It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence ...nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120
μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.
Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the ...mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-μm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.
Abstract
Eight EXOGAM-type, Ge-clover detectors of the EXILL array,
arranged on an octagon plan and acting as Compton polarimeters, were
used to measure directional-polarization correlations of
γ
...rays populated in various processes. Measurements of
γ
radiation following neutron-induced fission of
235
U and
241
Pu targets;
β
-
-decay following fission; (n,
γ
)
reactions on various stable targets and measurements using
radioactive sources of
60
Co,
133
Ba and
152
Eu were
performed at the PF1B cold-neutron facility of the Institut
Laue-Langevin in Grenoble. Digital electronics and a trigger-less
acquisition system allowed the collection of signals from Ge
detectors down to about 20 keV, providing measurements of linear
polarization down to 120 keV. The precise sensitivity
calibration was determined for the set of eight EXILL clover
polarimeters in a range from 100 keV to 10000 keV. A new formula
for calculating the directional-polarization correlations for the
upper transition in a
γγ
cascade is derived from first
principles. Directional-polarization correlations were measured for
a few dozens of transitions in a number of nuclei, including values
for twenty transitions in the
152
Sm nucleus measured for the
first time, and compared against the calculated values, to
illustrate the quality of the technique. The combined analysis of
angular and directional-polarization correlations is presented,
which helps the determination of spin-parity assignments to excited
levels.
Abstract
Ca
2+
influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca
2+
signaling is critical for understanding ...synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20–330) and arrangement (mostly linear 70 nm × 100–600 nm clusters) of Ca
2+
channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca
2+
imaging, we analyze presynaptic Ca
2+
signals at the nanometer scale and find confined elongated Ca
2+
domains at normal IHC AZs, whereas Ca
2+
domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca
2+
concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca
2+
-channel clusters of similar density but scalable length, thereby varying the number of Ca
2+
channels amongst individual AZs.
Background. In preclinical stroke models, improvement in motor performance is associated with reorganization of cortical motor maps. However, the temporal relationship between performance gains and ...map plasticity is not clear. Objective. This study was designed to assess the effects of rehabilitative training on the temporal dynamics of behavioral and neurophysiological endpoints in a rat model of focal cortical infarct. Methods. Eight days after an ischemic infarct in primary motor cortex, adult rats received either rehabilitative training or were allowed to recover spontaneously. Motor performance and movement quality of the paretic forelimb was assessed on a skilled reach task. Intracortical microstimulation mapping procedures were conducted to assess the topography of spared forelimb representations either at the end of training (post-lesion day 18) or at the end of a 3-week follow-up period (post-lesion day 38). Results. Rats receiving rehabilitative training demonstrated more rapid improvement in motor performance and movement quality during the training period that persisted through the follow-up period. Motor maps in both groups were unusually small on post-lesion day 18. On post-lesion day 38, forelimb motor maps in the rehabilitative training group were significantly enlarged compared with the no-rehab group, and within the range of normal maps. Conclusions. Postinfarct rehabilitative training rapidly improves motor performance and movement quality after an ischemic infarct in motor cortex. However, training-induced motor improvements are not reflected in spared motor maps until substantially later, suggesting that early motor training after stroke can help shape the evolving poststroke neural network.
This collection of essays is a memorial volume of Romance language etymological essays written by Prof. Carlton Cosmo Rice (1876-1945), a leading scholar of philology and linguistics at the time, and ...gathered by Urban T. Holmes.
Three annotated essays are examined and conjectures are made as to events probably occurring during the period. The essays are "Samuel Pepys in Paris," "Medieval Gardens," and "A Twelfth-Century ...Schoolmaster."
Lens-based fluorescence microscopy, which has long been limited in resolution to about 200 nanometers by diffraction, is rapidly evolving into a nanoscale imaging technique. Here, we show that the ...superresolution fluorescence microscopy called RESOLFT enables comparatively fast and continuous imaging of sensitive, nanosized features in living brain tissue. Using low-intensity illumination to switch photochromic fluorescent proteins reversibly between a fluorescent and a nonfluorescent state, we increased the resolution more than 3-fold over that of confocal microscopy in all dimensions. Dendritic spines located 10–50 μm deep inside living organotypic hippocampal brain slices were recorded for hours without signs of degradation. Using a fast-switching protein increased the imaging speed 50-fold over reported RESOLFT schemes, which in turn enabled the recording of spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours.
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► 3D superresolution (xy, 65 nm; z, 110 nm) of living brain using low light levels ► Imaging speed is increased 50-fold over reported schemes ► Long- and short-term changes of spine morphology and actin filaments observed ► No adverse photo-induced effects observed
Testa et al. introduce RESOLFT, a new superresolution fluorescence microscopy technique enabling continuous imaging of sensitive, nanosized features in living brain tissue using very low levels of light. Dendritic spines were recorded for hours without signs of degradation.
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