Multimodal single-molecule microscopy with continuously controlled spectral resolution

Jeffet, Jonathan; Ionescu, Ariel; Michaeli, Yael; Torchinsky, Dmitry; Perlson, Eran; Craggs, Timothy D.; Ebenstein, Yuval

doi:10.1016/j.bpr.2021.100013

Cited by 16 publications

(22 citation statements)

References 58 publications

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“…For the standard optical mapping analysis, the procedure has already been commercialized. We envision that an important step forward on the optical mapping track is to obtain multiple types of genetic and epigenetic information at the same time, using advanced labeling schemes along with modern imaging platforms, such as CoCoS microscopy (Jeffet et al, 2021a). Another avenue to pursue is an integrated lab-on-a-chip platform aimed at high-throughput single-cell optical mapping, where initial efforts have already been made by Marie et al (2018).…”

Section: Discussionmentioning

confidence: 99%

DNA in nanochannels: theory and applications

Frykholm

Müller

et al. 2022

Quart. Rev. Biophys.

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Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinitybased labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

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Section: Discussionmentioning

confidence: 99%

DNA in nanochannels: theory and applications

Frykholm

Müller

et al. 2022

Quart. Rev. Biophys.

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“…Jeffet et al introduced an Amici prism-based sSMLM system [ 32 ], which allows the user to tune spectral dispersion by rotating one of the prisms (Fig. 2 f).…”

Section: Implementations Of Ssmlmmentioning

confidence: 99%

Spectroscopic single-molecule localization microscopy: applications and prospective

2023

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Single-molecule localization microscopy (SMLM) breaks the optical diffraction limit by numerically localizing sparse fluorescence emitters to achieve super-resolution imaging. Spectroscopic SMLM or sSMLM further allows simultaneous spectroscopy and super-resolution imaging of fluorescence molecules. Hence, sSMLM can extract spectral features with single-molecule sensitivity, higher precision, and higher multiplexity than traditional multicolor microscopy modalities. These new capabilities enabled advanced multiplexed and functional cellular imaging applications. While sSMLM suffers from reduced spatial precision compared to conventional SMLM due to splitting photons to form spatial and spectral images, several methods have been reported to mitigate these weaknesses through innovative optical design and image processing techniques. This review summarizes the recent progress in sSMLM, its applications, and our perspective on future work. Graphical Abstract

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“…In addition, multi-color detection (>2 colors) could be implemented e.g. using prisms [74,75] to spectrally split individual colors in-situ, without the need for multiple separate detection channels. In summary, TIRF is sufficient to implement DyeCycling as demonstrated below, and zero-mode waveguides can further improve DyeCycling and extend it towards short timescales.…”

Section: Background Suppression By Localized Excitation and Detection...mentioning

confidence: 99%

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Vermeer

Schmid

2022

Preprint

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Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ″DyeCycling″, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

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Multimodal single-molecule microscopy with continuously controlled spectral resolution

Cited by 16 publications

References 58 publications

DNA in nanochannels: theory and applications

DNA in nanochannels: theory and applications

Spectroscopic single-molecule localization microscopy: applications and prospective

Can DyeCycling break the photobleaching limit in single-molecule FRET?

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