Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

Lee, Marissa K.; Rai, Prabin; Williams, Jarrod; Twieg, Robert J.; Moerner, W. E.

doi:10.1021/ja508028h

Cited by 111 publications

(137 citation statements)

References 45 publications

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“…Cells were fixed with the addition of 1% methanol (Fisher), followed by a 10 min incubation at room temperature then a 30 min incubation on ice, then washed three times with clean M2. The fixed cells were then resuspended in a mixture of 1 mL M2 and 50 μL of rhodamine spirolactam dye 9 (dissolved in DMSO (Fisher) at ~100 nM -1 μM), 55 then covered in foil and incubated at room temperature while shaking for 30 minutes. The cells were washed 5 times with clean M2, then resuspended in a small amount of M2 to produce a concentrated cell suspension.…”

Section: Labeling Cell Surface Amines With Rhodamine Spirolactam Dyementioning

confidence: 99%

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter 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: Labeling Cell Surface Amines With Rhodamine Spirolactam Dyementioning

confidence: 99%

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

Bayas

Wang

Lee

et al. 2017

Preprint

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“…The second class of localization microscopy label consists of single conventional dyes that can be made photoswitchable under reducing conditions,5 either by direct reduction of the dye6 or formation of thiol‐dye or phosphine‐dye adducts7 (i.e., d STORM dyes). The third class of localization microscopy labels are “caged” fluorophores where a synthetic fluorophore is appended with a photolabile group that can be removed or modified by illumination with short wavelength light to elicit a large change in fluorescence 3c, 8…”

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confidence: 99%

“…They typically exhibit higher photon counts than fluorescent proteins, leading to better localization precision. Additionally, caged fluorophores are activated by a photochemical reaction that is defined by the chemical structure of the compound, allowing sparse, controlled turn‐on of individual molecules 3c, 8. This eliminates the need for harsh, dye‐specific environmental conditions to elicit photoswitching, as required for d STORM dyes 1d, 5b, 6b–6d.…”

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confidence: 99%

“…Despite these advantages, caged fluorophores have not been utilized extensively in localization microscopy owing to their challenging syntheses and a limited color palette. Although a few red caged fluorophores have been developed,8e, 9 only dyes with green or orange emission have demonstrated utility in PALM;3c, 8 these dyes have not been used in multicolor localization microscopy experiments. Thus, facile chemistry towards caged fluorophores with red absorption and emission would expand the current collection of localization microscopy labels and enable multicolor super‐resolution imaging experiments using these versatile dyes.…”

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Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

et al. 2015

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The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.

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“…This approach has superior Fisher information and thus better localization precision than that of other approaches (178), and we have used the DH-PSF in two colors to co-image two different fluorescent protein fusions in Caulobacter (179) (159). The lower half of Figure 22 shows the application of the DH-PSF method to 3D surface imaging of Caulobacter labeled by the rhodamine spirolactam (172). Much remains to be done, as new point-spread function designs continue to appear (180)(181)(182), with the continuing goal to extract the maximum information from each tiny single-molecule emitter in the most efficient fashion.…”

Section: Figure 22 Here -mentioning

confidence: 99%

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

Moerner

2015

Rev. Mod. Phys.

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The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90's, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later superresolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized. The early days Introduction and early inspirationsI want to thank the Nobel Committee for Chemistry, the Royal Swedish Academy of Sciences, and the Nobel Foundation for selecting me for this prize recognizing the development of super-resolved fluorescence microscopy. I am truly honored to share the prize with my two esteemed colleagues, Stefan Hell and Eric Betzig. My primary contributions center on the first optical detection and spectroscopy of single molecules in the condensed phase(1), and on the observations of imaging, blinking and photocontrol not only for single molecules at low temperatures in solids, but also for useful variants of the green fluorescent protein at room temperature (2). This lecture describes the context of the events leading up to these advances as well as a portion of the subsequent developments both around the world and in my laboratory.In the mid-1980's, I derived much early inspiration from amazing advances that were occurring around the world where single nanoscale quantum systems were detected and explored for both scientific and technological reasons. Some of these were (i) the spectroscopy of single electrons or ions confined in vacuum electromagnetic traps (3-5), (ii) scanning tunneling microscopy (STM) (6) and atomic force microscopy (AFM) (7), and (iii) the study of ion currents in single membrane-embedded ion channels (8). But why not optical detection and ...

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Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

Cited by 111 publications

References 45 publications

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

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