Femtonics - Voltage test

Voltage Imaging

Genetically encoded voltage indicators (ASAP, JEDI2P, etc.) have recently attained a level of quality that renders them valuable tools for studying membrane potentials throughout cells. However, a significant challenge has persisted: the lack of sufficiently fast equipment to capture their dynamics. Until now, there has been a shortage of systems capable of imaging at speeds exceeding kHz, approaching the electrophysiological standard of 10 kHz. The Femtonics 3D AO Atlas addresses this gap by enabling high-speed, three-dimensional voltage imaging. Equipped with the real-time motion correction module, the 3D Real-Time Motion Correction, it achieves rapid imaging without motion artifacts and ensures high signal-to-noise ratio (SNR) for precise voltage imaging.

Indicators:

The rapidly evolving voltage sensor technology is going to determine the next decade of neuroscience. Voltage signals have faster kinetics and thus provide and require superior temporal resolution. The sensors report individual action potentials even within fast bursts. Conventional imaging techniques are not fast enough to capture these transients.

Figure 1: Visualizing the speed of the signal and the proof of necessity for Acousto-Optical scanning technology . A) Green: Ca²⁺ from a PV interneuron sampled at 30 Hz. Red: JEDI2p sampled at 8.4 kHz. Blue: Patch clamp electrophysiological recording of 5 evoked Action Potentials sampled at 20 kHz.
B) Same measurement down-sampled to 80, 40, and 21 Hz.

1. In vivo voltage imaging with FEMTO3D Atlas

One of today’s most interesting and challenging task is to measure voltage signals from neurons while the model animal is active. Even recently published in vivo voltage imaging studies have several limiting factors. Femtonics has overcome these limitations and allowed researchers to have higher success rate, higher SNR, higher number of recording locations in 3D, better focus on the dendrites and alternative ways to investigate and interact with neuronal population. All measurement techniques can be easily applied in head-fixed behaving animals too, due to the 3D Real-Time Motion Correction, which enables the high SNR in vivo as well.

Attila Losonczy, MD, PhD – Professor of Neuroscience; Principal Investigator at Columbia’s Zuckerman Institute

Increased SNR measurements due to 3D Real-Time Motion Correction

Figure 2: A) 3D model showing the relative locations of the measured neurons. B) Quality JEDI2P labelling in the visual cortex L2/3. C) Example traces showing the SNR with 1 kHz sampling from a behaving animal. Bottom: Chessboard scanning of 5 individual neurons from different Z planes. D) Average of individual APs. (n=17)

Increased number of somatic recordings

Figure 3: A) Chessboard ROIs in 600 x 600 x 600 um volume B) ASAP labelling in the in M1 region, red squares mark the recorded areas with low resolution. C) Voltage traces from 13 neurons in head-fixed conditions recorded with 400 Hz speed.

Dendritic voltage imaging

Figure 4: A) 3D model showing a chessboard and ribbon scans from the same neuron in visual cortex B) Recorded ROIs from different planes. C) Voltage traces the recorded areas D) Soma and the two different dendritic segment aligned.

Simultaneous Ca and Voltage imaging measurements

Figure 5: A) Experimental setup: mice were active on a treadmill while undergoing two-photon voltage imaging with Femto3DATLAS. Auditory stimulus served as activation signal in the V1 cortex. B) 3D model of the network which was recorded with line scans. C) JEDI2P labelled neurons in the field of view. Red lines mark the location of the line scans with 2.2kHz D) Averaged signal responses for the auditory stimulus from 17 neurons. Green: jRGECO calcium signals, Red: JEDI2P voltage signals excited on 1040nm. The black line shows the timing of the auditory stimulus.

Optical electrophysiology in drosophila

Figure 6: A) Schematics of Drosophila 2p imaging. B) ASAP labelled Drosophila neurons. Red lines over the somata are representing the line scan locations C) ASAP signal from the labelled neuron recorded with 6675.57 Hz.

2. In vitro voltage imaging with FEMTO3D Atlas

We offer optical electrophysiology solutions for problems that are impossible or close to impossible to solve with classical electrophysiology (i.e. multiple site dendritic patch, repeated bouton patch etc.) working today in the patch clamping world of electrophysiology and photoactivation, as multiple measurements sites can be recorded with up to 100 kHz by applying the DRIFT scanning in 3D. (Szalay et al. 2016)

Dr. Christopher D. Makinson – Assistant Professor of Neurological Sciences, Columbia University

Optical validation of voltage sensor

Figure 7: Validation of concept comparison of acousto-voltage imaging and electrophysiology measurements. A) Whole cell patch–clamp camera image and the maximum intensity projection of the patched neuron under two–photon microscopy B) Rise time comparison of the JEDI2P and the electrophysiological signal C) Half-width on the top and signal amplitude measurement comparisons on the bottom.

Optical somatic electrophysiology of cell cultures, organoids, brain slices

Figure 8: Combined two-photon and electrophysiology of HEK 293 cells. A) Camera image and fluorescent labelling of the field-of-interest. JEDI2P in the green channel. The measurements were done along the line marked with red. B) Fluorescent intensity change during ramp test on a HEK cell. The intensity decreased under the detection threshold C) Top is raw data, bottom is filtered. Somatically evoked ramp test. Blue: electrophysiology, red is voltage signals measured with 8.32 kHz.

3D Optical electrophysiology from dendrites and spines

Figure 9: A) 3D model of a single pyramidal neuron measured in vitro with 3D line scan. The red mark is showing the recorded region with a 3D line scan. B) Maximum intensity Z projection of a cortical pyramidal cell. White dots are indicating the measurement locations. C) Somatically evoked 5 backpropagating action potentials. Left: Fluorescent intensity map of JEDI2P from pyramidal cell’s apical dendrite. Right: Temporal integration of the intensity map at the given ROI. There were 24 ROIs from the measured dendritic area with 2.24 kHz. Bottom: Arrows indicating the somatically evoked events, blue: electrophysiology, red: integrated curves for the whole dendrite based on the JEDI2P signal.

Optical electrophysiology combined with uncaging

Figure 10: A) 3D reconstruction of a dendrite with the uncaging locations marked with yellow. The imaging as 3D line scan marked with blue B) Maximum Z-projection of a pyramidal neuron (ALEXA-594) and in the inset the scanning line (blue), uncaging locations (yellow, 15 locations, 0.3 ms ROI time). C) Examples of uncaging (DNI-Glu) evoked EPSP (top, JEDI-2P signal on dendrite, bottom somatically recorded electrophysiological signal).

Reference labs are located at: Stanford University, Baylor College of Medicine, Columbia University, Basel University, NIH, and BrainVisionCenter.

We are eager to demonstrate our capabilities online in vitro or in vivo. Book an appointment to see how easy it is to achieve such results.

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More developments are in the works that focus on voltage imaging such as disc scanning, 3-level image stabilization, immediate multi-channel analysis, etc. Our partners have an extremely high grant success rate with voltage imaging. We are ready to help you and work with you on your grant applications: CONNECT to you sales representative for grant application assistance!

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.

The microscope is fully equipped with high-bandwidth two-way communication ports realizing input and output options through BNC connected cables, providing both digital and analogue in- and outputs configured according to the needs of the future user constrained only by the total bandwidth of the electronic communication and driver cards. Communication is fully mediated by the software through different modules starting from the protocol editor through modules allowing scripting.

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, et al., Front. Cell. Neurosci. (2018)
Electrical behaviour of dendritic spines as revealed by voltage imaging. — Marko A. Popovic, Nicholas Carnevale, Balazs Rozsa, et al., Nature Communications (2015)
Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. — B. Chiovini, G. F. Turi, G. Katona, et al., Neuron (2014)
Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. — Balint Peter Kerekes, Kinga Toth, Balazs Rozsa, et al., Neurophotonics (2014)
Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. — B. Chiovini, G. F. Turi, G. Katona, et al., Neuron (2014)
Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. — Gergely Katona, Gergely Szalay, et al., Nature Methods (2012)
Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity. — B. Chiovini, G. F. Turi, G. Katona, et al., Neurochemical Research (2010)
See all publications

Voltage Imaging
Genetically encoded voltage indicators (ASAP, JEDI2P, etc.) have recently attained a level of quality that renders them valuable tools for studying membrane potentials throughout cells. However, a significant challenge has persisted: the lack of sufficiently fast equipment to capture their dynamics. Until now, there has been a shortage of systems capable of imaging at speeds exceeding kHz, approaching the electrophysiological standard of 10 kHz. The Femtonics 3D AO Atlas addresses this gap by enabling high-speed, three-dimensional voltage imaging. Equipped with the real-time motion correction module, the 3D Real-Time Motion Correction, it achieves rapid imaging without motion artifacts and ensures high signal-to-noise ratio (SNR) for precise voltage imaging. Read more

Optogen lenyíló

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

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…

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 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.

Simultaneous photoactivation and imaging

References

Activity Patterns in the Neuropil… – Rotem Rehani et al., eNeuro, 2019
Dendrite-Specific Amplification of Weak Synaptic Input… – Leiron Ferrarese et al., Cell Reports, 2018
Topography of a Visuomotor Transformation – Helmbrecht et al., Neuron, 2018
Linking Neurons to Network Function and Behavior… – Dal Maschio et al., Neuron, 2017
An optogenetic toolbox for unbiased discovery… – Förster et al., Nature Communications, 2017
See all publications

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.

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References

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