Abstract:Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze … Show more
“…A tool published by Merino et al . 44 , enables to obtain an objective parameter, in which the sample is also considered, that can be used to determine the optimal settings for the STED image acquisition. Using this tool, we demonstrated an extension in the frequencies that allowed identifying objects down to 110 nm; in contrast, with AT alone the size of the smallest structure detected in the lateral plane was 220 nm.Figure 2Comparison of the resolution between AT-Confocal and AT-STED modalities.…”
Section: Resultsmentioning
confidence: 99%
“…Additionally, in the super-resolution (SR) fluorescence microscopy domain, Stimulated Emission Depletion (STED) microscopy is a technique that combines a high resolution confocal microscope with a high power donut-shaped depletion laser. The STED configuration uses the excitation and depletion lasers simultaneously increasing the lateral resolution by reducing the emission area selectively depleting the fluorescent molecules under the donut-shaped beam 43 , 44 .…”
Soluble amyloid-β (Aβ) is considered to be a critical component in the pathogenesis of Alzheimer’s disease (AD). Evidence suggests that these non-fibrillar Aβ assemblies are implicated in synaptic dysfunction, neurodegeneration and cell death. However, characterization of these species comes mainly from studies in cellular or animal models, and there is little data in intact human samples due to the lack of adequate optical microscopic resolution to study these small structures. Here, to achieve super-resolution in all three dimensions, we applied Array Tomography (AT) and Stimulated Emission Depletion microscopy (STED), to characterize in postmortem human brain tissue non-fibrillar Aβ structures in amyloid plaques of cases with autosomal dominant and sporadic AD. Ultrathin sections scanned with super-resolution STED microscopy allowed the detection of small Aβ structures of the order of 100 nm. We reconstructed a whole human amyloid plaque and established that plaques are formed by a dense core of higher order Aβ species (~0.022 µm3) and a peripheral halo of smaller Aβ structures (~0.003 µm3). This work highlights the potential of AT-STED for human neuropathological studies.
“…A tool published by Merino et al . 44 , enables to obtain an objective parameter, in which the sample is also considered, that can be used to determine the optimal settings for the STED image acquisition. Using this tool, we demonstrated an extension in the frequencies that allowed identifying objects down to 110 nm; in contrast, with AT alone the size of the smallest structure detected in the lateral plane was 220 nm.Figure 2Comparison of the resolution between AT-Confocal and AT-STED modalities.…”
Section: Resultsmentioning
confidence: 99%
“…Additionally, in the super-resolution (SR) fluorescence microscopy domain, Stimulated Emission Depletion (STED) microscopy is a technique that combines a high resolution confocal microscope with a high power donut-shaped depletion laser. The STED configuration uses the excitation and depletion lasers simultaneously increasing the lateral resolution by reducing the emission area selectively depleting the fluorescent molecules under the donut-shaped beam 43 , 44 .…”
Soluble amyloid-β (Aβ) is considered to be a critical component in the pathogenesis of Alzheimer’s disease (AD). Evidence suggests that these non-fibrillar Aβ assemblies are implicated in synaptic dysfunction, neurodegeneration and cell death. However, characterization of these species comes mainly from studies in cellular or animal models, and there is little data in intact human samples due to the lack of adequate optical microscopic resolution to study these small structures. Here, to achieve super-resolution in all three dimensions, we applied Array Tomography (AT) and Stimulated Emission Depletion microscopy (STED), to characterize in postmortem human brain tissue non-fibrillar Aβ structures in amyloid plaques of cases with autosomal dominant and sporadic AD. Ultrathin sections scanned with super-resolution STED microscopy allowed the detection of small Aβ structures of the order of 100 nm. We reconstructed a whole human amyloid plaque and established that plaques are formed by a dense core of higher order Aβ species (~0.022 µm3) and a peripheral halo of smaller Aβ structures (~0.003 µm3). This work highlights the potential of AT-STED for human neuropathological studies.
“…The CW STED laser operates at the wavelength of 592 nm and imaging is performed using hybrid detectors. The system performance has been fully characterized and a complete analysis of the achieved transversal resolution can be found elsewhere (Merino et al, 2017). Sections with omission of antibodies or with secondary antibodies only were imaged to ensure specific and independent fluorophore visualization.…”
Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated α-synuclein phosphorylated at Ser129. Although phosphorylated α-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other α-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated α-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimer's disease and five healthy control subjects to analyse the presence of phosphorylated α-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or postsynaptic terminals were analysed. We found that phosphorylated α-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (<0.16 µm3). Between 19% and 25% of phosphorylated α-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated α-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated α-synuclein aggregation in synapses (pre > pre + post > postsynaptic) was observed. These results indicate that phosphorylated α-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated α-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated α-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.
“…Although nanoscopy techniques and STED, in particular, can theoretically achieve unlimited resolution, experimental constraints on biological samples considerably reduce the spatial resolution improvement to about 20 nm. Moreover, a series of factors related to cell labelling 5,6 and image acquisition [7][8][9][10][11] must be carefully assessed and adjusted depending on the biological mechanism under investigation. Examples of acquisition parameters that must be carefully adjusted in STED microscopy are the STED beam intensity, the excitation beam integration and the pixel dwell-time.…”
Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.
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