Pál Maák

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation–evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.

Random access three-dimensional two-photon microscopy

We propose a two-photon microscope scheme capable of real-time, three-dimensional investigation of the electric activity pattern of neural networks or signal summation rules of individual neurons in a 0.6 mm x 0.6 mm x 0.2 mm volume of the sample. The points of measurement are chosen according to a conventional scanning two-photon image, and they are addressed by separately adjustable optical fibers. This allows scanning at kilohertz repetition rates of as many as 100 data points. Submicrometer spatial resolution is maintained during the measurement similarly to conventional two-photon microscopy.

Improved design method for acousto-optic light deflectors

Abstract The purpose of this work is to find a reliable approach for one- and two-dimensional acousto-optic deflector design. The design method is based on an improved theoretical model of anisotropic acousto-optic interaction. The new feature of this model is the application of new approximations together with the inclusion of effects that were previously neglected, such as optical beam divergence, second order diffraction and optical activity. Configurations for maximum efficiency and maximum resolution are shown. Optimization of the two-dimensional deflector design is presented. The reliability of the model is demonstrated at wavelengths of 633 and 1064 nm through experimental results with devices designed based on the new model for TeO 2 deflectors.

Realization of true-time delay lines based on acoustooptics

A true-time optical delay line for short radiofrequency (RF) pulses using path length dispersion is proposed. It is an optical implementation of the linear phase-shift theorem of the Fourier transformation. Acoustooptic signal processing is used for conversion into the optical frequency domain and for spatial Fourier decomposition of the pulse. The processing of the pulse is obtained by differentially phase shifting the particular frequency components, followed by a heterodyne reconversion into the RF domain. The optical system is intended to be used for delaying, but also for shaping and filtering of RF pulses, mainly in phased array radar antennas. Theoretical analysis of the system principle is given together with experimental results, demonstrating 2-/spl mu/s time delay of 0.5-/spl mu/s-long pulses with maximum optical phase shift of 1.2/spl pi/. A detailed theoretical and experimental bandwidth analysis is carried out, pointing to the main technical problems and their solutions.

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals

Summary Understanding neural computation requires methods such as 3D acousto-optical (AO) scanning that can simultaneously read out neural activity on both the somatic and dendritic scales. AO point scanning can increase measurement speed and signal-to-noise ratio (SNR) by several orders of magnitude, but high optical resolution requires long point-to-point switching time, which limits imaging capability. Here we present a novel technology, 3D DRIFT AO scanning, which can extend each scanning point to small 3D lines, surfaces, or volume elements for flexible and fast imaging of complex structures simultaneously in multiple locations. Our method was demonstrated by fast 3D recording of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements, including in behaving animals. Finally, a 4-fold improvement in total excitation efficiency resulted in about 500 × 500 × 650 μm scanning volume with genetically encoded calcium indicators (GECIs).

Thermal behavior of acousto-optic devices: Effects of ultrasound absorption and transducer losses

In the present paper we analyze the electric and acoustic losses in acousto-optic devices, especially in their ultrasonic transducers and the related thermal effects. We include electric and acoustic losses into the classical electric equivalent model of the transducer, to explain the characteristics of the measured electric and thermal behavior. Measured temperature distributions on the acousto-optic crystal faces serve visualization of the conversion efficiency of the radio-frequency input to bulk acoustic waves. We show that the pronounced acoustic frequency dependence of the temperature distribution is in correlation with the frequency dependent losses in the transducer and in the bulk. We also demonstrate experimentally the effectiveness of our active and passive heat removing and temperature stabilization methods.

Application of the fast-Fourier-transform-based volume integral equation method to model volume diffraction in shift-multiplexed holographic data storage

Numerical simulation of diffraction on thick holographic gratings in shift-multiplexed optical data storage application is presented. The grating is generated by the interference of a spherical reference wave and a plane signal wave corresponding to a single pixel of the input data page. To describe diffraction on this weak-index-modulated grating, we use the volume integral equation in the first Born approximation. This description yields a convolution integral that can be efficiently evaluated by a 3D fast Fourier transform (FFT) technique. For a 51.2 microm recording layer thickness, a serial-divided single personal computer code was built based on parallel FFT coding principles. Diffracted electric field and Poynting-vector distributions are calculated for probe beams spatially shifted with respect to the reference beams. The shift selectivity curves show significant differences from previous analytical calculations based on paraxial propagation and infinite gratings, as they have monotonic decrease in all three directions instead of sinclike functions with Bragg nulls. With the chosen numerical aperture of 0.6 and linear polarization, both the scalar and vector calculations provided similar results within 5%.

Complex, 3D modeling of the acousto-optical interaction and experimental verification.

The acousto-optical crystals are frequently used, indispensable elements of high technology and modern science, and yet their precise numerical description has not been available. In this paper an accurate, rapid and quite general model of the AO interaction in a Bragg-cell is presented. The suitability of the simulation is intended to be verified experimentally, for which we wanted to apply the most convincing measurement methods. The difficulty of the verification is that the measurement contains unknown parameters. Therefore we performed an elaborated series of measurements and developed a method for the estimation of the unknown parameters.

Combination of a 2-D acousto-optic deflector with laser amplifier for efficient scanning of a Q-switched ND:YAG laser

Abstract A two-dimensional acousto-optic deflector has been combined with a large angular acceptance, laser diode-pumped Nd:YAG optical amplifier in order to obtain a scanning system with high angular resolution and with high and uniform optical transmission. Experiments have been carried out in order to optimize the set-up for intensity distribution and optical losses. The combination of newly developed nonlinear and active optical elements provides a relatively uniform intensity distribution over the scanned region corresponding to 300×300 discrete points in the back focal plane of a Fourier lens, at laser pulse energy levels of 1–5 mJ.