Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore

Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.Peer ReviewedPostprint (published version

Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer ReviewedPostprint (author's final draft

Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer Reviewe

Virtual grid to relocate the same region of interest.

<p>(A–B) During the first imaging session, the coordinates of two reference points are recorded (<i>P₁</i> and <i>P₂</i>, typically the corner coordinates of the sample chamber as shown in B) as well as the coordinates of the region of interest, <i>C</i>. During the subsequent imaging sessions, the new coordinates of the reference points are recorded (<i>P₁'</i> and <i>P₂'</i>) and these coordinates along with the previously recorded coordinates of the reference points and region of interest are used to calculate the new coordinates of the region of interest (<i>C</i>′). (C) Fiduciary markers (fluorescent beads) imaged on four subsequent days using the “virtual grid” approach to locate them. Scale bar 5 µm.</p

Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore

<div><p>Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.</p></div

Multi-color STORM imaging using a single fluorophore.

<p>(A) Microtubules (green) and mitochondrial outer membrane protein Tom20 (magenta) imaged sequentially using the same fluorophore activator-reporter pair (AlexaFluor405-AlexaFluor647). Arrows show the localized positions of fiduciary markers (fluorescent beads) that were used for image alignment. (B) Mitochondrial outer membrane protein Tom20 (magenta) and inner membrane protein ATP Synthase (green). (C) Three-color image of microtubules (green), mitochondrial outer membrane protein Tom20 (magenta) and mitochondrial inner membrane protein (ATP-synthase, orange) imaged sequentially using the same fluorophore activator-reporter pair (AlexaFluor405-AlexaFluor647). The discontinuous appearance of microtubules is due to the fact that we have used an anti-GFP antibody to label the GFP-α-tubulin and the endogenous α-tubulin is unlabelled in this scheme. The anti-GFP antibody was used since it offers a different antibody species to those used for ATP-synthase and Tom20. Scale bars, 1 µm (A–B), and 2 µm (C).</p

Multi-color STORM imaging using overlapping antibody species.

<p>(A) An image of ATP-synthase (localized to mitochondria) and LAMP2 (localized to lysosomes) both labelled using a mouse monoclonal primary and anti-mouse secondary antibody and imaged at the same time. (B) An image of Tom20, a mitochondrial outer membrane protein. Since Tom20 and ATP-synthase colocalize on mitochondria (arrows), the colocalization can be used to separate the initial image into separate colors. (C) ATP-synthase is identified as those molecules which colocalize with Tom20. Lysosomes are identified as those molecules which do not colocalize with Tom20. (D) A zoom-out of the three color Tom20 (magenta), ATP-Synthase (green), lysosome (orange) STORM image. (E) A five-color STORM image of mitochondrial outer membrane protein Tom20 (orange), mitochondrial inner membrane protein ATP-synthase (cyan), lysosomal protein Lamp2 (red), total tubulin (green) and acetylated tubulin (magenta). The five-color image is split between the two panels to more clearly display the different structures. The acetylated tubulin, ATP-synthase, and Lamp2 are all imaged using mouse primary antibodies. The acetylated tubulin colocalizes with total tubulin and ATP-synthase colocalizes with Tom20; Lamp2 does not colocalize with either total tubulin nor Tom20. Scale bars, 500 nm (C), 2 µm (D) and 5 µm (E).</p

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

<div><p>Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells <i>in situ</i> followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.</p></div

Conceptual application of multi-color STORM imaging using the same antibody species and same fluorophore.

<p>(A) Protein A (green) on the pre-synaptic structure (orange) and Protein B (green) on the post-synaptic structure (red) simultaneously colocalize with a third protein, Protein C (purple). (B) Protein C (purple) separately colocalizes with both Protein A (green) on the pre-synaptic structure (orange) and Protein B (green) on the post-synaptic structure (red) but rarely colocalizes with both proteins simultaneously. Sequential imaging using the same antibody species to label Protein A and B can be used to distinguish between these two scenarios (C) A second example following the same scenario as (A) and (B) but with mitochondrial inner and outer membrane proteins.</p

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors

Summary: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients’ antibodies cause cross-linking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients’ antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs. : Ladépêche et al. visualize NMDAR nano-organization in a model of NMDAR encephalitis. NMDARs organize in nano-objects, which show a time-dependent and subunit-specific change in their size and content upon patients’ antibody treatment. EphB2 receptor activation, which stabilizes NMDAR-protein interactions, partially antagonizes the alteration of NMDAR nano-organization caused by patients’ antibodies. Keywords: NMDAR encephalitis, antibody, autoimmune, pathogenic mechanism, STORM, super-resolution microscopy, synaptic proteins, GluN2 subunit