Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy

Diezmann, Alex von; Lee, Maurice Y.; Lew, Matthew D.; Moerner, W. E.

doi:10.1364/optica.2.000985

Cited by 91 publications

(89 citation statements)

References 56 publications

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“…Calibration scans were created by axially scanning a nanohole array (NHA) 45 filled with ~25 μL of ∼50 μM Alexa 568 (Life Technologies) and Atto 520 (Sigma) dyes using a piezoelectric objective scanner (Mad City Labs, C-focus).…”

Section: Data Analysis Proceduresmentioning

confidence: 99%

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

Bayas

Wang

Lee

et al. 2017

Preprint

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We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in E. coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase E's function in ribosomal RNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/228122 doi: bioRxiv preprint first posted online Dec. 3, 2017; In bacteria, the RNA degradosome mediates the majority of messenger RNA (mRNA) turnover and ribosomal RNA (rRNA) and transfer RNA (tRNA) maturation. 1 The RNA degradosome assembles on the C-terminal scaffold region of the RNase E endoribonuclease.2 RNase E in Escherichia coli (E. coli) and RNase Y, the RNase E homolog in Bacillus subtilis (B. subtilis),both associate with the cell membrane through a membrane-binding helix. [3][4][5][6] One proposed rationale behind the observed membrane association is to physically separate transcription within the nucleoid from RNA degradation, thus providing an inherent time delay between transcript synthesis and the onset of transcript decay, avoiding a futile cycle. Caulobacter crescentus (Caulobacter) is an alpha-proteobacterium widely studied as a model system for asymmetric cell division. Unlike E. coli or B. subtilis, Caulobacter contains a pole-tethered chromosome that fills the cytoplasm. 7-9 Surprisingly, Caulobacter RNase E does not have a membrane targeting sequence and is not membrane-associated. 2, 10 Furthermore, diffraction-limited (DL) images of RNase E exhibited a patchy localization throughout the cell, with RNase E directly or indirectly associating with DNA. 10Fluorescently labeled ribosomal proteins S2 and L1, proxies for ribosomes in E. coli and B. subtilis, respectively, were found enriched at the cell poles, spatially excluded from the nucleoid on the hundreds of nanometer scale. 11,12 Similar fluorescence imaging studies in Caulobacter revealed no apparent separation of ribosome...

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Section: Data Analysis Proceduresmentioning

confidence: 99%

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

Bayas

Wang

Lee

et al. 2017

Preprint

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“…Simplified PSF models such as the Gaussian model [27], the scalar diffraction based Airy model, the Gibson-Lanni model [28], or effective models based on Hermite functions [29] cannot meet this requirement. A solution is the use of an experimental reference PSF, or a spline fit of such a PSF as model PSF [30][31][32][33], or the use of one or multiple Look Up Tables (LUTs) to estimate the z-position [34]. We have shown previously that a vectorial PSF model can also be used for complex 3D and 3D+λ engineered PSFs [25].…”

Section: Introductionmentioning

confidence: 99%

High precision wavefront control in point spread function engineering for single emitter localization

et al. 2018

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“…Accurate localization of single molecules using a multichannel detection scheme requires that positions from one imaging channel be mapped into the other, using a process termed image registration. In order to avoid incurring systematic localization errors, which may lead to degraded resolution and imaging artifacts, accurate image registration requires specialized instrumentation, image-processing software and calibration procedures [35][36][37]. In contrast, our method conducts orientation measurements using a single imaging channel, sidestepping the need for image registration entirely.…”

Section: Introductionmentioning

confidence: 99%

Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements

Backer

Lee

Moerner

2016

Conference on Lasers and Electro-Optics

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Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groovebinding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.

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Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy

Cited by 91 publications

References 56 publications

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

High precision wavefront control in point spread function engineering for single emitter localization

Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements

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