Summary of the project context and objectives
We developed and prototyped a novel three-dimensional two-photon laser scanning microscope which can simultaneously image different brain regions in three spatial dimensions (3x3D system). The volumes imaged could exceed cubic millimeters. Such a 3x3D system is essential in understanding distributed brain computation since it allows the simultaneous recording of neuronal activity in multiple, distant brain volumes that are functionally connected. As each site is to be scanned with sub-millisecond temporal resolution in several hundred locations, the connectivity of distant neuronal microcircuits or even neuronal compartments such as dendrites or axons will be followed.
Participants of the project:
- Femtonics Ltd – IEM HAS – PPCU, Budapest
- Friedrich Miescher Institute for Biomedical Research (FMI), Basel
- Max Delbrück Center for Molecular Medicine (MDC), Berlin
- University of Szeged, Szeged
- Budapest University of Technology and Economics (BME), Budapest
Description of the work performed since the beginning of the project and the main results achieved so far
During the years of the 3x3Dimaging project, we have finished the development of the necessary technologies and built the prototype of the 3x3D microscope product. The project started with a kick-off meeting in Budapest on 07/31/2013 and ended on the same location with a review meeting on 01/17/2017. In the third year we encountered various technical challenges and faced unexpected complications when building the 3x3D microscope prototype. To correct these delays, we asked for a six-month non-cost project extension during which we not only completed the 3x3D microscope prototype but also performed the first biological measurements with the device. Although we have not been able to perform some of the planned biological measurements in their original form, many new, unexpected methods and results were achieved and published during the project in high impact journals such as Science, Nature Communications, Neuron, and Nature Neuroscience. The collaborative scientific research embedded in the project proved to be exceptionally fruitful.
WP1 Development of new acousto-optical (AO) deflectors for the 3x3D system
The task of this work package was to produce acousto-optic deflectors necessary for the construction of 3x3D microscopes and to develop new deflector configurations that allow higher optical and acoustic wavelength tunability and increase the scanning speed, the field of view, the z scanning range, and the resolution. We started to fabricate AO deflectors with the traditional technology, while also experimenting with and optimizing new bonding approaches. New glue types were found to increase the bonding strength as well as the mechanical and thermal resistance. We continued the production of deflectors in different sizes based on the new acousto-optic interaction configuration in order to increase the versatility of our commercially available devices. This allowed shorter configurations with a lower chromatic dispersion. Finally, the optical modeling of the new AO deflectors was improved and new parameters were introduced for handling the new crystal orientation described in the previous tasks. Besides optical modeling, the design of the scan-head hardware was also successfully carried out.
WP2 Development of the 3x3D microscope
We modeled and optimized the whole optical pathway including the scanning unit, the relay lens system, the objective, and the rest of the passive optical elements with an optical modeling software. Based on the results, a CAD model of the 3x3D microscope mechanics was developed. We also modeled the retinal adapter and showed that subcellular resolution is attainable with a relatively simple approach.Although our first approach has failed, we could still reach single cell resolution in the eye of living mice by using an alternative objective. We fabricated a deep brain imaging adapter and by using an existing microscope prototype we were able to image GCaMP6-expressing neurons in the mouse hippocampus. We tested the adaptive optical system in a simplified scenario.
WP3 Development of ultrafast lasers for deep imaging and efficient optogenetic activation
We have carefully examined three possible amplification schemes, and found that two of them exhibit similarly good properties. We implemented both solutions but the final laser power was not enough to feed the prototype microscope. As a corrective action, we purchased a newly developed fiber-coupled laser source with which the final testing of the 3x3D microscope was performed. As the original plan for effective ChR stimulation has been found inadequate, we developed two alternative methods; one has shown promise in proof-of-principle experiments and the other one has reached the product phase. A number of biological experiments have been performed using ChR stimulation.
WP4 Development of control hardware and 3D software
Three new types of electronic boards have been completed for the system: a digital I/O module, an analog I/O module, and an AO driver module. A new data bus system was designed to improve the reliability, the communication speed, and the versatility of the electronic system.We have developed novel methods for the measurement and analysis of neuronal network activity in multiple regions. The novel methods allow high-throughput data recording and analysis. In addition, transformation of the 3D volume data into 2D matrixes allows the use of currently available, welltested 2D methods for motion artefact compensation. Use of the new methods has also been published in the journal Neuron(Szalay et al, 2016 Neuron).
WP5 Neurophysiological and pharmaceutical demonstration in vitro and in vivo
The consortium started to develop the technology necessary for imaging Genetically Encoded Calcium Indicators (GECIs) is neuronal cells. FMI and Femtonics established a student exchange program to share the technology between the participants. The 3D technology was transferred successfully to IEM-HAS where the laboratory was converted to be viral-compatible and all the necessary licenses have been obtained.With a combination of techniques we reached large imaging depths in the cortex. We have also performed retinal imaging through the mouse eye. Moreover, we have developed single-cell initiated functionalized transsynaptic tracing methods in vivo in the mouse primary visual cortex and performed measurements with genetically encoded Ca-indicators to image the activity of single-cell connected neuronal networks in the cortex in 3D (Wertz et al, 2015, Science).
WP6 Demonstration of simultaneous 3D measurements and photo-activation in vivo at depth
We have developed novel software and hardware tools for optogenetic activation. All three biological laboratories have developed viral constructs to simultaneously express calcium indicators and photosensitive agents. These technologies have been used to in vivo application of Chr2 stimulation to reveal postsynaptic control of EPSP amplitude during active cortical state. We used the caged glutamate developed earlier on biological samples with a Femto3D-AO microscope (Chiovini et al, 2014 Neuron) to reveal postsynaptic computation due to concerted presynaptic network activity. Finally, we also performed the first 3D photostimulation and simultaneous calcium imaging experiments.
WP7 Development of the product prototype
This task was dependent on the system design in WP2. First,the prototypes of the AOD crystal holders and various smaller components were manufactured and tested.Then we successfully assembled the microscope prototype and corrected the unexpected optical and mechanical problems during the setting up and final testing of the device. The device has been designed and manufactured in such a high quality that it can also serve as a commercial demonstrator.
We maintained efficient networking and coordination among the members of the consortium.We organized General Assembly and Executive Board Meetings to coordinate the project. A student exchange program was established and maintained during the course of the project.
WP9 Dissemination, Collaboration and Exploitation
Numerous scientific publications were submitted during the course of the project and some of them were published in top journals of the field, such as Science, Nature Neuroscience, and Neuron. The student exchange programs were successfully continued and resulted in several common publications between the participants. The partners visited several conferences, and Femtonics Ltd exhibited on 9 conferences during the grant period. Press releases and media events were also used to advertise the project. Finally, several new patent applications were submitted.
Description of the expected final results and their potential impacts and use
Our technology will cause long lasting changes to Science, since new principles about neuronal circuit functions across distant brain regions will be discovered. To Technology, since the 3x3D system will enable large brain regions to be scanned in 3D at a cellular resolution. To Society, since the effects of retinal vision restoration in animal models of blindness can be tested in higher visual centers at an unprecedented resolution. To Theory, since theories about distributed brain computations can now be directly tested across multiple brain regions.
After the completion of the project the above list of impacts on Science, Technology, Society, and Theory will immediately be realizable since the 3x3D system will be made commercially available from Femtonics Ltd.
The development and testing of the 3x3D system requires a cross European approach, since there is no single country where all the required expertise for this project is present. It requires state-of-the-art knowledge and technologies in optics, scanning technologies, in vivo recordings, viral and genetic manipulations, physiology, and computational neuroscience.