Máté Veress

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.

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).

[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level].

INTRODUCTION Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials. AIM The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner. METHOD Authors developed new data acquisition methods and microscope hardware. RESULTS Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fluorescence transients. On the same principle, an acousto-optical deflector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access scanning of Ca(2+) transients in hundreds of neurons in the mouse visual cortex. CONCLUSIONS Region of interest scanning enables high signal-to-noise ratio and repetition speed, while keeping good depth penetration of the two-photon microscopes.

Modeling of in vivo acousto-optic two-photon imaging of the retina in the human eye

Our aim is to establish a novel combined acousto-optical method for in vivo imaging of the human retina with the two-photon microscope. In this paper we present modeling results based on eye model samples constructed with parameters measured on patients. We used effectively the potential of the electronic compensation offered by the acousto-optic lenses to avoid the use of adaptive optical correction. Simulation predicted lateral resolution between 1.6 µm and 3 µm on the retina. This technology allows the visualization of single cells and promises real time measuring of neural activity in individual neurons, neural segments and cell assemblies with 30-100 µs temporal and subcellular spatial resolution.

Theoretical and experimental analyses of the acoustic-to-optic phase transfer in specific acousto-optic devices

We present a comprehensive study of the acoustic-to-optic phase transfer during anisotropic Bragg diffraction. Our results refine the operating theory of widely used acousto-optic implementations such as pulse shapers, delay lines, and phase modulators.

Experimental and Theoretical Examination of the Phase Transfer from the Acoustic to Optical Waves during Strong Anisotropic Bragg Diffraction in Acousto-Optic Devices

The optical phase of a beam diffracted in an acousto-optic interaction may be directly controlled with the acoustic phase, but limitations occur in the case of commonly used AO devices, based on strong anisotropic AO interaction. In our study we present measurement and theoretical calculation of the phase shift of a laser beam diffracted in an AO deflector. The optical phase is a function of the acoustic and optical frequency and of the relative spatial positions of the interacting beams. We measured the phase shift with a heterodyne setup, where we measured the phase of the heterodyne signal relative to a fixed reference. We found that the phase shift introduced by the AO crystal is linear with the sound frequency, its gradient is of 10-20rad/MHz and depends mainly on the optical beams distance from the transducer. We show that this phase shift can be derived theoretically from the momentum matching condition that must be fulfilled at each optical and acoustic frequency pair.