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  • Review Article
  • Published:

Fluorescent probes for super-resolution imaging in living cells

Nature Reviews Molecular Cell Biology volume 9, pages 929–943 (2008)Cite this article

Key Points

  • The spatial resolution of conventional optical microscopy is limited by diffraction to ∼200 nm in biological imaging. In recent years, several microscopy techniques have successfully overcome this diffraction limit. These new methods are called super-resolution imaging techniques and include stimulated emission depletion (STED) and reversible saturable optically linear fluorescence transitions (RESOLFT), photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM) and stochastic optical reconstruction (STORM).

  • Far-field super-resolution techniques break the diffraction limit by spatially and/or temporally modulating the transition between bright and dark states of a fluorophore. In RESOLFT imaging, which includes STED, super resolution is achieved by narrowing the point spread function of an ensemble of fluorophores via stimulated emission depletion. By contrast, PALM, FPALM and STORM image single molecules, which are sequentially and stochastically switched on, imaged and localized with nanometer accuracy, and then switched off (or bleached). Many cycles of this allow reconstruction of a super-resolution image.

  • Two main classes of fluorophores have been used for super-resolution imaging: fluorescent proteins (FPs) and chemical (non-genetically encoded) probes, including small organic fluorophores and quantum dots. These probes can be reversibly or irreversibly photoactivated, or they can be irreversibly photoshifted.

  • For example, EosFP is an irreversible photoshiftable FP, which converts from green to red following illumination with ultraviolet or blue light. Another commonly used FP is the reversible photoactivatable FP Dronpa.

  • Among small-molecule probes, the photoswitchable cyanine dyes stand out for their excellent brightness, whereas photochromic rhodamines offer membrane permeability and thus the possibility of application to intracellular imaging. Photocaged compounds, such as caged Q-rhodamine and caged fluorescein, have also been used for super-resolution imaging.

  • Super-resolution biological imaging has revealed synaptic vesicle movement in live hippocampal neurons with 62 nm spatial resolution and 35 ms temporal resolution. Three-dimensional imaging of microtubules and clathrin-coated pits has been performed in fixed cells with 20 nm lateral and 50 nm axial resolution. Two-colour imaging of several adhesion complex proteins with ∼20–30 nm resolution revealed little overlap between proteins that were previously seen as colocalized using conventional microscopy.

  • Spatial and temporal resolution depend on the properties of the fluorophores used. Although live-cell imaging at 40–60 nm has been shown, the ultimate goal of video-rate imaging with molecular (1–5 nm) resolution will require the development of smaller, brighter, more photostable, membrane-permeable and genetically targetable fluorescent probes.

Abstract

In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1–5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

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Figure 1: Comparison of the spatial and temporal resolutions of biological imaging techniques.
Figure 2: Cellular features imaged by super-resolution techniques.

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Acknowledgements

The authors thank X. Zhuang, E. Betzig, R.Y. Tsien, T. Uttamapinant and P. Zou for useful feedback on the manuscript.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Marta Fernández-Suárez & Alice Y. Ting

  2. Center for Engineering in Medicine, Massachusetts General Hospital, 114 16th Street, Charlestown, 02129, Massachusetts, USA

    Marta Fernández-Suárez

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Glossary

Electron microscopy

A focused electron beam is used to illuminate the sample. Electron microscopes use electrostatic and electromagnetic lenses to form the image by focusing the electron beam in a manner that is similar to how a light microscope uses glass lenses to focus light.

Positron-emission tomography

An in vivo imaging technique that detects the location of positron-emitting isotopes by the pair of γ-rays that are emitted when the positrons encounter electrons. The most common scan is produced by imaging the metabolic activity of fluorodeoxyglucose, a radioactive analogue of glucose.

Magnetic resonance imaging

A medical imaging technique in which the magnetic nuclei (especially protons) of a subject are aligned in a strong, uniform magnetic field, absorb energy from tuned radio frequency pulses and emit radio frequency signals as their excitation decays.

Optical coherence tomography

An in vivo imaging technique that sends out femtosecond infrared pulses and uses optical interference to sense reflections from tissue inhomogeneities.

Confocal microscopy

A mode of optical microscopy in which a focused laser beam is scanned laterally along the x and y axes of a specimen in a raster pattern. Point-like illumination and point-like detection results in a focal spot that is narrower than that obtained in wide-field microscopy.

Wide-field microscopy

The most popular mode of light microscopy, in which the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto a camera.

Point spread function

(PSF). A measure of the performance of an optical system. The PSF defines the apparent shape of a point target as it appears in the output image. For a fluorophore, PSF is a Gaussian function, whose full-width at half maximum (FWHM) defines the spatial resolution of the imaging system.

Multiphoton microscopy

A form of laser-scanning microscopy that uses the simultaneous absorption of two or more photons of a long wavelength to excite fluorophores that are normally excited by a single photon of shorter wavelength. This is a nonlinear imaging technique that enables deep penetration into thick tissues and reduces light damage.

Optical sectioning

The imaging of thin sections of a sample without the need to mechanically slice it. This is achieved by eliminating the excitation and/or detection of fluorescence that originates in the out-of-focus planes. Effectively, the distance between the closest and furthest objects in focus is greatly reduced to yield a clean optical section.

Ground state depletion

A mode of RESOLFT microscopy (see RESOLFT) that exploits the saturation of fluorophore transition from the ground state to the dark triplet state. A laser beam with a light intensity distribution featuring one or more zeros switches some of the fluorophores to their triplet state T1 or another metastable dark state, while recording those that are still left or have returned to the ground state S0.

Saturated structured-illumination microscopy

A mode of RESOLFT microscopy (see RESOLFT) that exploits the saturation of fluorophore transition from the ground state S0 to the excited singlet state S1. This differs from STED in that ultrasharp dark regions of molecules are created with steeply surrounded regions of molecules in the bright state.

Reversible saturable optically linear fluorescence transitions

(RESOLFT). A mode of light microscopy that exploits the saturation of a reversible single photon transition from a dark state to a bright state, or vice versa. A light intensity distribution featuring zeros creates arbitrarily sharp regions of molecules in the dark or the bright states; the bright regions allow the assembly of a subdiffraction image. The spatial resolution is no longer limited by the wavelength of the light in use, but rather is determined by the saturation that can be realized.

Photoswitcher

A molecule that can reversibly switch between two molecular states on irradiation with light of a specific wavelength and intensity. Currently known fluorescent photoswitchers are photoactivatable molecules that switch between a dark and a fluorescent state upon illumination.

Total internal reflection fluorescence

A microscopy technique that is designed to probe the surface of fluorescently labelled living cells with an evanescent wave. This wave is generated by a light beam that strikes between two media of differing refractive indices at an angle beyond the critical angle.

Extinction coefficient

The (molar) extinction coefficient (εabs)of a species is defined by the equation A = εbc, where A is the absorbance of the solution, b is the path length and c is the concentration of the species. The fluorescence brightness of a species is proportional to the product of its molar extinction coefficient and fluorescence quantum yield.

Fluorescence quantum yield

The ratio of photons emitted to photons absorbed. The fluorescence brightness of a species is proportional to the product of its molar extinction coefficient and fluorescence quantum yield.

Nyquist criterion

The sampling frequency should be equal or larger than twice the largest frequency that is to be recorded.

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Fernández-Suárez, M., Ting, A. Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9, 929–943 (2008). https://doi.org/10.1038/nrm2531

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