Single-Molecule-Based Super-Resolution Images in the Presence of Multiple Fluorophores

Simonson, Paul D.; Rothenberg, Eli; Selvin, Paul R

doi:10.1021/nl203560r

Cited by 83 publications

(85 citation statements)

References 21 publications

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“…While this paper was in review, another paper published a similar approach to BaLM using bleaching of a single synthetic dye (44). As shown here, BaLM utilizes both the bleaching and blinking behaviors of synthetic dyes or fluorescent proteins and can be used in multi-spectral imaging.…”

Section: Methodsmentioning

confidence: 99%

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Burnette

Sengupta

Dai

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

197

159

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Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

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Section: Methodsmentioning

confidence: 99%

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Burnette

Sengupta

Dai

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

197

159

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“…The algorithm uses remote procedure calls to connect to a Python virtual machine implementing the DAOSTORM algorithm (Holden et al, 2011). This method uses DAOSTORM to detect and localize particles in both unprocessed and treated frames combining time averaging with frame subtraction in a process similar to a gSHRImP analysis (Simonson et al, 2011). Multiple detections caused by the same particle appearing in adjacent frames (distances smaller than 0.25 pixels) are merged and detections with low fitting accuracy are discarded.…”

Section: Quantification Of Lck Lat and Tcr Subcellular Distributionmentioning

confidence: 99%

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

Soares¹,

Henriques²,

Sachse³

et al. 2013

J Gen Physiol

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Abbreviations used: dSTORM, direct stochastic optical reconstruction microscopy; MAL, myelin and lymphocyte; Syt7, synaptotagmin-7; TIRF, total internal reflection fluorescence.M.-I. Thoulouze and A. Alcover contributed equally to this paper. Fig. 1, D and H). LAT vesicular compartment co-localized with Rab27a (35%) and Rab37 (30%; Fig. 1, M and N), two Rab molecules known to regulate cytokine and cytotoxic granule exocytosis in CD4 + and CD8 + T cells, respectively (Stinchcombe et al., 2001;Huse et al., 2006). TCR peripheral vesicles colocalized to the fast recycling Rab4b compartment (8%), whereas its core co-localized to Rab3d-(25%) and Rab8b-regulated (30%) exocytic compartments (Huse et al., 2006;Fukuda, 2008; Fig. 1, Q-S). Noteworthy, the secretory v-SNARE protein Ti-VAMP (Chaineau et al., 2009) co-localized with both LAT (30%) and TCR (30%) vesicular compartments (Fig. 1, O and W). Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse LAT and TCR vesicular release is regulated by calcium and synaptotagmin-7The traffic regulators present in LAT and TCR vesicles (Fig. 1) have been reported to be involved in stimulus-induced exocytosis at cytotoxic synapses (Ménager et al., 2007; MarcetPalacios et al., 2008). To pinpoint the molecular mechanism regulating Lck, LAT, and TCR release from vesicular compartments, we assessed the role of calcium, a known regulator of stimulus-dependent exocytosis (Stojilkovic, 2005;Pang and Südhof, 2010). To increase cytosolic calcium levels, we treated CD4 T cells either with thapsigargin or ionomycin. To accurately quantify Lck, LAT, and TCR distribution between intracellular vesicles and the plasma membrane, we stained for surface CD2 and then used it to automatically segment the plasma membrane. The resulting automated mask delimiting the internal and external outlines of the plasma membrane allowed us to discriminate and quantify the subcellular distribution (plasma membrane versus vesicular compartment) of our proteins of interest (materials and methods; unpublished data). We found LAT and TCR distribution to the vesicular compartment to be higher than anticipated by others (Bonello et al., 2004), 75% (Fig. 2, A, B, H, and I). In fact, through plasma membrane segmentation, we were able to distinguish plasma membrane-resident LAT and TCR from their intracellular compartments and subcortical vesicles, within the limits of the confocal microscopy resolution (unpublished data). Lck displayed a higher plasma membrane distribution with 25% in intracellular vesicles (Fig. 2, C and J; and not depicted).Consistent with LAT localization in Rab27a and Rab37 exocytic compartments, increased cytosolic calcium led to a clear release of its vesicular compartment into the plasma membrane (Fig. 2, A, I, and O; and not depicted). In agreement with TCR-segregated distribution into a Rab3d and Rab8b exocytic compartment and also into Rab4b rapid recycling endosomes, cytosolic calcium increase induced an inc...

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“…Given these observations, the less well-understood B. subtilis replisome appears more eukaryotic-like than the E. coli replisome, and a deeper in vivo understanding of how DNA polymerase recruitment and dynamics are performed in B. subtilis will provide valuable insight into how bacteria and eukaryotic organisms use a two-DNA polymerase system for replication. Here, we apply photobleaching-assisted microscopy (22,23), three-dimensional (3D) single-molecule superresolution microscopy (24), and single-particle tracking (25,26) to PolC in live B. subtilis cells to measure the stoichiometry, intracellular distribution, and dynamics of the essential DNA polymerase required for the majority of leading and lagging strand synthesis.…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

Liao

Schroeder

et al. 2016

Biophysical Journal

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PolC is one of two essential replicative DNA polymerases found in the Gram-positive bacterium Bacillus subtilis. The B. subtilis replisome is eukaryotic-like in that it relies on a two DNA polymerase system for chromosomal replication. To quantitatively image how the replicative DNA polymerase PolC functions in B. subtilis, we applied photobleaching-assisted microscopy, three-dimensional superresolution imaging, and single-particle tracking to examine the in vivo behavior of PolC at single-molecule resolution. We report the stoichiometry of PolC proteins within each cell and within each replisome, we elucidate the diffusion characteristics of individual PolC molecules, and we quantify the exchange dynamics for PolC engaged in lagging strand synthesis. We show that PolC is highly dynamic: this DNA polymerase is constantly recruited to and released from a centrally located replisome, providing, to our knowledge, new insight into the organization and dynamics of the replisome in bacterial cells.

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Single-Molecule-Based Super-Resolution Images in the Presence of Multiple Fluorophores

Cited by 83 publications

References 21 publications

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

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