We demonstrate quantitative multicolor 3D subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20–40 nm, a flexible locally-weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Simple surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, livecell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 × 12 μm).cell biology | wide-field microscopy | cytoskeleton | fluorescence microscopy | bacteria

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm.

We report the dynamic spatial organization of RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution 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, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in , 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. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in, with chromosomal readout serving as the template for this organization.

Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push–pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis–Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.

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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