Highly inclined thin illumination enables clear single-molecule imaging in cells

Tokunaga, Makio; Imamoto, Naoko; Sakata‐Sogawa, Kumiko

doi:10.1038/nmeth1171

Cited by 1,204 publications

(1,066 citation statements)

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“…Under optimal d STORM buffer conditions using a highly inclined and laminated optical sheet (HILO) setup,14 the AF647‐phalloidin gave high photon counts (median=5547) and a localization precision of 5.7 nm (Figure S2). In the initial trial of conjugate 17 we imaged in non‐degassed phosphate buffered saline (PBS).…”

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

et al. 2015

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“…For the interested reader, these include: HILO (by Tokunaga M.N. et al [55]. AFM cantilever lightsheet (by Gebhardt, J.C. et al [11]), lattice light-sheet (by Chen B.C.…”

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confidence: 99%

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Leake

2018

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High-speed single-molecule fluorescence microscopy in vivo shows that transcription factors in eukaryotes can act in oligomeric clusters mediated by molecular crowding and intrinsically disordered protein. This finding impacts on the longstanding puzzle of how transcription factors find their gene targets so efficiently in the complex, heterogeneous environment of the cell.Abbreviations CDF - cumulative distribution function; FRAP - fluorescence recovery after photobleaching; GFP - Green fluorescent protein; STORM - stochastic optical reconstruction microscopy; TF - Transcription factor; YFP - Yellow fluorescent protein

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“…For some cell-imaging applications, angles just below the critical angle are used -this technique is referred to as highly inclined illumination or HILO [4]. At the barely subcritical angles used, the intensities near the interface are similar to those seen in the evanescent wave.…”

Section: Intensity and Polarisationmentioning

confidence: 99%

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Knight

2014

Pure and Applied Chemistry

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Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

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Highly inclined thin illumination enables clear single-molecule imaging in cells

Cited by 1,204 publications

References 15 publications

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

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

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

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