This paper published in Nature Methods demonstrates the capabilities of the Femto3D-AO microscope developed by Gergely Katona et al. The acousto-optic two photon microscope is equipped with an electrically tunable lens focusing the excitation point very fast to any locations in 3D under the objective without mechanical restrictions. Scanning can be done in a near-cubic-millimeter range with high spatial and sub-millisecond temporal resolution. The paper describes two scanning strategies implemented on the system: continuous 3D trajectory scanning allows to precisely follow neural processes over several hundred micrometers to image the propagation of action potentials or dendritic spikes. Random access scanning mode supports imaging evoked or spontaneous calcium-activity in hundreds of neurons simultaneously in vivo.

Katona et al, Nature Methods (2012)


Katona et al introduce an innovative technique called 3D trajectory scanning or Roller Coaster scanning using galvanometer based two-photon microscope equipped with fast piezo objective positioner. The method enables measurements of neuronal calcium signals in long dendritic segments and individual spines. By 3D trajectory scanning, they detected and characterised network activity driven dendritic (i.e., NMDA) spikes generated by nonlinear synaptic integration in hippocampal CA1 stratum radiatum interneurons (RAD INs).

Katona et al, PNAS (2011)


Kerekes et al combined electrophysiological recording with two-photon imaging to study human neocortical slices prepared from epileptic and tumor patients and maintained in dual perfusion chamber. They demonstrated that calcium dynamics of human neurons measured in whole-cell, LFP and intracellular calcium levels, are comparable to those found in animals, and the combined method allows simultaneous optical and electrophysiological examination of large neuronal populations, individual neurons and neuronal assemblies with high spatial and temporal resolutions.

Kerekes et al, Neurophotonics (2014)


Roska’s lab (FMI, Basel) mapped the presynaptic network of a V1 orientation selective neuron by using a single-cell-iniciated, monosynaptically restricted, retrograde transsynaptic network tracing with rabies viruses. The labeled neurons expressed GCaMP6s, which allowed to image, in vivo, the visual motion-evoked activity of individual layer 2/3 pyramidal neurons and their whole presynaptic networks across layers in the primary visual cortex of the mice. For the measurement of this large number of neurons scattered across the  width of the cortex they used a Femto3D-AO microscope. They found that the neurons within each layer exhibited similar motion direction preferences, forming layer-specific functional modules. In one third of the networks, the layer modules were locked to the direction preference of the postsynaptic neuron, whereas for other networks the direction preference varied by layer.

Wertz et al, Science (2015)


Zheng et al demonstated in Neuron, that mapped basal Ca2+ in neurons and astrocytes with submicron resolution helps to unveil heterogeneous concentration landscapes that depend on age and preceding activity of the bain. Monitoring nanomolar-scale molecular interaction between OGB1 nanomolar sensitivity dye and Ca2+ was performed by Femto2D-Galvo equipped with FLIM module.

Zheng et al, Neuron, 2015

Marko A. Popovic et al showed new results about electrical behavior of dendritic spines in Nature Communications. The goal of their research was to generate and measure the spread of input signals from the head of an individual spine to the dendrite and in parallel to reveal the role and influence of the neck of the spine. To activate synapses they applied glutamate iontophoresis and two-photon glutamate uncaging. The uncaging experiments were performed with the commonly used MNI glutamate and a new compound, DNI-Glu which is around 7 times more efficient than the former, even in lower concentrations. The evoked electrical signals (EPSPs) were monitored by electrochromic voltage-sensitive dye simultaneously in the spine and the dendrite. The study provides direct evidence that the resistance of the spine neck is too low to electrically isolate synapses on spine heads, so the synapses of the head electrically behave as if they were located directly on the dendrites.

Popovic et al, NatCommun (2015)


A precisely controlled model of brain injury induced by cerebral ischemia was developed combined with fast in vivo two-photon calcium imaging and selective microglial manipulation revealing the influence of microglia on neuronal activity and survival in the injured brain. According to the model, selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases. All two-photon experiments were performed on a Femto2D-Dual microscope.

Szalay et al, NatCommun (2016)


Understanding neural computation requires methods that can simultaneously read out activity on both somatic and dendritic scales. As shown by Katona el al. AO point scanning can effectively record fluorescent signal from up to 1000 points from in vitro preparation or from anesthetized animal. But the maximal scanning rate is limited by the switching time of the AO deflectors. In this article presents 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 around our ROIs. The scanning abilities were 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. Also, when measuring from awake, behaving animals relatively big motion artifacts can occur caused by vessel pulsing, respiration or locomotion. Extending the ROIs by with maintained temporal resolution allows of line motion compensation and the elimination of most of the motion artifacts. All two-photon experiments were performed with Femto3D-AcoustoOpctic microscope.

Szalay et al, Neuron (2016)


Using fast 3D two-photon imaging (Femto3D-AO) in combination with electrophysiological recordings and a caged glutamate compound (DNI-Glu-TFA), Chiovini et al demonstrated that a single activation of clustered glutamatergic inputs in the distal dendrites of FS-PV INs, which generate a depolarizing hump and reproduce the hot spots associated with spontaneous SPW-R events, is capable of generating secondary membrane oscillations in the ripple frequency range.

Chiovini et al, Neuron (2014)


Scientific papers


Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function. Anne-Kathrin Theis, Balázs Rózsa, Gergely Katona, Dietmar Schmitz1 and Friedrich W. Johenning, Front. Cell. Neurosci. (2018)

Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic Spines. Marina Mikhaylova, Julia Bär, Bas van Bommel, Philipp Schätzle, PingAn YuanXiang, Rajeev Raman, Johannes Hradsky, Anja Konietzny, Egor Y. Loktionov, Pasham Parameshwar Reddy, Jeffrey Lopez-Rojas, Christina Spilker, Oliver Kobler, Syed Ahsan Raza, Oliver Stork, Casper C. Hoogenraad, Michael R. Kreutz, Neuron (2018)

Precisely Timed Nicotinic Activation Drives SST Inhibition in Neocortical Circuits. Joanna Urban-Ciecko, Jean-Sebastien Jouhanneau, Stephanie E. Myal, James F.A. Poulet, Alison L. Barth, Neuron (2018)

Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging. Kaiyu Zheng, Thomas P Jensen & Dmitri A Rusakov, Nature Protocols (2018)

Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells. Claudio Elgueta, Felix Leroy, Alex H. Vielma, Oliver Schmachtenberg & Adrian G. Palacios, Nature Scientific Reports (2018)

Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease. Gyula Batta, Lilla Soltész, Tamás Kovács, Tamás Bozó, Zoltán Mészár, Miklós Kellermayer, János Szöllősi & Peter Nagy, Nature Scientific Reports (2018)

Nano-engineered microcapsules boost the treatment of persistent pain. Olga Kopach, Kayiu Zheng, Luo Dong, Andrei Sapelkin, Nana Voitenko, Gleb B. Sukhorukov & Dmitri A. Rusakov, Drug Delivery (2018)

High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis. Denes Palfi, Balazs Chiovini, Gergely Szalay, Attila Kaszas, Gergely F. Turi, Gergely Katona, Peter Abranyi-Balogh, Milan Szori, Attila Potor, Orsolya Frigyesi, Csilla Lukacsne Haveland, Zoltan Szadai, Miklos Madarasz, Aniko Vasanits-Zsigrai, Iboly Molnar-Perl, Bela Viskolcz, Imre G. Csizmadia, Zoltan Mucsi and Balazs Rozsa, Organic & Biomolecular Chemistry (2018)


Cell-type–specific inhibition of the dendritic plateau potential in striatal spiny projection neurons. Kai Du, Yu-Wei Wu, Robert Lindroos, Yu Liu, Balázs Rózsa, Gergely Katona, Jun B. Ding, and Jeanette Hellgren Kotaleski, PNAS (2017)

Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging. Dal Maschio M, Donovan JC, Helmbrecht TO, Baier H, Neuron (2017)

An optogenetic toolbox for unbiased discovery of functionally connected cells in neural circuits. Dominique Förster, Marco Dal Maschio, Eva Laurell, and Herwig Baier, Nature Communication (2017)

Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue. Jensen TP, Zheng K, Tyurikova O, Reynolds JP, Rusakov DA, Cell Calcium (2017)

Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex. Daniel Hillier, Michele Fiscella, Antonia Drinnenberg, Stuart Trenholm, Santiago B Rompani, Zoltan Raics, Gergely Katona, Josephine Juettner, Andreas Hierlemann, Balazs Rozsa & Botond Roska, Nature Neuroscience (2017)

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo. Szabo Z, Heja L, Szalay G, Kekesi O, Furedi A, Szebenyi K, Dobolyi A, Orban T, Kolacsek O, Tompa T, Miskolczy Z, Biczok L, Rozsa B, Sarkadi B, Kardos J., Sci Rep (2017)

Imaging membrane potential changes from dendritic spines using computer-generated holography. Tanese D, Weng JY, Zampini V, De Sars V, Canepari M, Rozsa B, Emiliani V, Zecevic D., Neurophotonics (2017)


Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals. Gergely Szalay, Linda Judak, Gergely Katona, Katalin Ocsai, Gabor Juhasz, Mate Veress, Zoltan Szadai, Andras Feher, Tamas Tompa, Balazs Chiovini, Pal Maak, Balazs Rozsa, Neuron (2016)

Temporal asymmetries in auditory coding and perception reflect multi-layered nonlinearities. Thomas Deneux, Alexandre Kempf, Aurélie Daret, Emmanuel Ponsot, and Brice Bathellier, Nature Communications (2016)

Accurate spike estimation from noisy calcium signals for ultrafast three-dimensional imaging of large neuronal populations in vivo. Thomas Deneux, Attila Kaszas, Gergely Szalay, Gergely Katona, Tamas Lakner, Amiram Grinvald, Balazs Rozsa & Ivo Vanzetta, Nature Communications (2016)

Astrocytic GABA transporter activity modulates excitatory neurotransmission. Boddum K, Jensen TP, Magloire V, Kristiansen U, Rusakov DA, Pavlov I, Walker MC, Nature Communications (2016)

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Szalay G, Matinecz B, Lenart B, Kornyei Z, Orsolits B, Judak L, Csaszar E, Fekete R, West BL, Katona G, Rozsa B, Denes A, Nature Communications (2016)

Correlated Synaptic Inputs Drive Dendritic Calcium Amplification and Cooperative Plasticity during Clustered Synapse Development. Kevin F.H. Lee, Cary Soares, Jean-Philippe Thivierge, Jean-Claude Béïque, Neuron (2016)

Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus. Jeffrey Lopez-Rojas, Martin Heine, Michael R. Kreutz, Nature (2016)


A Visual Pathway for Looming-Evoked Escape in Larval Zebrafish. Incinur Temizer, Joseph C. Donovan, Herwig Baier, Julia L. Semmelhack, Curr Biol (2015)

In Vivo Monosynaptic Excitatory Transmission between Layer 2 Cortical Pyramidal Neurons. Jean-Sebastien Jouhanneau, Jens Kremkow, Anja L. Dorrn, James F.A. Poulet, Cell Reports (2015)

Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes. Williamson A, Ferro M, Leleux P, Ismailova E, Kaszas A, Doublet T, Quilichini P, Rivnay J, Rozsa B, Katona G, Bernard C, Malliaras GG., Advanced Materials (2015)

Matching Cell Type to Function in Cortical Circuits Luc Estebanez, Jens Kremkow, James F.A. Poulet, Neuron (2015)

Time-Resolved Imaging Reveals Heterogeneous Landscapes of Nanomolar Ca2+ in Neurons and Astroglia Kaiyu Zheng, Lucie Bard, James P. Reynolds, Claire King, Thomas P. Jensen, Alexander V. Gourine, Dmitri A. Rusakov, Neuron (2015)

Electrical behaviour of dendritic spines as revealed by voltage imaging. Marko A. Popovic, Nicholas Carnevale, Balazs Rozsa; Dejan Zecevic, Nature Communications (2015)

Tonic endocannabinoid-mediated modulation of GABA release is independent of the CB1 content of axon terminals Nora Lenkey, Tekla Kirizs, Noemi Holderith, Zoltan Mate, Gabor Szabo, E. Sylvester Vizi, Norbert Hajos, Zoltan Nusser, Nature Communications (2015)

Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules Wertz A, Trenholm S, Yonehara K, Hillier D, Raics Z, Leinweber M, Szalay G, Ghanem A, Keller G, Rozsa B, Conzelmann KK, Roska B, Science (2015)

Unitary GABAergic volume transmission from individual interneurons to astrocytes in the cerebral cortex. Rozsa M, Baka J, Borde S, Rozsa B, Katona G, Tamas G, Brain Struct Funct. (2017)

A Calcium-Dependent Mechanism of Neuronal Memory Gabriel Gasque, PLoS Biology Journal (2015)

Ryanodine Receptor Activation Induces Long-Term Plasticity of Spine Calcium Dynamics Friedrich W. Johenning, Anne-Kathrin Theis, Ulrike Pannasch, Martin Rückl, Sten Rüdiger, Dietmar Schmitz, PLoS Biology Journal (2015)

Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling Igor Delvendahl, Lukasz Jablonski, Carolin Baade, Victor Matveev, Erwin Neher, and Stefan Hallermann, PNAS (2015)

Quantitation of various indolinyl caged glutamates as their o-phthalaldehyde derivatives by high performance liquid chromatography coupled with tandem spectroscopic detections: derivatization, stoichiometry and stability studies.Vasanits-Zsigrai A, Majercsik O, Toth G, Csampai A, Haveland-Lukacs C, Palfi D, Szadai Z, Rozsa B, Molnar-Perl I, J Chromatogr A. (2015)

Molecular Tattoo: Subcellular Confinement of Drug Effects Miklos Kepiro, Boglarka H. Varkuti, Anna A. Rauscher, Miklos S.Z. Kellermayer, Mate Varga, Andras Malnasi-Csizmadia, CellPress (2015)

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin Mouloud Bouhadfane, Attila Kaszas, Balazs Razsa, Ronald M Harris-Warrick, Laurent Vinay, Frederic Brocard, eLife (2015)

Joint CP-AMPA and group I mGlu receptor activation is required for synaptic plasticity in dentate gyrus fast-spiking interneurons. Thomas Hainmüller, Kerstin Krieglstein, Akos Kulik, and Marlene Bartos, PNAS (2015)

Local Postsynaptic Voltage-Gated Sodium Channel Activation in Dendritic Spines of Olfactory Bulb Granule Cells Wolfgang G. Bywalez, Dinu Patirniche, Vanessa Rupprecht, Martin Stemmler, Andreas;V.M. Herz, Denes Palfi, Balazs Rozsa, Veronica Egger, Neuron (2015)

MRZ-99030 - A novel modulator of Aβ aggregation: II - Reversal of Aβ oligomer-induced deficits in long-term potentiation (LTP) and cognitive performance in rats and mice. Rammes G, Gravius A, Ruitenberg M, Wegener N, Chambon C, Sroka-Saidi K, Jeggo R, Staniaszek L, Spanswick D, O'Hare E, Palmer P, Kim EM, Bywalez W, Egger V, Parsons CG, Neuropharmacology (2015)


Cortical fosGFP Expression Reveals Broad Receptive Field Excitatory Neurons Targeted by POm Jean-Sebastien Jouhanneau, Leiron Ferrarese, Luc Estebanez, Nick J. Audette, Michael Brecht, Alison L. Barth, James F.A. Poulet, Neuron (2014)

Ultrafast Action Potentials Mediate Kilohertz Signaling at a Central Synapse. Andreas Ritzau-Jost, Igor Delvendahl, Annika Rings, Niklas Byczkowicz, Harumi Harada, Ryuichi Shigemoto, Johannes Hirrlinger, Jens Eilers ,Stefan Hallermann, Neuron (2014)

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. B Chiovini, G F Turi, G Katona, A Kaszas, D Palfi, P Maak, G Szalay, M F Szabo, Z Szadai, Sz Kali and B Rozsa, Neuron (2014)

Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Balint Peter Kerekes, Kinga Toth, Attila Kaszas, Balazs Chiovini, Zoltan Szadai, Gergely Szalay, Denes Palfi, Attila Bago, Klaudia Spitzer, Balazs Rozsa, Istvan Ulbert, Lucia Wittner, Neurophotonics (2014)

Spine neck plasticity regulates compartmentalization of synapses J Tønnesen, G Katona, B Rozsa, U V Nägerl, Nature Neuroscience (2014)


Role of RIM1α in short- and long-term synaptic plasticity at cerebellar parallel fibres. Kintscher M, Wozny C, Johenning FW, Schmitz D, Breustedt J., Nature Communications (2013)

The RIP1-Kinase Inhibitor Necrostatin-1 Prevents Osmotic Nephrosis and Contrast-Induced AKI in Mice Andreas Linkermann, Jan-Ole Heller, Agnes Prokai, Joel M. Weinberg, Federica De Zen, Nina Himmerkus, Attila J. Szabo, Jan H. Bräsen, Ulrich Kunzendorf and Stefan Krautwald, JASN (2013)

Differential Subcellular Targeting of Glutamate Receptor Subtypes during Homeostatic Synaptic Plasticity, Cary Soares, Kevin F. H. Lee, Wissam Nassrallah and Jean-Claude Béïque, The Journal of Neuroscience (2013)

Silicon carbide quantum dots for bioimaging, David Beke, Zsolt Szekrenyes, Denes Palfi, Gergely Rona, Istvan Balogh, Pal Andor Maak, Gergely Katona, Zsolt Czigany, Katalin Kamaras, Balazs Rozsa, Laszlo Buday, Beata Vertessy and Adam Gali, Journal of Materials Research, (2013)


Release probability of hippocampal glutamatergic terminals scales with the size of the active zone, Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe & Zoltan Nusser, Nature Neuroscience (2012)

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes, Gergely Katona, Gergely Szalay, Pál Maak, Attila Kaszas, Mate Veress, Daniel Hillier, Balazs Chiovini, E Sylvester Vizi, Botond Roska & Balazs Rozsa, Nature Methods (2012)

Experimental evidence for sparse firing in the neocortex Alison L. Barth, James F.A. Poulet, Trends in Neurosciences, (2012)


Complementary Sensory and Associative Microcircuitry in Primary Olfactory Cortex, HF Wiegand, P Beed, MHK Bendels, C Leibold, D Schmitz, FW. Johenning, J. Neurosci (2011)

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons, G. Katona, A. Kaszas, G. F. Turi, N. Hajos, G. Tamas, E.S. Vizi, B. Rozsa, PNAS (2011)

Synaptic Mechanisms Underlying Sparse Coding of Active Touch Crochet S, Poulet JF, Kremer Y, Petersen CC., Neuron (2011)


Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity, B. Chiovini, GF. Turi, G. Katona, A. Kaszas, F. Erdelyi, G. Szabo, H. Monyer, A. Csakanyi, ES. Vizi, B. Rozsa, Neurochemical Research (2010)

An Embedded Subnetwork of Highly Active Neurons in the Neocortex, L. Yassin, BL. Benedetti, JS. Jouhanneau, JA. Wen, JFA. Poulet, AL. Barth, Neuron (2010)


Differential distribution of NCX1 contributes to spine-dendrite compartmentalization in CA1 pyramidal cells. A. Lorincz, B. Rozsa, G. Katona, ES. Vizi, G. Tamas, PNAS (2007)

Random access 3D two-photon microscopy, B. Rozsa, G. Katona, ES. Vizi, Z. Varallyay, A Saghy, L. Valenta, P. Maak, J. Fekete, A. Banyasz, R. Szipocs, Applied Optics (2007)

Dendritic nicotinic receptors modulate Backpropagating action potentials and long-term plasticity of interneurons. B. Rozsa, G. Katona, A. Kaszas, R. Szipocs, ES Vizi, Eur J Neurosci. (2007)