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The 2015 super-resolution microscopy roadmap
Stefan Hell, Steffen Sahl, Mark Bates, Xiaowei Zhuang, Rainer Heintzmann, Martin J Booth, Joerg Bewersdorf, Gleb Shtengel, Harald Hess, Philipp Tinnefeld, Alf Honigmann, Stefan Jakobs, Ilaria Testa, Laurent Cognet, Brahim Lounis, Helge Ewers, Simon J Davis, Christian Eggeling, David Klenerman, Katrin Willig, Giuseppe Vicidomini, Marco Castello, Alberto Diaspro, Thorben Cordes, Steffen J Sahl, Philip Tinnefeld, David Klenerman, Katrin I Willig
TL;DR
Super-resolution microscopy has revealed nanoscale neuronal structures, but its broader use in neuroscience remains constrained by challenges in imaging integrated, intact living tissue. This roadmap reviews major approaches, their applications, and the requirements for connecting molecular-scale observations with brain function.
Problem
Super-resolution neuroscience must extend demonstrations from dissociated cultures and thin fixed slices to integrated, ultimately intact living samples.
Method
The roadmap synthesizes STED and single-molecule localization approaches, their nanoscale applications, and requirements for imaging living brain tissue.
Results
STED and localization methods have visualized nanoscale molecular organization, spine morphology, synaptic compartmentalization, and actin dynamics in neuronal preparations.
Takeaways & Limitations
Widespread neuroscience application requires linking molecular-scale subcellular information to macroscopic organ function through multidisciplinary collaboration.
Takeaways & Limitations
Light scattering and absorption limit penetration in thick brain tissue, while two-photon excitation increases photobleaching and complicates single-molecule super-resolution.
Abstract
from arXiv · showhide
Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.
Status
Understanding brain processes requires investigating organization at subcellular scales, while advances in neuroscience have expanded knowledge of cellular mechanisms underlying neuronal communication and their evolution during life.
- Status: Subcellular investigations of brain organization are key to understanding brain processes.The brain’s efficient functioning reflects its multiscale complexity.
- Status: Neuroscience has greatly expanded knowledge of cellular mechanisms involved in neuronal communication and their evolution during life.This growing understanding has largely come from discovering molecules.
- Status: uPAINT enabled high-density single-molecule trajectories and super-resolution imaging of endogenous glutamate receptors on a live neuron.The image includes a synapse and has a 500 nm scale bar.
Current and Future Challenges
Super-resolution microscopy generally controls the number of emitting molecules within specific imaging volumes, with STED and single-molecule localization methods achieving major applications in neuroscience. These approaches have enabled nanoscale studies of molecular organization and live-neuron synaptic structures.
- Methodological foundations: Super-resolution methods mostly rely on controlling the number of emitting molecules in specific imaging volumes.The passage identifies molecular-emission control as a common basis of these methods.
- Methodological foundations: Two major neuroscience approaches are STED, which optically shapes localized fluorescence-emission volumes, and single-molecule localization methods including PALM, STORM, and PAINT.The passage lists (f)PALM, (d)STORM, and (u)PAINT as single-molecule localization methods.
- Neuroscience applications: Single-molecule localization methods have proven powerful for studying nanoscale molecular organizations.The supplied passage begins an example involving nanoscale molecular organization but is truncated.
- Neuroscience applications: STED microscopy visualizes dendritic spine shapes in living neurons with remarkable resolution and reveals spine-neck plasticity regulation of synaptic compartmentalization.The passage also states that STED further deciphered the dynamic organization of actin.
- Neuroscience applications: Nanometer-resolution imaging of synaptic structures and their molecular contents constitutes a breakthrough.This conclusion is presented as an example of the achievements described in the passage.
Advances in Science and Technology to Meet Challenges
Super-resolution imaging in intact living brain tissue is emerging because thick biological samples limit visible-light penetration through scattering and absorption. These effects are especially detrimental to STED, which requires a zero-intensity depletion region.
- Super-resolution imaging in intact living brain tissues requires several conditions to address major neuroscience questions.The passage states that this capability is conditioned on several requirements.
- Light scattering and absorption limit visible-light penetration in thick biological samples, making tissue super-resolution imaging only emerging.These optical effects constrain imaging depth in tissues.
- Scattering is particularly detrimental to STED microscopy because depletion requires a zero-intensity region.STED depends on realizing this zero-intensity region for depletion.
Concluding Remarks
Widespread neuroscience applications of super-resolution imaging require linking molecular-scale subcellular information to global organ function in a well-defined functional state. Achieving this will require a multidisciplinary collaboration spanning several scientific and clinical fields.
- Concluding Remarks: The main challenge is connecting molecular-scale subcellular information, such as synaptic processes, with global organ function at macroscopic scales.This connection must be made in a well-defined functional state.
- Concluding Remarks: Physicists, chemists, computer scientists, neurophysiologists, and neuropathologists will need to work together to achieve this integration.The paper characterizes the task as comparable to the complexity of the brain.
- Concluding Remarks: STED microscopy can reveal long-term-potentiation effects on the morphology of synaptic spines and heads.The example is shown with a 500 nm scale bar.