Super-resolution imaging of human brain samples with STORM
To assess STORM imaging on human brain tissue, we first aimed to resolve well-defined histological structures such as neocortical axon tracts. Frozen samples of prefrontal cortex from control subjects were cut by standard cryostat methods (Fig. S1) and immunostained with an anti-neurofilament primary antibody and a secondary antibody conjugated with the photo-switchable fluorophore Alexa Fluor (AF) 647. Coverslips with immunostained brain sections were bathed in a switching buffer (Fig. S1) and placed on the stage of an inverted motorized microscope equipped with a high-power laser module, a total internal reflection fluorescence (TIRF) system, and an electron multiplying charge coupled device (EMCCD) single-photon sensitive camera (Fig. 1A, Fig. S2).
STORM is based on separating stochastically the emission of photo-switchable fluorescent molecules in time, so that each individual emitter can be distinguished from the others that reside within the same diffraction-limited volume. For each acquisition, thousands of frames were recorded, and each frame was computationally processed to detect activated fluorescent molecules, determine their center position, and report it as a single pixel on a corresponding reconstruction picture. The final super-resolved STORM image was obtained by merging all the reconstruction pictures in a single overlap image (Fig. 1B, Video S1). The total acquisition and reconstruction time for one super-resolved image lasted from 5 to 10 minutes, depending on the number of acquired frames.
With the aim of assessing the final resolution of the technique, we imaged axon processes with conventional wide field fluorescence microscopy, STORM, and transmission electron microscopy (TEM) in prefrontal cortex samples of age-matched control subjects. Although neurofilament fibrils (~ 10 nm) could not be defined as well as when using TEM, the caliber of axons imaged with STORM was lower than when using conventional wide field fluorescence microscopy (Fig. S3). For the same prefrontal axons, the apparent diameters and areas of longitudinally- and transversally-sectioned axons were more than 50% smaller when measured with STORM compared to wide field fluorescence microscopy (p < 0.001 for the two parameters) (Fig. 1C-E, Tables S1 and S2). In contrast, there was no significant difference when comparing apparent diameters (p = 0.441) and areas (p = 0.596) measured with STORM and TEM (Fig. 1D,E, Tables S1 and S2). STORM is thus a reliable and resolutive technique allowing to image and analyze histological brain structures at the nanoscale level.
3D-STORM and two-color STORM imaging of human brain samples
Three-dimensional (3D) and multichannel imaging provide fundamental insights into the architectural organization of nanoscale structures. To perform 3D-STORM in human brain sections we used an astigmatism-based method . After setting a cylindrical lens in the detection path of the microscope, the z coordinate of each fluorescent molecule was determined from the ellipticity of its image. Prefrontal cortex samples of control subjects were immuno-stained for neurofilaments (NF), and after reconstruction, axons were visualized in 3D with high resolution and an imaging depth of ~ 0.5 µm (Fig. 2A, Video S2). The cylindrical shape of axon tracts was discernible and both longitudinal and transversal sections of the 3D modelled axons were similar in size to those obtained with 2D-STORM.
Then, to assess two-color STORM imaging on cortex samples, we immuno-stained human brain sections with two primary antibodies detecting nearby structures, the pre- and post-synaptic Bassoon and Homer 1 proteins, and with secondary antibodies conjugated with the photoswitchable fluorophores AF 647 and AF 532 (Fig. 2B, Fig. S4). In conventional fluorescence microscopy, Bassoon and Homer 1 signals appeared diffuse and overlapping, and the synaptic clefts could not be precisely defined. In contrast, two-color STORM accurately distinguished between pre- and post-synaptic protein clusters separated by the synaptic cleft, and defined the size, orientation and organization of the synapses. Interestingly, multichannel imaging combining conventional wide field fluorescence microscopy and STORM was also achievable, allowing super-resolution imaging of brain structures using both photoswitchable and non-photoswitchable fluorophores. Although less specific than two-color STORM, this technique provides valuable information about the layout of adjacent structures, as for example the myelin sheath around axonal tracts (Fig. 2C, Fig. S4).
Together, these results demonstrate the possibility to perform 3D and two-color STORM imaging with a nanoscale resolution on human brain samples, providing valuable information to current topics in neuroscience, such as axonal organization, myelination and synaptic plasticity in the human brain.
Understanding the pathophysiology of neurodegenerative disorders to identify novel therapeutic prospects is another major challenge in neuroscience. Since most of these diseases are defined by specific intra- or extra-cellular protein aggregates in distinctive anatomical brain regions, novel insights must be disclosed on the precise characterization of the corresponding lesions. To this end, we performed STORM imaging of Amyloid-β (Aβ), Tau, α-synuclein and TDP-43 aggregates in samples from patients affected with neurodegenerative disorders.
STORM imaging of Amyloid-β and Tau proteinopathy
Alzheimer’s disease (AD) is the leading cause of dementia. The two main hallmarks of the disease are extracellular deposits of Aβ peptides, some of which constituting the core of senile plaques, and intraneuronal aggregates of hyperphosphorylated tau protein (p.Tau) called neurofibrillary tangles (NFT) . Since aggregates can measure up to 100 µm, keeping an overall view of the whole lesions is critical to study Aβ and Tau pathology in the brain, while high resolution imaging is mandatory to characterize the nanoscale organization of the misfolded proteins.
Towards this goal, we imaged entire senile plaques and degenerating neurons with STORM. Tissue samples from the prefrontal, parietal and temporal cortex of AD patients were immunostained for Aβ and p.Tau (phospho Ser202, Thr205). Auto-fluorescence quenchers were used to reduce the signal of lipofuscin, an autofluorescent pigment present in senescent neurons . Nevertheless, lipofuscin signal was specifically detectable in neuron but did not preclude STORM acquisitions.
STORM images of ~ 30 µm diameter senile plaques and ~ 15 µm degenerating neurons with NFTs were acquired. While the Aβ fibrils and the paired helical filaments of Tau could not be identified as with TEM, STORM images provided highly resolved details of the nanoscale distribution and size of Aβ and p.Tau aggregates (Fig. 3, Fig. S5). Aggregated Aβ branches were reticulated and cross-linked in the extracellular matrix, and their widths ranged from 60 to 240 nm (140.8 ± 39.6 nm, mean ± SD) (Fig. S6). Intraneuronal p.Tau NFTs appeared denser, with a honeycombed structure in the soma and a filamentous organization in the axon. The presence of unstained spots within the aggregates suggested the inclusion of other components, such as proteins or organelles. These results emphasize that STORM can be used to image Aβ and p.Tau aggregates in brain samples from AD patients with high resolution.
STORM imaging of Lewy Pathology
Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are two neurodegenerative diseases characterized by the presence of intra-neuronal phosphorylated α-synuclein (p.α-syn) immuno-reactive inclusions, called Lewy bodies (LB) . The structure of LBs varies according to their localization within the central nervous system. Two main LB types can be observed by immunohistochemistry: typical Lewy bodies (TLB) with a pale core surrounded by a dense halo mainly found in the brainstem, and smaller cortical Lewy bodies (CLB), lacking the central core and mainly detected in the neocortex. Accumulation of p.α-syn in dystrophic axons called Lewy neurites (LN) can also be observed. To date, the structure of LBs remains unclear, as the resolution of conventional fluorescence microscopy is too low to characterize their internal architecture and TEM does not provide sufficient information about their protein content and organization.
To characterize LB organization at the nanoscale level with a molecular staining approach, we performed STORM imaging on brain samples from PD and DLB patients. Substancia nigra and prefrontal cortex sections were immuno-stained with an anti-p.α-syn (phospho Ser129) antibody and images of TLBs, CLBs and LNs were acquired. The ring shaped appearance of TLBs were observed both with conventional and super-resolution imaging, although only the STORM images defined precisely their architecture (Fig. 4A). The pale core appeared unstained, while the peripheral dense halo was made of reticulated p.α-syn. Likewise, STORM imaging of CLBs revealed dense honeycomb structures that could not be observed by conventional fluorescence microscopy (Fig. 4B,C). As for p.Tau aggregates, the unstained cores and spots observed in LBs might correspond to protein partners or trapped organelles. Indeed, Lewy bodies are known to be multiprotein complexes composed of more than 100 proteins, including p.Tau . We thus performed two-color STORM imaging of LBs using antibodies against p.Tau and p.α-syn, to precisely define the internal architecture of the lesion and specifically distinguish one protein from the other (Fig. 5A). STORM imaging accurately measured the width of aggregated p.α-syn branches and the area of the unstained cores observed in CLBs, averaging 281.2 ± 95.8 nm and 0.133 ± 0.130 µm2 respectively (mean ± SD) (Fig. 5B). Finally, two-color STORM imaging of LNs revealed the internal organization of the neurites with a core of aggregated p.α-syn bounded to neurofilaments (Fig. 5C, Fig. S7). These very first STORM images of p.α-syn aggregates hold great promise for characterizing the composition and spatial organization of Lewy pathology in human brain.
STORM imaging of TDP-43 neuronal inclusions
Transactive response DNA-binding protein 43 (TDP-43), encoded by TARDBP gene, is a DNA/RNA binding protein predominantly located in the nucleus of cells under physiological condition, while the accumulation of misfolded TDP-43 in the cytosol and axons of degenerating neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) [11, 12]. TDP-43 aggregates are called neuronal cytoplasmic inclusions (NCI) when located in the cytosol, and dystrophic neurites (DN) when located in axons. The physiopathological mechanism leading to neuronal degeneration in both ALS and FTLD associates the cytoplasmic toxicity of aggregated TDP-43 and the nuclear loss of function of the protein. In frontal cortex sections from a control subject, STORM imaging revealed the presence of dense nuclear TDP-43 clusters while other nuclear zones were unstained, whereas the TDP-43 signal appeared diffuse and heterogeneous in conventional wide field fluorescence microscopy (Fig. 6A, Fig. S8). NCI and DN imaging with STORM in frontal and temporal cortex of patients affected with FTLD revealed compact and granular cytoplasmic structures, slightly denser and less reticulated than p.Tau and p.α-syn aggregates (Fig. 6B,C, Fig. S8). Interestingly, empty vacuoles were also defined in the core of NCI and DN, suggesting again the presence of additional unstained components within these aggregates. Thus, STORM imaging of TDP-43 allowed to resolve the physiological distribution of the protein in the nuclear compartment, and its spatial organization within pathological cytoplasmic aggregates.