Fluorescence nanoscopy in cell biology

Key Points

  • Fluorescence nanoscopy (also known as super-resolution microscopy) methods have expanded optical imaging to reach the nanometre resolution range, typically 20–50 nm and even down to the 1 nm level.

  • Diffraction-unlimited nanoscopy methods, which neutralize the resolution-limiting role of diffraction, separate fluorophores by transiently transferring them between (at least) two discernible states, typically an 'on' and an 'off' state of fluorescence.

  • The counting of molecules in nanoscale settings such as within organelles is a crucially important development, along with labelling strategies to reliably pinpoint the locations and spatial proximities of all the molecules investigated in an imaging experiment.

  • Dynamic nanoscopy and extensions of nanoscopy imaging to tissue and in vivo contexts are further frontiers.

  • Examples taken from mitochondrial biology and neurobiology illustrate the capabilities and discovery potential of nanoscale molecule-specific imaging with focused light.

Abstract

Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

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Figure 1: Dissecting animal cells with fluorescence nanoscopy.
Figure 2: Examples of fluorescence nanoscopy in bacteria and yeast.
Figure 3: The state of the art in fluorescence nanoscopy: basic working principles and comparisons of 3D resolution.
Figure 4: Nanoscopy of neurons.
Figure 5: Nanoscopy of mitochondria.
Figure 6: Sizes of commonly used binding probes: a challenge for nanoscopy.
Figure 7: Super-resolution microscopy in vivo mouse and fruitfly nanoscopy.

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Acknowledgements

S.J. and S.W.H. acknowledge funding through the Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).

Author information

All three authors contributed equally to all four aspects of preparing the article (researching data for the article, substantial contributions to the discussion of the content, writing, and reviewing and editing of the manuscript before submission).

Correspondence to Steffen J. Sahl or Stefan W. Hell or Stefan Jakobs.

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Competing interests

S.W.H. is a co-founder of Abberior Instruments GmbH and Abberior GmbH, companies commercializing super-resolution microscopy systems and fluorophores for super-resolution applications, respectively.

Supplementary information

Supplementary information S1 (box)

The diffraction limit of optical microscopy (schematic). (PDF 278 kb)

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Glossary

Numerical aperture

(NA). Measure of the opening angle under which light is collected by an objective lens. The NA (n· sinα, with n being the refractive index and α the semi-aperture angle) determines the tightest focusing possible and thus establishes the resolution of diffraction-limited microscopy.

Fluorophore states

States with defined properties. In the context of nanoscopy, useful pairs of states are pairs for which one of them gives a signal ('on'), whereas the other one does not ('off'), as this allows fluorophores to be distinguished even when they are located in closer proximity to each other than the diffraction limit.

Stimulated emission depletion

(STED). The stimulated emission process transfers the excited fluorophore to its ground state. The stimulating photon induces the generation of a stimulated identical photon, which is not detected. The STED light thus exits the specimen, providing a clean fluorophore off-switch. The near-infrared light used in STED is hardly absorbed by the cell.

Reversible saturable/switchable optical linear (fluorescence) transitions

(RESOLFT). The general conceptual framework for coordinate-targeted nanoscopy. The term is mostly used in reference to approaches using reversibly switchable fluorescent proteins (RSFPs, see below) or photochromic organic compounds.

Photo-activated localization microscopy/stochastic optical reconstruction microscopy

(PALM/STORM). Coordinate-stochastic nanoscopy concepts based on the switching and localization of single molecules. Conceptually similar techniques include fluorescent PALM (fPALM) and ground state depletion with individual molecule return (GSDIM).

Points accumulation for imaging in nanoscale topography

(PAINT). A coordinate-stochastic nanoscopy concept based on separating fluorophores by registering only the bound ones ('on'), with the diffusing fluorophores remaining undetected ('off').

Nanoscopy with minimal photon fluxes

(MINFLUX). A concept that allows precise localization of fluorophores with minimal fluxes of emitted photons. MINFLUX nanoscopy combines coordinate-targeted and coordinate-stochastic aspects.

Multiple off-state transitions for nanoscopy

(MOST). A concept that synergistically combines two or more state-transfer mechanisms to, for example, protect the fluorophore from pathways related to photobleaching and improve signal-to-background in coordinate-targeted nanoscopy.

MINFIELD

A method for increasing the signal (photobleaching reduction) in coordinate-targeted nanoscopy. Using scan fields below the diffraction limit around an intensity minimum (for example, at the centre of a doughnut shape) avoids subjecting the fluorophores to the excess intensities of switching light at the maxima of the off-switching pattern.

Optical sectioning

Used to obtain an image with sufficient contrast that is not compromised by fluorescence originating in other axial planes of the specimen. For example, a confocal pinhole can act to reject the out-of-plane background. Other sectioning strategies include selective excitation or photoactivation by multi-photon absorption or light sheets.

Deconvolution

An algorithm to reverse the effects of convolution in the image formation process. By removing the optical blur, a sharper image is computed based on the (ideally) exact knowledge of the blurring (formalized by the so-called point spread function (PSF)). Because knowledge of this PSF is in practice imperfect, and registered images are compromised by noise, artefacts can easily arise in the deconvolution process. Deconvolution is not equivalent to methods that actually improve the spatial resolution by a (on-off) state transition.

Structured illumination microscopy

(SIM). A diffraction-limited method that produces up to 2-fold improved resolution and requires the acquisition of several images of a specimen with shifted illumination patterns and computation of a reconstructed image. Further improvements in resolution can be realized if on-off transitions (as in reversible saturable/switchable optical linear (fluorescence) transitions) are incorporated.

AiryScan

A diffraction-limited method that combines conventional confocal laser scanning microscopy with fast widefield detection or other detector designs to achieve close to a doubling of resolution after mathematical processing. Also known as image scanning microscopy (ISM).

Lattice light-sheet microscopy

A diffraction-limited method that uses a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images that can provide information about dynamic biological processes.

Super-resolution optical fluctuation imaging

(SOFI). A method that analyses on-off fluctuations of fluorescence signals (but not strictly at the single-molecule level as in photo-activated localization microscopy and stochastic optical reconstruction microscopy) by examining correlations in time to improve resolution typically 2- to 3-fold in comparison with epifluorescence.

4Pi

Optical arrangement for coherent excitation and/or collection of fluorescence emissions featuring two juxtaposed lenses of high numerical aperture to expand the solid angle as much as possible, which enables very high axial resolution in nanoscopy (<10 nm).

Single-particle averaging

Computational methods that infer a structure by sorting and averaging data from a large dataset of images showing the same object.

Epitopes

Parts of a protein that are detected by an antibody or other binding probe.

Bio-orthogonal labelling

Chemical labelling reactions that can occur inside living cells without interfering with endogenous biochemical processes.

Genetic code expansion

A process that enables the site-specific incorporation of an amino acid that is not among the 20 common proteinogenic amino acids into a protein.

Click chemistry

A term that encompasses several chemical reactions that facilitate the fast, specific and irreversible attachment of a probe such as a fluorophore to a specific biomolecule.

Labelling coverage

The fraction of epitopes decorated by a binding probe such as an antibody out of all epitopes potentially available for decoration by this binding probe.

Fluorescence fluctuation spectroscopy

A set of methods, in particular fluorescence correlation spectroscopy (FCS), which allow the determination of timescales of dynamic processes. By analysing the (self-) similarity (so-called correlations) of the signal from an observed spot over time, information on, for example, molecular diffusion can be obtained.

Dwell time

Duration for which a scanning nanoscope collects signal at a given position (pixel or voxel).

Reversibly switchable fluorescent proteins

(RSFPs). Fluorescent proteins that can be reversibly switched by light irradiation between long-lived non-fluorescent 'off' and fluorescent 'on' states. RSFPs can be efficiently transferred between the two states at even a low light dose. Because the established state difference remains in place for milliseconds to hours, in RSFP-based reversible saturable/switchable optical linear (fluorescence) transitions nanoscopy, much lower light intensities are needed to break the diffraction barrier than in stimulated emission depletion nanoscopy.

Adaptive optics

Optical strategies to compensate for the effects of aberration and ensure more optimal focusing by deliberately modifying the phase across the light wavefront, often in response to a measurement to characterize the presence of aberrations, which is used as feedback.

Refractive index

A dimensionless number expressing the factor by which light is slowed down when travelling through a material compared with in vacuum. The refractive index of the immersion medium of an objective lens co-determines its numerical aperture.

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Sahl, S., Hell, S. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18, 685–701 (2017). https://doi.org/10.1038/nrm.2017.71

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