Researchers have actually established a brand-new imaging and virtual reconstruction technology called LIONESS, which uses high-resolution imaging of live brain tissue, imagining it in real-time 3D nanoscale detail. LIONESS integrates sophisticated optics, synthetic intelligence, and a collective interdisciplinary technique, conquering the limitations of previous imaging techniques and leading the way for a better understanding of brain tissue dynamics and complexity.
Collaborative efforts at ISTA yield an extraordinary “live” view of the brains complexity.
The human brain, with its complex network of roughly 86 billion neurons, is perhaps amongst the most intricate specimens scientists have ever encountered. It holds an immense, yet currently immeasurable, wealth of info, positioning it as the peak of computational devices.
Understanding this level of complexity is challenging, making it vital for us to use sophisticated technologies that can decipher the minute, complex interactions happening within the brain at tiny levels. Hence, imaging emerges as a pivotal instrument in the realm of neuroscience.
The brand-new imaging and virtual reconstruction technology developed by Johann Danzls group at ISTA is a huge leap in imaging brain activity and is appropriately named LIONESS– Live Information Optimized Nanoscopy Enabling Saturated Segmentation. LIONESS is a pipeline to image, rebuild, and evaluate live brain tissue with a comprehensiveness and spatial resolution not possible previously.
LIONESS delineates the intricacy of dense brain tissue.” With LIONESS, for the first time, it is possible to get a detailed, dense restoration of living brain tissue. By imaging the tissue numerous times, LIONESS enables us to observe and determine the dynamic cellular biology in the brain take its course,” says first author Philipp Velicky. In this innovative method, it accomplishes a resolution of around 130 nanometers, while being mild enough for imaging of living brain tissue in real-time. Brain structure and activity are extremely dynamic; its structures evolve as the brain performs and learns new jobs.
LIONESS defines the intricacy of thick brain tissue. a: Complex neuronal environment b: LIONESS can image and rebuild the sample in a method that clarifies many dynamic structures and functions in live brain tissue. Credit: Johann Danzl
” With LIONESS, for the very first time, it is possible to get an extensive, thick restoration of living brain tissue. By imaging the tissue numerous times, LIONESS allows us to observe and determine the vibrant cellular biology in the brain take its course,” says first author Philipp Velicky. “The output is a reconstructed image of the cellular plans in 3 dimensions, with time making up the 4th measurement, as the sample can be imaged over minutes, hours, or days,” he adds.
Cooperation and AI the Key
The strength of LIONESS depends on improved optics and in the two levels of deep knowing– a technique of Artificial Intelligence– that make up its core: the very first improves the image quality and the second recognizes the various cellular structures in the thick neuronal environment.
The pipeline is an outcome of a cooperation in between the Danzl group, Bickel group, Jonas group, Novarino group, and ISTAs Scientific Service Units, in addition to other worldwide collaborators. “Our approach was to put together a vibrant group of scientists with special combined proficiency across disciplinary boundaries, who work together to close an innovation gap in the analysis of brain tissue,” Johann Danzl of ISTA states.
A pipeline to reconstruct live brain tissue. Acquisition of Microscopy with enhanced laser focus– Image Processing (DL)– Segmentation (DL)– 3D visual analysis. Credit: Johann Danzl
Surpassing difficulties
Previously it was possible to get reconstructions of brain tissue by using Electron Microscopy. This technique images the sample based upon its interactions with electrons. Regardless of its ability to capture images at a few nanometers– a millionth of a millimeter– resolution, Electron Microscopy needs a sample to be repaired in one biological state, which requires to be physically sectioned to acquire 3D details. No vibrant info can be acquired.
Another previously understood technique of Light Microscopy permits observation of living systems and record undamaged tissue volumes by slicing them “optically” rather than physically. Nevertheless, Light Microscopy is severely hindered in its dealing with power by the really properties of the light waves it utilizes to generate an image. Its best-case resolution is a few hundred nanometers, much too coarse-grained to capture crucial cellular details in brain tissue.
Utilizing Super-resolution Light Microscopy researchers can break this resolution barrier. Current operate in this field, called SUSHI (Super-resolution Shadow Imaging), revealed that applying dye molecules to the spaces around cells and applying the Nobel Prize-winning super-resolution method STED (Stimulated Emission Depletion) microscopy exposes super-resolved shadows of all the cellular structures and therefore pictures them in the tissue.
LIONESS can image and reconstruct the sample in a method that clarifies numerous vibrant structures and functions in live brain tissue. Credit: Julia Lyudchik ISTA
It has actually been impossible to image entire volumes of brain tissue with resolution improvement that matches the brain tissues complex 3D architecture. This is because increasing resolution likewise involves a high load of imaging light on the sample, which may harm or fry the subtle, living tissue.
Herein lies the prowess of LIONESS, having actually been developed for, according to the authors, “quick and moderate” imaging conditions, hence keeping the sample alive. The method does so while supplying isotropic super-resolution– suggesting that it is equally great in all 3 spatial dimensions– that permits visualization of the tissues cellular elements in 3D nanoscale resolved information.
LIONESS gathers just as little info from the sample as needed during the imaging action. This is followed by the very first deep knowing action to fill in extra information on the brain tissues structure in a process called Image Restoration. In this ingenious way, it accomplishes a resolution of around 130 nanometers, while being gentle enough for imaging of living brain tissue in real-time. Together, these actions permit a second step of deep knowing, this time to make sense of the incredibly intricate imaging information and recognize the neuronal structures in an automatic manner.
ISTA Scientist Johann Danzl in his lab at the Institute of Science and Technology Austria. Credit: Nadine Poncioni|ISTA
Homing In
” The interdisciplinary technique allowed us to break the intertwined limitations in fixing power and light exposure to the living system, to make sense of the complex 3D data, and to pair the tissues cellular architecture with molecular and functional measurements,” states Danzl.
For virtual reconstruction, Danzl and Velicky coordinated with visual computing experts: the Bickel group at ISTA and the group led by Hanspeter Pfister at Harvard University, who contributed their know-how in automated division– the procedure of immediately acknowledging the cellular structures in the tissue– and visualization, with further assistance by ISTAs image analysis staff researcher Christoph Sommer. For sophisticated labeling techniques, neuroscientists and chemists from Edinburgh, Berlin, and ISTA contributed.
Subsequently, it was possible to bridge practical measurements, i.e. to read out the cellular structures together with biological signaling activity in the same living neuronal circuit. This was done by imaging Calcium ion fluxes into cells and determining the cellular electrical activity in cooperation with the Jonas group at ISTA. The Novarino group contributed human cerebral organoids, often nicknamed mini-brains that imitate human brain advancement. The authors underline that all of this was facilitated through specialist support by ISTAs first-class clinical service units.
Brain structure and activity are extremely dynamic; its structures evolve as the brain carries out and learns new jobs. This element of the brain is typically referred to as “plasticity”. Observing the changes in the brains tissue architecture is important to opening the tricks behind its plasticity. The new tool established at ISTA shows possible for comprehending the practical architecture of brain tissue and possibly other organs by exposing the subcellular structures and catching how these may alter with time.
Reference: “Dense 4D nanoscale restoration of living brain tissue” by Philipp Velicky, Eder Miguel, Julia M. Michalska, Julia Lyudchik, Donglai Wei, Zudi Lin, Jake F. Watson, Jakob Troidl, Johanna Beyer, Yoav Ben-Simon, Christoph Sommer, Wiebke Jahr, Alban Cenameri, Johannes Broichhagen, Seth G. N. Grant, Peter Jonas, Gaia Novarino, Hanspeter Pfister, Bernd Bickel and Johann G. Danzl, 10 July 2023, Nature Methods.DOI: 10.1038/ s41592-023-01936-6.
The research study was funded by the Austrian Science Fund, Gesellschaft für Forschungsförderung NÖ (NFB), H2020 Marie Skłodowska-Curie Actions, the H2020 European Research Council, the Human Frontier Science Program, the Simons Foundation, the Wellcome Trust, and the National Science Foundation.