Investigating Cellular Structures at the Nanoscale with Organic Fluorophores

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure

Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging

In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity

Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells

A Blueprint for Cost-Efficient Localization Microscopy

Holm T, Klein T, Loeschberger A, et al. A Blueprint for Cost-Efficient Localization Microscopy. ChemPhysChem. 2014;15(4):651-654

Impact of hypotonicity on the myo-inositol permeability <i>P</i>_ino in HEK293 cells.

<p>The <i>P</i>_ino values were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">Eq. 2</a> from the rates of secondary swelling, using the volumetric data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g002" target="_blank">Fig. 2A</a>. The fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a> to the data yielded a <i>C</i>₅₀ value of 144 ± 10 mOsm, i.e. the tonicity at which the myo-inositol permeability was half-activated. In the inset, the same <i>P</i>_ino data are plotted as function of the cell volume at the time point of <i>myo</i>-inositol application. Curve fitting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a>) shows that <i>P</i>_ino was half-activated as cells swelled by about 26% (<i>v</i>₅₀ = 1.26±0.02).</p

<i>d</i>STORM imaging of immunolabeled SLC5A3 protein in the plasma membrane of HEK293 cells under isotonic and hypotonic conditions.

<p>Images of the same cells in transmitted light (TL) are also shown. From the <i>d</i>STORM images (reconstructed from 15,000 single frames), the surface membrane density of SLC5A3 localizations [loc/μm²] were identified in individual cells. The bar graph shows the impact of hypotonic stress on the surface membrane density of SLC5A3 protein localizations. The data are means (±SD) from 8–16 individual cells for each osmotic condition and hypotonic stress duration. The differences in the mean values between the isotonic control and the two hypotonic samples were statistically significant (as denoted by *; <i>P</i> < 0.05), according to the Mann-Whitney test conducted using the Software Origin 9 (Microcal, Northampton, MA). The difference between the two hypotonic samples (10 vs 20 min) was not significant (n.s.).</p