Although biological imaging is mostly performed in aqueous media, it is hardly ever considered that water acts as a classic fluorescence quencher for organic fluorophores. By investigating the fluorescence properties...

In spite of their relatively low fluorescence quantum yield, cyanine dyes such as Cy3, Cy5, or Cy7 are widely used in single-molecule fluorescence applications due to their high extinction coefficients and excellent photon yields. We show that the fluorescence quantum yield and lifetime of red-emitting cyanine dyes can be substantially increased in heavy water (D2 O) compared with water (H2 O). We find that the magnitude of the quantum yield increase in D2 O scales with the emission wavelength, reaching a particularly high value of 2.6-fold for the most red-emitting dye investigated, Cy7. We further demonstrate a higher photon yield in single-molecule superresolution experiments in D2 O compared to H2 O, which leads to an improved localization precision and hence better spatial resolution. This finding is especially beneficial for biological applications of fluorescence microscopy, which are typically carried out in aqueous media and which greatly profit from the red spectral range due to reduced cellular auto-fluorescence.

A new photoactivatable fluorophore based on a quinone-cyanine-7 (QCy7) scaffold was synthesized and spectroscopically characterized. The fluorophore exhibits fluorescence in the near-infrared spectral window and a large Stokes shift. Its application in super-resolution microscopy as an antibody bioconjugate is demonstrated.

A new one- and two-photon activatable fluorophore based on ATTO565 was developed using a photolabile linker that simultaneously acts as a quencher. It is especially interesting for protein and peptide applications because it can be incorporated by standard peptide chemistry. The application of the new fluorogenic construct in super-resolution microscopy of antibody conjugates is shown.

Array tomography (AT) is a method for mapping the expression patterns of many proteins at high resolution in three dimensions. Using ultrathin serial tissue sections, multiple cycles of immunohistochemistry, optical imaging, and 3D reconstruction, AT can map the expression of multiple proteins at submicrometer resolution across large tissue volumes. Using antibodies to synaptic proteins, AT has been used to reconstruct synaptic architecture in brain tissue, and we have developed automated routines for counting synapses. To validate our algorithms and immunostaining, we examined AT samples by both light and electron microscopy in order to correlate synaptic protein immunoreactivity and ultrastructure. We cut a 30-section array of 70 nm serial sections of Lowicryl-embedded mouse hippocampus and ran two cycles of immunostaining and light microscopy. It was then stained with heavy metals and a subregion of six serial sections imaged by field-emission scanning electron microscopy (SEM). After deconvolving, stitching, aligning and merging AT and SEM data, we manually scanned the SEM volume and detected 98 synapses, largely by the presence of electron-dense post-synaptic densities. We then overlaid fluorescence signals from anti-PSD-95 and anti-synapsin antibodies (Cell Signaling) and compared them to the ultrastructurally-defined synapses. Finally, we ran light-level data through our automated synapse-detection algorithm which detects the co-localization of PSD-95 and synapsin local maxima.To determine false negatives, we examined how many SEM-identified synapses were detected by the algorithm. An optimized algorithm detected 72% of SEM-identified excitatory synapses. False-negatives were caused by failure of either immunostaining (12%) or the algorithm (16%). To determine false positives, we examined how many algorithm-detected synapses corresponded to SEM-identified synapses. 81% were validated, while 19% were falsepositives often due to the close-packing of multiple synapses. We expect to extend these analyses to other markers and algorithms. 2013-Pos Board B743Structural and Dynamic Study of Caveolin-1 Membrane Microdomains by Single Molecule Imaging Ramunas Stanciauskas, Fabien Pinaud. Within the plasma membrane, the protein caveolin-1 (cav-1) forms various diffraction limited microdomains that have yet to been precisely characterized in terms of structure and dynamics. Using single molecule and super-resolution imaging techniques we study the membrane distribution and the dynamics of caveolae and cav-1 scaffolds at the nanometer scale in both fixed and live cells. Cav-1 knock out MEF cells were rescued with cav-1 fusions to SNAP, ptagRFP or mCherry tags co-expressed at equimolar amounts with a cav-1-split-GFP fusion for 3D super-resolution imaging and quantitative posttranslational modifications of individual cav-1 proteins with single molecule sensitivity. At the plasma membrane, 3D super-resolution imaging by dSTORM and PALM revealed the expected~100 nm globular shape of caveolae as well as single layer cav-1 d...