PALM and STORM: Unlocking live‐cell super‐resolution

Henriques, Ricardo; Griffiths, Caron; Rego, E. Hesper; Mhlanga, Musa M.

doi:10.1002/bip.21586

Cited by 168 publications

(130 citation statements)

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“…It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches.…”

Section: Single-molecule Observations Using Cell Extractsmentioning

confidence: 99%

“…A variety of chemical strategies are used for protein labeling [22][23][24]. These approaches can be reliably practiced with purified proteins, but many of these schemes suffer from an undesirably low quality of conjugation when they are performed intracellularly or within crude cell extracts.…”

Section: Real-time Observation Of Spliceosome Assemblymentioning

confidence: 99%

“…The first is a series of 'protein-directed' labeling techniques including DHFR [28], SNAP-tag [29], CLIP-tag [29], and Halo-tag [30]-based conjugations. The conjugations between these proteins and their substrates exhibit high selectivity and high affinity, even within cells and cell extracts [24]. The second technology is a series of 'enzyme-mediated' labeling strategies such as biotin ligase-mediated [31], phosphopantetheine transferase-mediated [32], lipoic acid ligase-mediated [33], and formylglycine-generating enzyme-mediated [34,35] approaches.…”

Section: Box 1 Labeling Proteins With Organic Fluorophores Within Celmentioning

confidence: 99%

“…Due to this limitation, the use of cell extracts represents a promising alternative that has been successfully adopted to perform in vitro splicing of labeled pre-mRNA at the singlemolecule level [26,27]. Although it was anticipated that the kinetics of the assembly and action of the spliceosome would be discovered soon after the development and use of single- A variety of chemical strategies are used for protein labeling [22][23][24]. These approaches can be reliably practiced with purified proteins, but many of these schemes suffer from an undesirably low quality of conjugation when they are performed intracellularly or within crude cell extracts.…”

mentioning

confidence: 99%

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Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Section: Single-molecule Observations Using Cell Extractsmentioning

confidence: 99%

Section: Real-time Observation Of Spliceosome Assemblymentioning

confidence: 99%

Section: Box 1 Labeling Proteins With Organic Fluorophores Within Celmentioning

confidence: 99%

mentioning

confidence: 99%

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Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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“…The two major implementations of SMLM -fluorescence photoactivation localization (PALM 10 ), stochastic optical reconstruction (STORM 11 ) and their variants 12 -utilize probes undergoing irreversible or reversible transitions between dark and emissive excited states and their corresponding ground states. 13,14 In addition to the generic requirement of brightness (large absorption cross-section and quantum yield), high contrast (on/off ratio), and photostability, probes must exhibit thermal stability of both dark and emitting states and, in many applications, a high resistance to fatigue during photoswitching, i.e.…”

Section: Introductionmentioning

confidence: 99%

Photoswitchable fluorescent diheteroarylethenes: substituent effects on photochromic and solvatochromic properties

Gillanders

Giordano

Dı́az

et al. 2014

Photochem Photobiol Sci

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Photoswitchable fluorescent diheteroarylethenes are promising candidates for applications in superresolution molecular localization fluorescence microscopy thanks to their high quantum yields and fatigue-resistant photoswitching characteristics. We have studied the effect of varying substituents on the photophysical properties of six sulfone derivatives of diheteroarylethenes, which display fluorescence in one (closed form) of two thermally stable photochromic states. Electron-donating substituents displace the absorption and emission spectra towards the red without substantially affecting the fluorescence quantum yields. Furthermore, ethoxybromo, a very electron-donating substituent, stabilizes the excited state of the closed isomer to the extent of almost entirely inhibiting its cycloreversion. Multi-parameter Hammett correlations indicate a relationship between the emission maxima and electron-donating character, providing a useful tool in the design of future photochromic molecules. Most of the synthesized compounds exhibit small bathochromic shifts and shorter fluorescence lifetimes with an increase in solvent polarity. However, the ethoxybromo-substituted fluorescent photochrome is unique in its strong solvatochromic behaviour, constituting a photoactivatable ( photochromic), fluorescent and highly solvatochromic small organic compound. The Catalán formalism identified solvent dipolarity as the principal basis of the solvatochromism, reflecting the highly polarized nature of this molecule.

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Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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PALM and STORM: Unlocking live‐cell super‐resolution

Cited by 168 publications

References 56 publications

Bringing single-molecule spectroscopy to macromolecular protein complexes

Bringing single-molecule spectroscopy to macromolecular protein complexes

Photoswitchable fluorescent diheteroarylethenes: substituent effects on photochromic and solvatochromic properties

Imaging the adenovirus infection cycle

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