improving the acquisition rate of two-photon imaging with the goal of recording multiple spikes, simultaneously and in depth.

Summary of project

Two-photon microscopy is now commonly used in neurophysiology. A old but challenging task that has not been achieved yet, consists in recording spike activity in a large number of identified neurons, Controlling precisely the activity of an entire small neuronal network is an essential step in the study of cerebral function. Two-photon microscopy of fast fluorescent activity reporters provides imaging of neuronal activity with a micrometric spatial resolution in vivo, and up to a depth of 500m.
A drawback of conventional two-photon microscopes is the inertia of the mechanical scanners leading to a slow image acquisition rate. Various attempts have been made to overcome this issue. For example, Lillis et al [1] have described a targeted path scanning approach for monitoring the dynamics of few spatially extended neurons. A more efficient method employs acousto-optic deflectors to quasi-instantaneously scan the excitation beam between specific cells. [2] [3] Other techniques involve the use of array detectors.[4] [5] However the resulting increase in acquisition speed achieved by any of these techniques is detrimental to the image quality in terms of either signal to noise ratio, field of view, point spread function (PSF) or imaging depth.
We have designed a novel two photon microscope able to image multiple sites simultaneously at temporal resolution in the order of 1ms without loss in imaging depth and spatial resolution. This technique is based on (1) the separation of a laser beam into multiple sub-beams each targeted to user-defined sites (or cells), (2) the amplitude modulation of each sub-beam with a different temporal pattern, (3) the collection of fluorescence signals from all sites onto a single fast and high sensitivity photodetector, and (4) the demodulation of signals to extract the fluorescence emitted at each site. A proof of principle was already achieved on non-biological samples at low frequencies leading to a patent taken at the European patent office.
The PhD student will be responsible for (1) the construction and alignment of the optical system, (2) the development of a user friendly interface to drive it, (3) the characterization of its performances in brain tissue, (4) the optimization of its functionalities in particular by testing various methods of amplitude modulation (fast SLM, standard and custom made DMD), (5) testing and using the microscope to record the activity of a small neuronal network, in collaboration with a neuroscientist.
The student should be highly motivated by working at the interface between physics and biology. He/she should have a background in either optics, programming (preferentially in LabView, Matlab or C++), or electronics, signal and image processing.

References
1. Lillis, K.P., et al., Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution. J Neurosci Methods, 2008. 172(2): p. 178-84.
2. Otsu, Y., et al., Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope. J Neurosci Methods, 2008. 173(2): p. 259-70.
3. Reddy, G.D. and P. Saggau, Fast three-dimensional laser scanning scheme using acousto-optic deflectors. J Biomed Opt, 2005. 10(6): p. 064038.
4. Nikolenko, V., et al., SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators. Front Neural Circuits, 2008. 2: p. 5.
5. Niesner, R., et al., The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. Biophys J, 2007. 93(7): p. 2519-29.