Abstract:A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several comm… Show more
“…As the chromosome preparations used in this study were extremely flat, we did not see an advantage to apply Airyscan imaging in addition to SIM. However, the interested reader can find a comparison between Airyscan and SIM to investigate the surface texture and 3D shape of pollen [ 30 ], and the Airyscan advantages described when imaging thicker samples [ 31 ].…”
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
“…As the chromosome preparations used in this study were extremely flat, we did not see an advantage to apply Airyscan imaging in addition to SIM. However, the interested reader can find a comparison between Airyscan and SIM to investigate the surface texture and 3D shape of pollen [ 30 ], and the Airyscan advantages described when imaging thicker samples [ 31 ].…”
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
“… Note: The STORM acquisition software provided here is available as an open-source resource under the MIT license. Alternatives: Commercial instrumentation and software may be used in lieu of custom instrumentation and specific codebases we reference here ( Albrecht et al, 2021 ; Chang, 2015 ; Tröger et al., 2020 ). Figure 8 Automated image acquisition (A) Screenshot of the hal4000.py user interface showing a ROI in the 561 nm channel (WGA) within a single ultrathin section of mouse retina.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Alternatives: Commercial instrumentation and software may be used in lieu of custom instrumentation and specific codebases we reference here ( Albrecht et al, 2021 ; Chang, 2015 ; Tröger et al., 2020 ).…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Furthermore, this protocol requires technical skills in histology and ultrasectioning ( Harris et al., 2006 ; Micheva and Smith, 2007 ). Additionally, users should familiarize themselves with principles of single-molecule localization microscopy (SMLM) ( Lelek et al, 2021 ) and operation of available commercial instrumentation ( Albrecht et al, 2021 ; Chang, 2015 ; Tröger et al., 2020 ).…”
Section: Before You Beginmentioning
confidence: 99%
“…Resources for building SMLM instruments have been published ( Bates et al, 2013 ; Dempsey, 2013 ; Ma et al, 2017 ). Commercially available instruments for SMLM are also available ( Albrecht et al, 2021 ; Chang, 2015 ; Tröger et al., 2020 ).…”
Summary
Here, we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models.
For complete details on the use and execution of this protocol, please refer to
Sigal et al. (2015)
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