The 2015 super-resolution microscopy roadmap (1711.04999v1)

Abstract: 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.

• The paper presents a comprehensive review of super-resolution techniques that surpass diffraction limits to reveal synaptic structures.
• The paper details advancements in STED and single-molecule localization methods that precisely map dendritic spines and receptor dynamics.
• The paper highlights challenges like imaging depth and speed in live tissues and advocates for multidisciplinary strategies to enhance neural imaging.

Overview of Super-Resolution Microscopy in Neurosciences: Focus on Synapses

The paper authored by Laurent Cognet and Brahim Lounis provides a comprehensive review of the impact and future potential of super-resolution microscopy techniques in the field of neuroscience, with particular emphasis on the paper of synapses. The investigation of synaptic structures at the nanoscale level is pivotal to advancing our comprehension of neuronal communication processes that drive brain functionality and plasticity.

Super-Resolution Microscopy Techniques

The paper discusses several key super-resolution microscopy (SRM) techniques that have been instrumental in enhancing the resolution beyond the diffraction limit of light microscopy, which traditionally cannot resolve structures such as synapses measuring at submicron scales. Techniques like STED (stimulated emission depletion) microscopy and single-molecule localization methods such as (f)PALM, (d)STORM, and (u)PAINT are covered.

STED microscopy enhances resolution by optically confining fluorescence emission, allowing for the visualization of dendritic spine shapes with high precision. Single-molecule localization techniques have aided in elucidating nanoscale molecular organizations, enabling the discovery of mobile neurotransmitter receptors and their role in synaptic transmission, significantly enriching our understanding of synaptic functionality.

Challenges and Future Directions

Despite advancements, SRM in living tissues faces considerable challenges. The paper highlights obstacles related to imaging depth and speed, especially considering light scattering and absorption in thick biological samples that impair STED performance. Two-photon microscopy offers a potential solution due to its ability to penetrate deeper using near-infrared excitations, but it introduces complications like enhanced photobleaching rates.

It is advocated that future improvements should focus on developing near-infrared probes with better photophysical properties to fully exploit biological transparency windows, and advancing wide-field super-resolution imaging to accommodate the dynamics of living samples in three dimensions. Further, adapting super-resolution methodologies to more integrated, intact neural samples remains a pivotal goal. This endeavor will necessitate multidisciplinary collaboration across physics, chemistry, computational science, and neurophysiology to overcome the intricate challenges of brain imaging.

Implications for Neuroscience

The capabilities of SRM tools extend significant implications for various neuroscience domains, including developmental neuroscience, aging studies, and neurodegenerative disease research. The detailed synaptic-level insights gained could enhance the understanding of synaptic processes within the context of the broader brain organ function.

To maximize these implications, combining SRM with other imaging modalities and manipulating the brain's physiological state with techniques such as optogenetics are suggested as promising future directions. This integrative approach aims not only to image but to manipulate and interpret brain functions in both physiological and pathophysiological states.

Conclusion

Laurent Cognet and Brahim Lounis have presented a critical reflection on super-resolution microscopy's current state and projected developments within neuroscience. The research subtly underscores the existing technological constraints while emphasizing the groundbreaking molecular and cellular insights gained thus far. The advancement of super-resolution techniques holds potential not only for elaborating synaptic structures but also in linking microscopic details to macroscopic brain functions, potentially revolutionizing our understanding of neurological processes.