Laurent Bourdieu

Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors

Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

Constrained synaptic connectivity in functional mammalian neuronal networks grown on patterned surfaces

The use of ordered neuronal networks in vitro is a promising approach to study the development and the activity of small neuronal assemblies. However, in previous attempts, sufficient growth control and physiological maturation of neurons could not be achieved. Here we describe an original protocol in which polylysine patterns confine the adhesion of cellular bodies to prescribed spots and the neuritic growth to thin lines. Hippocampal neurons in these networks are maintained healthy in serum free medium up to 5 weeks in vitro. Electrophysiology and immunochemistry show that neurons exhibit mature excitatory and inhibitory synapses and calcium imaging reveals spontaneous activity of neurons in isolated networks. We demonstrate that neurons in these geometrical networks form functional synapses preferentially to their first neighbors. We have, therefore, established a simple and robust protocol to constrain both the location of neuronal cell bodies and their pattern of connectivity. Moreover, the long term maintenance of the geometry and the physiology of the networks raises the possibility of new applications for systematic screening of pharmacological agents and for electronic to neuron devices.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

Late Emergence of the Vibrissa Direction Selectivity Map in the Rat Barrel Cortex

In the neocortex, neuronal selectivities for multiple sensorimotor modalities are often distributed in topographical maps thought to emerge during a restricted period in early postnatal development. Rodent barrel cortex contains a somatotopic map for vibrissa identity, but the existence of maps representing other tactile features has not been clearly demonstrated. We addressed the issue of the existence in the rat cortex of an intrabarrel map for vibrissa movement direction using in vivo two-photon imaging. We discovered that the emergence of a direction map in rat barrel cortex occurs long after all known critical periods in the somatosensory system. This map is remarkably specific, taking a pinwheel-like form centered near the barrel center and aligned to the barrel cortex somatotopy. We suggest that this map may arise from intracortical mechanisms and demonstrate by simulation that the combination of spike-timing-dependent plasticity at synapses between layer 4 and layer 2/3 and realistic pad stimulation is sufficient to produce such a map. Its late emergence long after other classical maps suggests that experience-dependent map formation and refinement continue throughout adult life.

A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view.

Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.

Measuring aberrations in the rat brain by coherence-gated wavefront sensing using a Linnik interferometer

Aberrations limit the resolution, signal intensity and achievable imaging depth in microscopy. Coherence-gated wavefront sensing (CGWS) allows the fast measurement of aberrations in scattering samples and therefore the implementation of adaptive corrections. However, CGWS has been demonstrated so far only in weakly scattering samples. We designed a new CGWS scheme based on a Linnik interferometer and a SLED light source, which is able to compensate dispersion automatically and can be implemented on any microscope. In the highly scattering rat brain tissue, where multiply scattered photons falling within the temporal gate of the CGWS can no longer be neglected, we have measured known defocus and spherical aberrations up to a depth of 400 µm.

Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.

A radial map of multi-whisker correlation selectivity in the rat barrel cortex

In the barrel cortex, several features of single-whisker stimuli are organized in functional maps. The barrel cortex also encodes spatio-temporal correlation patterns of multi-whisker inputs, but so far the cortical mapping of neurons tuned to such input statistics is unknown. Here we report that layer 2/3 of the rat barrel cortex contains an additional functional map based on neuronal tuning to correlated versus uncorrelated multi-whisker stimuli: neuron responses to uncorrelated multi-whisker stimulation are strongest above barrel centres, whereas neuron responses to correlated and anti-correlated multi-whisker stimulation peak above the barrel–septal borders, forming rings of multi-whisker synchrony-preferring cells.

Fast optimization wavefront shaping with acousto-optic deflectors (Conference Presentation)

Since a decade, wavefront shaping techniques has allowed to coherently manipulate speckle patterns. It opens the possibility to focus light through complex media and ultimately to image in them, provided that the medium can be considered as stationary during the process. However, scattering by tissues evolves over millisecond timescales, creating a fast decorrelation of the speckle pattern, thus limiting the use of this technique for in vivo microscopy. Therefore, focusing through biological tissues requires fast wavefront shaping devices, sensors and algorithms. It has been demonstrated by Akemann et al that an Acousto-Optic Deflector (AOD) time locked on the output laser pulses of a regenerative amplifier can be used as an arbitrary 1D beam shaper: the locally modulated acousto-optic phase grating allows the spatial control of the laser pulse wavefront, with refresh rate of several tens up to several hundreds of kHz, limited by the size of the AOD aperture. We have investigated through simulations and experiments, the use of two crossed AODs to implement 2D spatial wavefront shaping, and perform focusing by optimization through a scattering media. We have used different algorithms adapted to this grating modulator and analyzed in each case the AOD bandwidth used, the speed of convergence and the maximum intensity enhancement. In particular, we have shown that two crossed 1D modulators provide larger enhancement than a single 2D wavefront shaper with the same number of pixels. We will present our latest results towards achieving the ultimate optimization, limited by the AOD speed of 40 kHz.