Network Imaging
All sensations and behaviors are encoded in the dynamic activity patterns of neural networks. In other words, complex networks of many individual neurons respond to environmental features, visual or auditory stimuli, reward, or punishment, etc. Every neural network extends over 3D space and, in most cases, across more than one cortical layer in the brain. Two-photon microscopy has made it possible for neuroscientists to reach the deep regions of the brain (down to 850 µm) and study them at a high spatiotemporal resolution by using various technical solutions.
- imaging of thousands of cells
- maximal scanning speed: 30 kHz
- high SNR
- motion correction in behaving animal models
- simultaneous 3D photostimulation with 3D imaging
- available by the FEMTO3D Atlas
The FEMTO3D Atlas microscope with its exceptional ability enables the precise spatial and real-time complexity of neuronal coding in a neural network to be resolved by scanning a large number of cells distributed in a near cubic millimeter 3D volume.
The imaging of cell populations in 3D is the fastest with FEMTO3D Atlas
3D random-access point scanning, Cell3DFinder
Several thousand cells can be measured near-simultaneously (30 kHz) by 3D random-access point scanning by restricting the imaging to sub-regions of the 3D volume, which results in ultra-high scanning speed and signal-to-noise ratio. The Cell3DFinder software module finds cell centers in the image stack of a 3D neural network automatically and can display the data as a set of points. The figure shows the changes in Ca2+ concentration of 2000 cells from the visual cortex of a GCaMP-expressing mouse as a function of time. See more: Wertz et al., Science, 2015; Katona et al., Nat Meth, 2012.
Imaging of multiple somata distributed in 3D in behaving animals
3D chessboard scanning using 3D Anti-motion technology
Chessboard scanning is a planar extension of random-access point scanning using the Anti-motion technology, where random-access points are extended to small squares by drifting the laser beam. These squares can be located anywhere in a near cubic millimeter volume while including the somata with their surrounding areas. Up to 300 somata can be measured simultaneously. The chessboard arrangement of the squares helps to visualize somata, analyze the data on their activity, and correct for motion artifacts. The video shows in vivo neuronal activity, Ca2+ transients from 100 somata from a neural network in the V1 region of a mouse, labeled with GCaMP6. See also Szalay et al., Neuron, 2016.
Imaging of sparsely distributed interneurons in a large neuronal network during behavior in real-time in 3D
3D chessboard scanning in a large neuronal network
Whole neural networks of even sparsely distributed brain cells (such as interneurons) can be followed in real-time in a 3D volume of nearly a cubic millimeter using chessboard scanning. In the study depicted in the figure below, the calcium activity from nearly 250 interneurons was measured simultaneously, across every CA1 hippocampal layer, in an 800 × 800 × 500 µm volume with the chessboard scanning mode, while the mouse was performing behavioral tasks on a treadmill. Using these recordings, the molecular identity of the imaged cells was also determined from data acquired from post hoc immunohistochemistry. Such a novelty – identifying distinct interneuron types and their activity dynamics simultaneously in a large neural network – could not have been achieved with traditional recording techniques. See more: Geiller et al., Neuron, 2020. Watch the interview with one of the authors.
Imaging of multiple somata during large motions
3D multi-cube scanning using 3D Anti-motion technology
Multi-cube scanning is a spatially extended mode of chessboard scanning, where a Z dimension is added to the aforementioned squares to cover the whole extent of the somata of neurons from any neural network, therefore preserving all fluorescent information even during large amplitude movements. Learn more: Szalay et al., Neuron, 2016.
Fastest frame scanning with rotational freedom
High-speed arbitrary frame scanning
Cells assembled into a neural network can be acquired with a frame rate of 40 Hz in a 500×500 µm field-of-view (FOV) by the High-speed arbitrary frame scanning mode of the Atlas. Beyond the high speed, another important and unique advantage of this mode is that the selected plane can be rotated to any angle in space. Moreover, to cover more cells in the neural network, the scanning plane can be extended into a volume element as an alternative of the Bessel beam technology, which allows you to follow the actions of networks in a 3D tissue.
The video demonstrates the rotation of the selected plane in High-speed arbitrary frame scan mode. The left panel shows the relative position of the objective’s focal plane with blue and the actual two-photon focal plane with pink. The movement of the pink plane exemplifies the possibilities Atlas offers you to orient the plane in a three dimensional tissue. The right panel shows the neurons of a network in the actual scanned plane during rotation in live mode.
FEMTOSmart offers numerous imaging opportunities, and, thanks to its modularity, researchers can study the cells of a neural network from a wide variety of perspectives.
Imaging of somata as ROIs in 2D
FEMTOSmart Galvo with flexible ROI scanning possibilities
The FEMTOSmart Galvo, with its flexible scanning patterns such as 2D random-access point, 2D multiple-line, and folded-frame scanning, supports the manual selection of individual cells in a neural network in a 2D plane. By skipping the measurement of the entire field, it is possible to reach a high scanning speed and maintain a high signal-to-noise ratio. Using the TravellingSalesman additional software module, it is possible to determine the shortest pathway visiting defined points arbitrarily dispersed in the field of view. The short round-trip time results in a higher measurement repetition speed, up to 100 Hz for about 30 cells.
Multiple-line scanning
Fast scanning of the entire FOV or an entire volume
FEMTOSmart Resonant equipped with Piezo objective positioner kit
Fast-frame scanning by FEMTOSmart Resonant combined with fast Z-focusing performed by a Piezo objective positioner kit is a well-known approach for studying any three-dimensional neural network. In this case, the entire field of view is imaged continuously by the fast scanner, while the objective positioner moves between planes. More than one (cortical) layer might be recorded simultaneously this way, or the frames may be assembled into volumes resulting in a four-dimensional set of data.
References
Large-Scale 3D Two-Photon Imaging of Molecularly Identified CA1 Interneuron Dynamics in Behaving Mice Mice Tristan Geiller, Bert Vancura, Satoshi Terada, Eirini Troullinou, Spyridon Chavlis, Grigorios Tsagkatakis, Panagiotis Tsakalides, Katalin Ócsai, Panayiota Poirazi, Balázs J. Rózsa, Attila Losonczy, Neuron (2020)
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)
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)
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)
Dendritic Imaging
Dendrites and dendritic spines are made of fine, thin, and vulnerable processes therefore they are difficult to study. However, performing in vivo imaging with the two-photon laser technology, we are able to collect signals from femtoliter volumes of deeper regions of the brain, while avoiding phototoxicity at the same time. Besides this, spatially confined scanning of axons, dendrites, and spines of a cell as ROIs allows detecting even subthreshold signals with a high signal-to-noise ratio (SNR). We have several configurations with which we are able to visualize dendritic arborization and perform functional measurements under in vivo and in vitro conditions.
With the FEMTO3D Atlas microscope, events less than a millisecond apart can be separated during in vivo imaging, and, therefore, the propagation speed of regenerative activity can be determined at multiple sites of the dendritic tree. High-speed arbitrary frame scanning, 3D random-access point scanning, and its advanced versions support all kinds of functional dendritic imaging.
The video shows a 3D recording of Ca2+ signals at multiple dendritic sites by 3D random-access point scanning. See more in Chiovini et al., Neuron, 2014.
Fast drift along the dendrite
3D trajectory scanning
3D random-access point scanning extended by drifting the focal point along short 3D trajectories allows in vivo imaging without interruption at multiple, long dendritic branches. Imaging is continuous during the drift, so this scanning mode gives a more detailed spatial resolution without changing the overall scanning time: this remains as high as during point scanning. As a result, the function of thin dendritic segments, or even spines and single action potentials can be revealed. See more in Katona et al., Nat Meth, 2012, Chiovini et al., Neuron, 2014.
In vivo imaging of hundreds of spines of a neuron
3D multi-line scanning
The 3D multi-line scanning method is similar to the 3D trajectory scanning; however, it was developed for the in vivo imaging of spines during movements. As it scans along short lines, the spines are still covered by them if the animal model is moving. In the figure, each scanning line is associated with one spine in a layer II/III pyramidal cell labeled with GCaMP6. The direction of the drift is set to meet the average trajectories calculated from brain motion, helping to eliminate motion artifacts. More than 160 pre-selected spines were examined simultaneously during the research, and four representative Ca2+ transients are shown before and after motion correction, demonstrating the improved SNR. Learn more: Szalay et al., Neuron, 2016.
Dendritic and spine activity during small motions
3D ribbon scanning
An extension of 3D multi-line scanning performed by Anti-motion technology is 3D ribbon scanning, which makes it possible to perform in vivo imaging of ribbon-shaped surfaces containing dendrites and their neighboring areas. The video shows 3D ribbons encompassing seven dendritic segments with their spines of a GCaMP6-labeled layer II/III pyramidal neuron measured in vivo within the brain of a behaving mouse. The seven ribbons were projected into a 2D image, ordering dendrites above each other for better visualization, and activity was recorded from 20 selected spines and visualized in the form of classical Ca2+ transients. Learn more: Szalay et al., Neuron, 2016.
Dendritic and spine activity during large motions
3D snake scanning using 3D Anti-motion technology
3D snake scanning is a volume extension of ribbon scanning and contains the entire 3D environment of the dendrite. It, therefore, supports in vivo imaging of dendrites in larger animals, or behavioral protocols, where the amplitude of motion can be large. The video shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal brain cell. Learn more: Szalay et al., Neuron, 2016.
Fast frame scanning of long dendrites
High-speed arbitrary frame scanning gives you the freedom to tilt and rotate the scanning plane in any direction. With this feature, in vivo imaging of long dendrites can be performed in a single plane. To examine multiple neurons’ apical dendrites simultaneously, the tilted frame can be extended into a volume element.
The video shows the apical dendrites of the neurons getting aligned with the scanning plane, which means the spatio-temporal activity patterns of these long segments passing multiple cortical layers can be recorded without requiring lengthy and painstaking optical sectioning.
The fastest systems in 2D in vivo imaging
2D Multiple-Line Scanning by FEMTOSmart GALVO
The adaptable X and Y mirrors of the galvanometric scanner in the FEMTOSmart Galvo, coupled with the special electronic boards from Femtonics, can follow the tortuous protrusions of the dendritic arbor precisely using the multiple-line scanning method. By limiting scanning to the dendritic segments and their spines, and omitting the space between them, both the scanning speed (up to 2 kHz) and the SNR can be increased. The Multiple-line scanning mode is a cost-effective, alternative solution in in vivo imaging, compared to the scanning methods of acousto-optic technology-based systems.
In vivo imaging of dendrites and spines with motion correction
Folded frame scanning by FEMTOSmart GALVO
Using folded frame scanning, the in vivo imaging of an area along a pre-selected line can be performed. The selected regions can take many shapes, from areas around straight lines to complex bent curves. This advanced scanning method is useful for following events along curved dendrites with spines and can also be advantageous for dendritic measurements in behaving animal models, where motion artifacts are a common problem. The images can be motion corrected offline by the control software, as long as the dendrite remains in the scanned area.
Follow dendrites in 3D
3D trajectory scanning by FEMTOSmart GALVO equipped with Piezo Objective Positioner
The microscope equipped with a Piezo Objective Positioner kit and controlled by the Roller Coaster software module enables the user to perform in vivo imaging of dendritic arborization in a 3D sample using 3D trajectory scanning. These tools with this advanced scanning mode enable you to collect signals from a 25 µm range with a 150 Hz speed, which is fast enough to follow changes in the Ca2+-level and to resolve biological functionality through a 3D sample.
References
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)
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)
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)
Matching Cell Type to Function in Cortical Circuits. Luc Estebanez, Jens Kremkow, James F.A. Poulet, Neuron (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)
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)
Voltage Imaging
Keeping up with action potentials
Voltage sensors, combined with the achievable 100kHz sampling rate of the FEMTO3D Atlas, are capable of detecting ultrafast transients, such as action potentials. The FEMTO3DAtlas, equipped with the AO technology and our real-time motion correction feature, is currently the only scanning device which can keep up with the speed of firing neurons!
Voltage imaging with the FEMTO3D Atlas
The rapidly evolving voltage sensor technology will determine the next decade of bioimaging. These molecules have faster kinetics than calcium sensors and can provide superior temporal resolution, capable of differentiating individual action potentials even within bursts.
Fast evolving voltage sensors, combined with the impressive 100kHz sampling rate of the FEMTO3D Atlas acousto-optic (AO) two-photon microscope, are suited for detecting ultrafast transients, such as action potentials in a scanning volume of a cubic millimeter. The Atlas, powered by the AO technology, is currently the only imaging device that can keep up with the speed of firing neurons during complex, in vivo behavioral experiments. Moreover, the novel Femtonics FocusPinner real-time 3D motion correction feature eliminates motion artifacts in real time, and provides an unprecedented signal-to-noise ratio!
Learn more about
- the Femto3D ATLAS multiphoton microscope or about the acousto-optic technology
- network imaging and dendritic imaging, or other applications of the Femto3D ATLAS
- combining the Femto3D ATLAS with behavioral studies
The investigation of neuronal circuit mechanisms requires experimental approaches that give access to the electrical activities of a high number of neurons across a large volume of brain tissue with a temporal resolution consistent with the millisecond timescale of synaptic communication. Our technology makes it possible: contact our team to learn more!
Behavioral studies
Behavioral processes are paired with patterns of brain activation. Mapping the neural basis of a behavioral distinction, such as visuomotor learning, memory retrieval, associative learning or spatial navigation can be established by controlled multiform experimental manipulation. This can be a change in stimulus information, task instruction or reward and punishment which alter the underlying pattern of brain activation. Monitoring or evoking behavioral data and parallel imaging of neuronal circuits in a living animal’s brain can reveal the correspondence between changes at the cellular and behavioral levels.
Our 3D Anti-motion scanning technology by the FEMTO3D Atlas enables cell activity to be captured while an animal is moving in virtual reality and performing tasks. The acousto-optic drift scanning technology is useful for correcting tissue motions caused by behavior. To preserve signals, scanning points are extended to drifted lines which are precisely fitted to each other, resulting in surface or 3D volume elements. These elements cover not only the pre-selected ROIs but also the neighboring areas giving an opportunity to preserve all fluorescent information during motions and decrease the artefacts by more than one order of magnitude in behaving animals.
Surface and volume scanning possibilities supporting motion correction
The scanning modes using 3D Anti-motion technology
The surface scanning methods are optimized for speed, while the methods based on volume imaging are optimized for large amplitude movements, maintaining the 10–1,000 Hz sampling rate necessary to resolve neural activity at the individual ROIs. Each scanning mode is useful for different neurobiological aims: ribbon scanning, snake scanning, 3D multiple-line scanning are optimal for various dendritic measurements, while chessboard scanning and multi-cube scanning are best used for somatic recordings.
Learn more about the 3D Anti-motion technology and fast 3D AO imaging in behaving animals from Szalay et. al, Neuron (2016)
Examples of behavioral neuroscience experiments conducted by the FEMTO3D Atlas, or by the previous versions of the microscope.
Simultaneous recording of neuronal activity and mouse behavior during visual stimulation in a virtual reality (VR) environment
The top panel shows the schematic diagram of the experiment. Two-photon imaging was performed by using the 3D Multiple-line scanning mode of the FEMTO3D Atlas, while the mouse was placed in a virtual reality setup, where the environment changed accordig to mouse’s movements. At specific locations, the animal received a reward (reward zone) or a punishment (aversive zone). The bottom panel demonstrates the selected neurons and location of imaging and their calcium activity aligned with the collected motion data. Aversive and reward zones are marked with red and green, respectively.
Ca2+ imaging of VIP interneurons during an auditory discrimination task
Left panel: schematic diagram of the experiment. VIP interneurons were imaged by the Chessboard scanning mode of the FEMTO3D Atlas, while the mouse was performing an auditory discrimination task. In this “classical” behavioral experimental protocol, water-deprived mice were trained to associate water reward with a „go” tone, and punishment with a „no-go” tone. Altogether 811 VIP cells were imaged from 18 brain regions. On the right panel, each row of the raster plot represents the average neuronal response for Hit and False alarms, and the graph shows the Ca2+ responses of one VIP interneuron averaged for Hit (thick green), False alarm (thick red), Miss (thin green), and Correct rejection (CR, thin red).
Hippocampal interneuron activity recordings during random foraging, context remapping and goal-oriented learning experiments
In these behavioral tests, water restricted mice were trained to run on a fabric belt enriched with tactile cues, and to operantly lick and receive water rewards depending on the type of the experiment. The velocity of the animal was recorded, and hippocampal interneurons were imaged simultaneously by the Chessboard scanning mode of the FEMTO3D Atlas.
The figure shows that more than 200 interneurons were imaged simultaneously during the random foraging behavioral task, and the locomotion state of the animal (velocity, locomotion, immobility) was associated with the Ca2+ activity of the subsequently characterized interneuron types.
Learn more: Geiller et al. Neuron (2020)
Extremely large space under the objective
The FEMTOSmart product line was primarily developed to perform in vivo imaging. This system’s special feature is the elevated body which can move in X, Y, and Z dimensions: it offers plenty of space under the objective for free and easy positioning of the sample. This feature supports functional behavioral studies of model organisms with different weights and sizes, from flies to even non-human primates, while they are moving in a virtual-reality environment. The microscopes can be assembled using galvanometric scanner supporting flexible ROI scanning and resonant scanner supporting fast frame and volume scanning possibilities. Many optional modules are available to the microscopes enabling the scientists to perform optogenetics and other studies in larger animals even during behavior.
Examples of behavioral neuroscience experiments
Simultaneous two-photon imaging, photostimulation and behavior recording in Zebrafish
Dal Maschio et al. described a method for high-resolution interrogation of circuit function, which combines simultaneous two-photon functional imaging, 3D photostimulation, and quantitative behavioral tracking in zebrafish larvae. Tail movements were initiated by photostimulation, while the induced behavior and neuronal network activity were recorded. The schematic diagram of the experiment is shown on the left panel of the figure. The right panel shows the calcium activity of 486 imaged neurons and the kinematics of the tail in response to stimulation. Red lines indicate photostimulation onset and offset.
Learn more: dal Maschio et al. Neuron (2017)
Two-photon imaging during short-term memory testing in Drosophila
Böhme et al. used two-photon imaging to record calcium responses during a short-term memory test in Drosophila. In this classical aversive olfactory conditioning experiment, flies were trained by pairing an odor with an electric shock. During the simultaneous two-photon imaging and behavioral experiment, calcium activity was evoked in the neurons by odors. See the left panel of the figure for the schematic diagram. On the right panel, averaged odor responses are shown in control and mutant Drosophila. Five responses per odor were averaged per animal.
Learn more: Böhme et al. Nat Commun (2019)
Partners in behavior instrumentation
Luigs & Neumann treadmill with virtual reality
The Luigs & Neumann treadmill was designed to investigate the integration of sensory and spatial information in the brain of mice. The system allows tactile, visual or olfactory stimulation of a head-fixed mouse, while the animal is moving a treadmill belt and navigating in a virtual environment. In parallel all established optical and electrophysiological measurements or manipulations for head-fixed animals can be performed e.g. to study navigation strategies.
Phenosys virtual reality system
We offer VR systems for investigating cognition, navigation, learning, memory, operant conditioning, etc.. Phenosys JetBall-TFT consists of a TFT surround monitoring system focusing on 200° around the animal, and a spherical treadmill which make unobscured field or maze designs possible. This device can be coupled with optional operant devices, and allows a restrained animal to navigate in virtual space.
Neurotar virtual reality system
Neurotar Mobile HomeCage provides a real and familiar moving VR environment. A head-fixed, awake rodent walks freely on a flat-floored, air-lifted cage that moves according to the animal’s locomotion, while exploring and navigating during in vivo recordings and imaging experiments.
Head holders for rodents
Head holder stands and head plates fix the rodent’s head in various positions, adapted to the conditions and enabling precise measurements in the brain, the eye and other parts of the head. Two types of head holders are available with different dimensions for surgery, studying of anesthetized rodents behaving animal models moving in VR systems.
Behavioral experiment control
Bpod is an open-source behavior-recording system and real-time environment controller for rodent experiments. Using high-level programming environments (MATLAB/Python), it provides a low-latency closed-loop link between behavioral events, stimulus delivery, and stimulation. Using its built-in liquid reward delivery system, it can be used to power go/no-go discrimination, two alternative forced choice, and CS/US behavioral paradigms. Bpod data acquisition is fully synchronized with the microscope, and the behavioral events can be precisely aligned to the recording of Ca2+ transients.
Software-controlled triggering of stimuli
Control software of Femtonics microscopes
Different forms of stimulation can be triggered or driven directly using the analog and digital output signals of the microscope, planned and controlled from the measurement control software. Visual, auditory, tactile, whisker or odor stimuli can be generated this way. The MES command line access and MESc API allow custom scripting of the measurement control including third party devices to form a complex environment for behavior experiments.
References
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)
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)
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)
Matching Cell Type to Function in Cortical Circuits. Luc Estebanez, Jens Kremkow, James F.A. Poulet, Neuron (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)
Synaptic Mechanisms Underlying Sparse Coding of Active Touch. Crochet S, Poulet JF, Kremer Y, Petersen CC., Neuron (2011)
3P imaging
Three-photon (3P) microscopy allows noninvasive structural and functional imaging by making cells visible in deep tissues with high spatial resolution and better contrast compared to two-photon excitation. The optical design required for 3P excitation establishes higher axial resolution than that of two-photon microscopy. The spectral window enables three-photon excitation of a variety of fluorophores, such as the current generations of protein-based genetically encoded calcium indicators (e.g. GCaMP6) and the repetition rate of the laser source is adequate for imaging Ca2+ transients produced from neural activity.
The video shows an approximately 1000 μm thick 3P z-stack from a mouse cortex. Purple: blood vessels, green: GFP labelled pericytes. Footage courtesy of Dr. Severin Filser.
3P range microscopy
FEMTOSmart Galvo equipped with 3P range microscopy optional module allows excitation by a wavelength range of 1,200-1,700 nm. The longer excitation wavelengths are scattered less in the biological tissues, which allows extending the penetration depth, reduces out-of-focus excitation, and increases the signal-to-noise ratio. The figure, prepared using a FEMTOSmart Galvo with a 3P range microscopy module, shows spontaneous neuronal activity in the GCaMP6f-labeled visual cortex of a mouse: the cells were excited using a 1300 / 1400 nm laser wavelength for 3P excitation.
Third Harmonic Generation
Third Harmonic Generation is a specific effect of 3P excitation, that results from the conversion of three incoming photons into one emitted photon with tripled energy and thus, the emission of the light of one-third the wavelength. THG occurs at structural interfaces that are formed between aqueous fluids and lipid-rich structures, for instance, biological membranes, and between water and large protein aggregates such as collagen bundles or muscle fibers.
The figure shows a THG image of a section of a mouse kidney: kidney cells were excited at 1500 nm, and the emitted photons were collected at the green channel (~500 nm) of the detector system.
“We have found a massively great partner in Femtonics and acquiring the groundbreaking 3P (three-photon) FEMTO SMART system ensures never-seen experimental results in deep imaging. We are impressed how fast they are reacting at our requests and able to think together with us on scientific solutions. Their technical level and support are professional, and it is always good to have a trustful partner in daily scientific life.”
Dr. Severin Filser
DZNE – German Center for Neurodegenerative Diseases, Germany
Fluorescence-lifetime imaging microscopy – FLIM
Fluorescence-lifetime imaging microscopy (FLIM) delivers both structural (intensity image) and functional (lifetime image) information and minimizes the imaging effect of photon scattering in thick layers of sample. FLIM provides an accurately quantitative assessment of changes of many biophysical parameters in a target molecular microenvironment, without being affected by the intensity of the emission, quenching or uneven fluorophore distribution. The most frequent FLIM applications are the measurement of molecular environment parameters, molecular interaction and distance measurements by Förster resonance energy transfer (FRET), besides studying the metabolic state of cells and tissues via their autofluorescence. Two-photon fluorescence-lifetime imaging microscopy (2P-FLIM) meets the needs of neuroscientific research because of the combined advantages of two-photon excitation and fluorescence-lifetime imaging: superior depth penetration and decreased photo-bleaching, owing to long excitation wavelengths, and inherent optical sectioning.
The FEMTOSmart for FLIM
The FEMTOSmart Galvo upgraded with the FLIM extension is suitable for performing in vivo and in vitro 2P-FLIM studies in a wide range of model organisms. In this complex system, up to two hybrid detectors (combination of GaAsP cathode and an avalanche detector) with two photon counting channels provide a high detection efficiency and a fast timing response, with less noise. As afterpulsing is essentially absent in these detectors, the dynamic range of fluorescence decay recordings is considerably increased. The special scanning modes and patterns of the FEMTOSmart Galvo provide high spatiotemporal resolution during measurements. The setup is perfectly suitable to monitor glutamate release and intracellular calcium concentration, to perform FRET measurements, or to quantify the ratio between reduced and oxidized NADH and FAD molecules, in the research of neurodegenerative diseases or to reveal metabolic changes during tumorigenesis in cancer research.
Tornado imaging for the direct monitoring of single-quantum, single-synapse glutamate release in situ
A FEMTOSmart Galvo with a FLIM extension is suitable to follow single action potential-evoked presynaptic Ca2+ dynamics, with nanomolar sensitivity, in individual small, presynaptic boutons in acute brain slices. Jensen et al., from the laboratory of Prof. Dmitri Rusakov, have advanced a two-photon excitation FLIM technique. They used the flexible Tornado scanning mode of our galvanometric scanner-based microscope, which provides pixel sampling in a spiral, recording the signal’s spatiotemporal dynamics more favourably, and improves the signal-to-noise ratio. With this method, they performed time-resolved imaging of Ca2+ sensitive fluorescence lifetime of Oregon Green BAPTA-1. In parallel, intensity Tornado imaging made it possible to monitor single-quantum, single-synapse glutamate release in situ directly, using the locally expressed extracellular optical glutamate sensor iGluSnFr. This study paves the way for simultaneous registration of presynaptic Ca2+ dynamics and transmitter release in an intact brain at the level of individual synapses.
(Related article: Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue, Thomas P. Jensen, Kaiyu Zheng, Olga Tyurikova, James P. Reynolds, Dmitri A. Rusakov, Cell Calcium, 2017)
FLIM imaging reveals that distinct sites in individual boutons release glutamate in a probabilistic fashion
In a subsequent publication from the same laboratory, Rama et al. describe how they used the FEMTOSmart Galvo with the FLIM extension to monitor spike-evoked glutamate release and presynaptic calcium entry in the mossy fiber boutons of granule cells, with the use of the optical glutamate sensor SF-iGluSnFR and the intracellular Ca2+ indicator Cal-590, and the above mentioned Tornado scanning mode. These presynaptic boutons feature multiple release sites and are densely packed with synaptic vesicles to sustain a highly facilitating “detonator” transmission. In this study, multiplexed imaging showed that distinct sites in individual boutons release glutamate in a probabilistic fashion, while also showing use-dependent short-term facilitation. The results help to reveal whether glutamate release sites act
independently or rather synchronously, and to understand the fundamentals of synaptic transmission and hippocampal network function in a wider context.
(Related article: Glutamate Imaging Reveals Multiple Sites of Stochastic Release in the CA3 Giant Mossy Fiber Boutons, Sylvain Rama, Thomas P. Jensen, Dmitri A. Rusakov, Frontiers in Cellular Neuroscience, 2019)
References
Fluorescence lifetime imaging reveals regulation of presynaptic Ca2+ by glutamate uptake and mGluRs, but not somatic voltage in cortical neurons. Olga Tyurikova, Kaiyu Zheng, Elizabeth Nicholson, Yulia Timofeeva, Alexey Semyanov, Kirill Volynski, Dmitri A. Rusakov, Journal of Neurochemistry (2020)
Local Resting Ca2+ Controls the Scale of Astroglial Ca2+ Signals. Claire M. King, Kirsten Bohmbach, Daniel Minge, Andrea Delekate, Kaiyu Zheng, James Reynolds, Cordula Rakers, Andre Zeug, Gabor C. Petzold, Dmitri A. Rusakov, Christian Henneberger, Cell Reports (2020)
Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ homeostasis at multiple synapses in situ. Thomas P. Jensen, Kaiyu Zheng, Nicholas Cole, Jonathan S. Marvin, Loren L. Looger, Dmitri A. Rusakov, Nature Communications (2019)
Polymer microchamber arrays for geometry-controlled drug release: a functional study in human cells of neuronal phenotype. Olga Kopach, Kayiu Zheng, Olga A. Sindeeva, Meiyu Gai, Gleb B. Sukhorukov, Dmitri A. Rusakov, Biomaterials Science (2019)
Glutamate Imaging Reveals Multiple Sites of Stochastic Release in the CA3 Giant Mossy Fiber Boutons. Sylvain Rama, Thomas P. Jensen, Dmitri A. Rusakov, Front. Cell. Neurosci. (2019)
A genetically encoded fluorescent sensor for in vivo imaging of GABA. Jonathan S. Marvin, Yoshiteru Shimoda, Vincent Magloire, Marco Leite, Takashi Kawashima, Thomas P. Jensen, Ilya Kolb, Erika L. Knott, Ondrej Novak, Kaspar Podgorski, Nancy J. Leidenheimer, Dmitri A. Rusakov, Misha B. Ahrens, Dimitri M. Kullmann & Loren L. Looger, Nature Methods (2019)
Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging. Kaiyu Zheng, Thomas P Jensen & Dmitri A Rusakov, Nature Protocols (2018)
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)
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)
Principles of Fluorescence Spectroscopy, 3rd ed. Lakowicz, J.R., Springer (2006)
Fluorescence lifetime maesurement and biological imaging. Berezin, M.Y. & Achilefu, S., Chem. Rev. (2010)
Non-invasive imaging of skin physiology and percutaneous penetration using 5D (space, time and anisotropy) fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy. Roberts, M.S., Dancik, Y., Prow, T.W., et al., Eur. J. Pharm. Biopharm. (2011)
Blood flow tracking
Following blood flow deep in the living tissue
Abnormalities in hemodynamics are linked to various disease states, such as stroke, Alzheimer’s, etc., however, to better understand these associations, more precise optical techniques are needed for interrogating blood flow velocity in vivo in 3D. Acousto-optic (AO) multiphoton microscopy is a superior technique to perform blood flow tracking at the cellular level, at a high spatiotemporal resolution deep in the living 3D tissue. The unique scanning modes of the FEMTO3D Atlas allow you to record continuously along intricate 3D patterns, and its extensions make it possible to follow multiple parameters at the same time, like blood flow and cellular activity. These applications can be useful in various fields, such as neuroscience, immunology, dermatology, oncology and more.
FEMTO3D Atlas Dichro with two laser sources for dual excitation
This extension of the FEMTO3D Atlas platform enables fast 3D region of interest (ROI) scanning in a large 3D volume, as well as a quasi-simultaneous detection of neuronal activity and blood flow using two different laser sources. The Femtonics software package provides built-in ROI and curve analysis options, meeting all needs.
Microscope equipment
- two pairs of acousto-optic crystals for XY and Z rapid focusing (3D imaging)
- 4D Beam Conditioning Units for the stabilization of the laser beams
- two two-photon laser sources of different wavelengths
- a Dichro system for fast switching between the two lasers line for quasi-simultaneous dual excitation
Technical procedure for acquisition and analysis
Here we injected a red dextran intravenously to make blood vessels appear in red. The passing red blood cells (RBCs) show as shadows in the red stream (Fig1.A). This way, the speed of the blood stream can be measured by following an RBC in a time-dependent manner (Fig 1.B).
Fig 1. How to measure blood flow velocity using 3D two-photon imaging. A. An example image and blood stream visualization from one 3D ribbon in a ~200 um depth. B. A montage image showing the stream of individual red blood cells (yellow circles and arrows). The average speed in a certain part of capillaries or vessels can be calculated from the values of the speed of the red blood cells (v1, v2…vn).
Rapid ROI imaging by the state-of the-art FEMTO3D Atlas AO platform enables 3D ribbon scanning, following vessels through multiple cortical layers in a large brain volume. The total length of the ribbons can reach up to ~5 mm, while maintaining a resolution and speed sufficient to measure the velocity of blood flow (Fig 2.)
Fig 2. Maintaining a proper scanning speed for 3D blood stream measurement, the FEMTO3D Atlas is able to scan an up to 5 mm long ribbon network. A. 3D reconstruction of the ribbon pattern laid down to measure blood flow in mouse cortex. B. Blood stream visualization of a 4.78 mm long 3D ribbon-network; scanning speed: 7 Hz.
Simultaneous measurement of neuronal activity and blood flow velocity
Research of cardiac diseases may be supported by a combined method of simultaneous Ca2+ activity and blood flow imaging. This provides a novel opportunity of acquiring local information about neuronal and cardiovascular status. Using the FEMTO3D Atlas Dichro with two laser-sources of different wavelength, blood cell movement and neuronal activity can be measured at the same time. Fast switching between the two laser sources makes it possible to image quasi-simultaneously on two different emission wavelengths. This method can be useful for studying the detrimental consequences of stroke on vascular condition, neuronal damage, and even regeneration processes (Fig 3.).
Fig 3. Simultaneous imaging of neuronal activity and blood flow. Visual cortex neuron appears in green; red indicates a vessel (as a vertical band).
Fig 4. In vivo imaging of blood flow in zebrafish eye. RED: tdTomato labelled blood cells. GREEN: GCaMP6 labelled neurons
Optogenetics
The essence of optogenetics is introducing light-activated recombinant ion channels such as channelrhodopsin (ChR2) or halorhodopsin (NpHR) into excitable cells. Light activation of these molecules leads to an influx of ions which induces turning neurons on or off selectively. Halorhodopsin and channelrhodopsin together enable multicolor optical activation, silencing, and desynchronization of neural activity, creating a powerful neuroengineering toolbox. Photostimulation can be induced using visible or infrared light, while imaging is performed by a femtosecond IR laser. Switching between the stimulation and imaging is done at a sub-millisecond scale. Importantly the detectors are protected during the stimulation by a built-in gating system.
3D PHOTOSTIMULATION AND IMAGING
Photostimulation can be interlaced with imaging by selecting a scan area covering one or more somata situated anywhere in the 3D field of view by the FEMTO3D Atlas Dichro extension. Stimulation can be performed by the 3D chessboard scanning mode which is an advanced experimental approach, that extends 3D random-access point scanning into small local planes. Distributing the stimulation energy homogenously onto the somata of the selected cells allows maximum stimulation efficiency, thereby minimizing the possibility of photodamage to the targeted cells. The laser beam used for photostimulation excites the channelrhodopsin expressed in the cells, eliciting a controlled burst of action potentials. For calcium imaging a second scanning pattern with larger regions can be selected, enveloping cell bodies with the surrounding areas, in which the evoked activity can be followed even during sample movements. Timing of the photostimulation and the imaging can be precisely controlled by various protocols from the software GUI. This way the connectivity of the network elements can be examined, and correlated with the network function.
Figure: ChrimsonR-mRuby2 and GCaMP6f expressing cells were stimulated and imaged by the 3D chessboard scanning method in the cortex of a mouse in vivo. Red square: stimulation of a cell at 10 x 10 µm, green squares: imaging of cells at 25 x 25 µm/cell in the presented plane (continuous line) and at other Z depths (dotted line). Stimulation was performed with 10 x 1 ms pulses (10 Hz repetition rate) for higher efficiency, imaging was performed with 12 Hz.
Full-field illumination
FEMTOSmart Resonant equipped with LED light source
The entire FOV can be stimulated with the LED source above the objective. LEDs are available at different wavelengths, exciting ChR2 at 473 nm or NpHR at 561 nm. The light source provides homogeneous illumination, and the light impulses are precisely timed and highly repeatable.
The FEMTOSmart Resonant microscope follows the changes over the whole field of view at a resolution of 31 frames per second.
Stimulation along ROIs
FEMTOSmart Galvo equipped with Multiple beam path extension
To stimulate cells or subcellular components selectively, the best solution is using the FEMTOSmart Galvo equipped with a secondary laser (Multiple Beam Path) to steer the laser beam rapidly through optimized scanning patterns such as point, line, spiral, zigzag. While scanning along points and lines, which allow stimulation on in precise locations on spines or dendrites, the spiral and zigzag patterns covering larger region enable more molecules to be stimulated simultaneously on the soma resulting activation. We offer a continuous laser tuned to 473 or 561 nm for ChR2 or NpHR activation, respectively. Precise two-photon activation of these molecules is also a viable option.
Simultaneous photoactivation and imaging
FEMTOSmart Dual equipped with Multiple beam path extension
The FEMTOSmart Dual microscope contains a galvo and a resonant scanner which function in tandem to combine the advantages of both the galvo and resonant microscopes. This is the best solution for simultaneous photostimulation and high-speed imaging.
References
Activity Patterns in the Neuropil of Striatal Cholinergic Interneurons in Freely Moving Mice Represent Their Collective Spiking Dynamics. Rotem Rehani, Yara Atamna, Lior Tiroshi, Wei-Hua Chiu, José de Jesús Aceves Buendía, Gabriela J. Martins, Gilad A. Jacobson and Joshua A. Goldberg, eNeuro, 2019
Dendrite-Specific Amplification of Weak Synaptic Input during Network Activity In Vivo. Leiron Ferrarese, Jean-Sébastien Jouhanneau, Michiel W.H. Remme, Jens Kremkow, Gergely Katona, Balázs Rózsa, Susanne Schreiber, James F.A. Poulet, Cell Reports, 2018
Topography of a Visuomotor Transformation. Thomas O. Helmbrecht, Marco dal Maschio, Joseph C. Donovan, Styliani Koutsouli, Herwig Baier, Neuron, 2018
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)
Uncaging
Uncaging means the activation of biochemically masked (‘caged’) molecules via photolysis, which mimics the physiological release of bioactive compounds. This technique is widely used in neuroscience, where the bioactive molecule is usually glutamate or another neurotransmitter. Using two-photon photostimulation with ultrafast pulsed IR laser light, very precise release of these compounds can be elicited in extremely small volumes. Two-photon imaging is a powerful opportunity to follow the changes evoked in dendrites or spines, even the distribution of receptors on the neurons can be investigated.
In vitro uncaging at well-defined points
FEMTOSmart Galvo equipped with a Multiple beam path extension
The accuracy of the excitation point, and the highly flexible scanning patterns, mean that the FEMTOSmart Galvo is the best choice for uncaging experiments. Multiple-point scanning (yellow points on the figure) is used for stimulation around spines, while multiple-line scanning (arrows) makes high-speed imaging along the dendrites possible. The secondary laser beam (Multiple beam path optional module), essential for the stimulation, is coupled to the existing light path. Thus the stimulation and the imaging can be performed near simultaneously using the galvo scanner. The microsecond-scale switching time between the stimulation and imaging is established by using of Pockels cell.
Specialized software functions for photostimulation mapping
Stimulus mapping and analysis software module
This module of MES control software allows performing photostimulation mapping experiments at a range of locations and shapes. The locations can be picked manually one-by-one, along a line or in a raster, and various stimulation patterns can be selected like point, spiral, x, zigzag. It forms datasets quickly by evaluating fluorescence changes images at defined time intervals. Analysis tool for stimulation mapping can create (multichannel) map images formed by the elicited responses after a mapping experiment.
Compounds for uncaging experiments
DNI-GLU-TFA synthesized by FemtoChemistry
This dinitro-indoline-masked form of glutamate releases the bioactive glutamate more rapidly than any other commercially available compound. It was developed for high-quantum yield requiring less irradiation for release, so its effective concentration is lower than other caging scaffolds. DNI-Glu is a compound developed in-house, only available from Femtonics. See also Chiovini et al., Neuron, 2014.
Do you want to learn more about all of our caged neurotransmitters?
References
Dendritic inhibition differentially regulates excitability of dentate gyrus parvalbumin-expressing interneurons and granule cells. Claudio Elgueta & Marlene Bartos, Nat. Commun (2019)
Dendritic NMDA receptors in parvalbumin neurons enable strong and stable neuronal assemblies. Jonathan H Cornford Is a corresponding author, Marion S Mercier, Marco Leite, Vincent Magloire, Michael Häusser, Dimitri M Kullmann, eLIFE (2019)
Altered dendritic spine function and integration in a mouse model of fragile X syndrome. Sam A. Booker, Aleksander P. F. Domanski, Owen R. Dando, Adam D. Jackson, John T. R. Isaac, Giles E. Hardingham, David J. A. Wyllie & Peter C. Kind, Nat. Commun. (2019)
Parsing Out the Variability of Transmission at Central Synapses Using Optical Quantal Analysis. Cary Soares, Daniel Trotter, André Longtin, Jean-Claude Béïque and Richard Naud, Front. Synaptic Neurosci. (2019)
Endocannabinoid Signaling Mediates Local Dendritic Coordination between Excitatory and Inhibitory Synapses. Hai Yin Hu, Dennis L.H. Kruijssen, Cátia P. Frias, Balázs Rózsa, Casper C. Hoogenraad, Cell Reports (2019)
A compact holographic projector module for high-resolution 3D multi-site two-photon photostimulation. Mary Ann Go, Max Mueller, Michael Lawrence Castañares, Veronica Egger, Vincent R. Daria, PLOS One (2019)
Coincidence Detection within the Excitable Rat Olfactory Bulb Granule Cell Spines. S. Sara Aghvami, Max Müller, Babak N. Araabi and Veronica Egger, J. Neurosci. (2019)
Disentangling astroglial physiology with a realistic cell model in silico. Leonid P. Savtchenko, Lucie Bard, Thomas P. Jensen, James P. Reynolds, Igor Kraev, Nikolay Medvedev, Michael G. Stewart, Christian Henneberger & Dmitri A. Rusakov, Nat. Commun. (2018)
Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function. Anne-Kathrin Theis, Balázs Rózsa, Gergely Katona, Dietmar Schmitz and Friedrich W. Johenning, Front. Cell. Neurosci. (2018)
High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis. Dénes Pálfi, Balázs Chiovini, Gergely Szalay, Attila Kaszás, Gergely F. Turi, Gergely Katona, Péter Ábrányi-Balogh, Milán Szőri, Attila Potor, Orsolya Frigyesi, Csilla Lukácsné Haveland, Zoltán Szadai, Miklós Madarász, Anikó Vasanits-Zsigrai, Ibolya Molnár-Perl, Béla Viskolcz, Imre G. Csizmadia, Zoltán Mucsi and Balázs Rózsa, Org. Biomol. Chem. (2018)
Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue. Jan Tønnesen, V.V.G. Krishna Inavalli, U. Valentin Nägerl, Cell (2018)
Metaplasticity at CA1 Synapses by Homeostatic Control of Presynaptic Release Dynamics. Cary Soares, Kevin F.H. Lee, Jean-Claude Béïque, Cell Reports (2017)
Imaging membrane potential changes from dendritic spines using computer-generated holography. Dimitrii Tanese, Ju-Yun Weng, Valeria Zampini, Vincent de-Sars, Marco Canepari, Balazs J. Rozsa, Valentina Emiliani, Dejan Zecevic, Neurophotonics (2017)
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)
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)
Electrical behaviour of dendritic spines as revealed by voltage imaging. Marko A. Popovic, Nicholas Carnevale, Balazs Rozsa; Dejan Zecevic, Nature Communications (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)
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)
Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity. Joju George, Cary Soares, Audrey Montersino, Jean-Claude Beique, Gareth M Thomas, eLIFE (2015)
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)
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)
The kinetics of multibranch integration on the dendritic arbor of CA1 pyramidal neurons. Sunggu Yang, Valentina Emiliani and Cha-Min Tang, Front. Cell. Neurosci. (2014)
Differential Subcellular Targeting of Glutamate Receptor Subtypes during Homeostatic Synaptic Plasticity. Cary Soares, Kevin F. H. Lee, Wissam Nassrallah and Jean-Claude Béïque, J. Neurosci. (2013)
GnRH Neurons Elaborate a Long-Range Projection with Shared Axonal and Dendritic Functions. Michel K. Herde, Karl J. Iremonger, Stephanie Constantin and Allan E. Herbison, J. Neurosci. (2013)
Dendritic Hold and Read: A Gated Mechanism for Short Term Information Storage and Retrieval. Mariton D. Santos, Michael H. Mohammadi, Sunggu Yang, Conrad W. Liang, Joseph P. Y. Kao, Bradley E. Alger, Scott M. Thompson, Cha-Min Tang PLOS One (2012)
Parallel optical control of spatiotemporal neuronal spike activity using high-speed digital light processing. Jason Jerome, Robert C. Foehring, William E. Armstrong, William J. Spain and Detlef H. Heck, Front. Syst. 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)
Electrophysiology
Two-photon Ca2+ imaging and electrophysiological data acquisition provide excellent tools for the study of neural cell and network activity. Using the two methods simultaneously, it is possible to follow potential fluctuations and relate them to changes in their Ca2+ levels. While the two-photon Ca2+ imaging technique shows events in multiple small areas (distinguishable parts of dendrites and spines) at high spatial resolution (less than 1 µm) but low temporal resolution (>100 ms), electrophysiology can cover changes over larger areas (cells or extensive areas) at low spatial resolution (~150 µm), and the changes can be followed considerably faster (<1 ms).
Dendritic patch-clamp
With intracellular patch-clamp recording it is possible to observe and manipulate membrane potential fluctuations in neurons. All Femtonics microscopes can be equipped with electrophysiological devices, and the following settings facilitate the experiments:
- automatic software module moves the tip of the pipette to the selected dendritic location,
- pipette movement and distance to target location can be monitored in real-time,
- guide points can be positioned by the user according to the scanned transmitted or two-photon images,
- real-time transmitted infrared and two photon images can be shown separately or in overlay,
- only one simple calibration step is required before using a new recording pipette.
Processing of electrophysiological data
The electrophysiology software module acquires electrophysiology signals using the A/D converters of the microscope. The telegraphing function communicates the conversion parameters from the amplifier to the A/D converters managed by the control software. Electrophysiology signal acquisition time is aligned with the imaging data. The electrophysiology GUI also includes a seal test window.
References
Multiple Two-Photon Targeted Whole-Cell Patch-Clamp Recordings From Monosynaptically Connected Neurons in vivo. Jean-Sébastien Jouhanneau and James F. A. Poulet, Front. Synaptic Neurosci. (2019)
Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function. Anne-Kathrin Theis, Balázs Rózsa, Gergely Katona, Dietmar Schmitz and Friedrich W. Johenning, Front. Cell. Neurosci. (2018)
Electrical behaviour of dendritic spines as revealed by voltage imaging. Marko A. Popovic, Nicholas Carnevale, Balazs Rozsa; Dejan Zecevic, Nature Communications (2015)
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)
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)
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)
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)
Deep functional imaging
One of the most useful advantages of two-photon microscopy is the possibility to examine not just the surface of the tissue but the deeper layers of the sample as well.
Confocal microscopy can penetrate the tissue, but it is limited to a few dozens of micrometers.
In deeper regions, despite the longer imaging wavelength the scattering of the photons is marginal, especially the high Numerical Aperture (NA) photons which means the imaging intensity is decreases substantially. To resolve this concern, it is important to decrease the diameter of the imaging laser beam. If the laser beam is more narrow, the scattering is minimized, which increases the quality of the image at any depth in the tissue (Figure 1).
Figure 1. Schematics of the effects of over and underfilling on deep imaging. During overfilling the high NA photons are likely to scatter out before reaching the focal point. On the other hand, in the case of underfilling, photons are focused into a narrower beam, so less photons will be scattered.
The motorized beam expander, which is located at the laser exit port in our 4D Beam Conditioning Unit (4D-BCU), is equipped with a sliding-lens mechanism. This mechanism makes it effortless to adjust the beam diameter at the objective misalignment-free and wavelength-independent. Our analysis software package allows the user setting the beam diameter during anatomical volume recordings and functional imaging.
Features:
- fully integrated into 4D-BCU and MES
- convenient, direct control of beam diameter at objective pupil
- the low pointing provided by the sliding lens mechanism is fully compensated by the beam stabilization system of the Atlas scanhead
- wavelength-independent beam diameter adjustment
- calibrated for the specific laser
- optimized and AR coated for the 750-1050 nm wavelength range
- minimal focus shift under the objective, easily compensated by objective movement or AO focusing
- can be programmatically adjusted during z-stacks and metaprotocols
- can be installed as an upgrade – contact us for details
Benefits:
- precise control of effective excitation numerical aperture
- penetration depth up to 1 mm
- lower power losses caused by scattering during deep imaging
- fine tuning of focal spot (PSF) size (to compensate for motion artifacts and/or increase the volume where imaging or photostimulation happens)
- lower power losses caused by aberrations (effect of uncorrected cover glass)
- higher transmission of medium and high magnification objectives
- larger effective Acousto Z (remote) focusing range due to higher transmission even for higher |Z| values
Depth over everything
With the help of proper staining, the Deep Functional Imaging (DFI) opens up possibilities to look into the tissue up to the physical limits. Longer the wavelength, deeper the imaging. Measured from the surface, 1 millimeter of depth is possible with an optimized beam diameter (Figure 2). Even live process can be visualized during in vivo awake experiments.
Figure 2. Example of Deep Functional Imaging (DFI) in the neocortex. (Left) overfilling example from -850 um with ROI examples. (Right) Underfilling with the same parameters.
Labelling: GCaMP8 (green)
Increased SNR during functional imaging
The more intensity we get, the more information we are able to collect during imaging. Hippocampal imaging is a great example to highlight the potential of DFI. Without burning the tissue, a higher SNR can be achieved. This means elongated acquisitions and more detailed recordings are possible.
Figure 3. Increased SNR with DFI in the hippocampal CA1 region. Calcium imaging in transgenic gcamp6f mouse. The videos were taken under the same conditions, with the exception of the beam diameter.