Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes
Introduction
Fluorescence microscopy is one of the most widely used tools in modern biomedical research. Inherently non-invasive, fluorescence imaging enables the observation of specific components or processes in living cells, tissues, and whole organisms. Fluorescent probes are available with a range of colors that span the visible spectrum, and a variety of labeling techniques such as immunofluorescence and in situ hybridization allow fluorophores to be specifically coupled to molecules of interest, enabling the simultaneous visualization of multiple targets by multicolor imaging. Furthermore, the revolutionizing development of fluorescent proteins and other genetically encoded fluorescent labels has allowed specific proteins in living cells to be observed in real time [1].
The limited resolution of fluorescence microscopy, however, leaves many biological structures too small to be studied in detail. Subcellular structures span a range of length scales from micrometers to nanometers, while the light microscope is classically limited to a resolution of ∼200 nm in the lateral direction and ∼500 nm in the axial direction. Other imaging techniques such as electron microscopy (EM) have achieved much higher spatial resolutions [2], and the ability of these methods to visualize biological samples with molecular resolution has had a tremendous impact on our understanding of biology. The use of electron-dense tags for molecule-specific labeling in EM, however, has limitations such as low labeling efficiency and the small number of species that can be simultaneously observed, making it difficult to map out molecular interactions in cells. Moreover, the sample preparation methods used for EM currently preclude the imaging of live samples. To achieve image resolutions comparable to EM but with the labeling specificity and live-cell compatibility provided by fluorescence microscopy would open a new window for the study of the nanoscale structure and dynamics of cells and tissues. With this goal in mind the classical limit of optical resolution has been tested, giving way in recent years to a number of new ideas which we collectively refer to as ‘super-resolution’ imaging techniques. In this review we focus primarily on a newly developed concept for super-resolution imaging that is based on the nanoscale localization of photo-switchable fluorescent probes.
Section snippets
The diffraction limit of resolution
It was recognized by Abbe in the 19th century that the spatial resolution of optical microscopy is limited by the diffraction of light [3]. It is due to diffraction that a point source of light, when imaged through a microscope, appears as a spot with a finite size. The intensity profile of this spot defines the point spread function (PSF) of the microscope. The full width at half maximum (FWHM) of the PSF in the lateral (x–y) and axial (z) directions is given approximately by Δx, Δy ≈ λ/(2NA),
Optical resolution beyond the diffraction limit
The first demonstration of optical image resolution substantially below the diffraction limit was by near-field scanning optical microscopy [9, 10, 11], though the requirement of a near-field scanning probe has limited the application of this technique to imaging near the sample surface. The idea of far-field light microscopy with diffraction-unlimited resolution is a relatively recent one. Hell and co-workers introduced Stimulated Emission Depletion (STED) fluorescence microscopy and related
Super-resolution imaging by single molecule localization
The position of an isolated fluorescent emitter, although its image appears as a diffraction-limited spot, can be precisely determined by finding the centroid of its image. The precision of this localization process is given approximately by , where s is the standard deviation of the PSF and N is the number of photons detected [25]. This concept has been used to track small particles with nanometer-scale accuracy [26, 27]. Recently it has been shown that, even when the emitter is a single
Multicolor imaging
One of the major advantages of fluorescence microscopy is its capacity for multicolor imaging, allowing the relative organization and interactions between different biological structures or molecules to be visualized, for example, through the colocalization of differently colored probes [1]. This aspect of fluorescence imaging is essential for probing interactions between biomolecules and could provide invaluable insights into biological processes if realized in super-resolution. Multicolor
Three-dimensional imaging
Among the most useful aspects of fluorescence microscopy is its ability to provide a 3D image of the sample. To extend this capability to super-resolution imaging by fluorophore localization, a means to determine the lateral as well as the axial positions of the activated fluorophores is required. Such a method has been demonstrated by Huang et al., in which the axial position of the fluorophore is determined on the basis of astigmatism in the image, as illustrated in Figure 3a [54].
Live-cell imaging
An important advantage of fluorescence microscopy is its capacity for time-resolved imaging of living organisms. Extending this capability to nanoscale resolutions will provide exciting new insights into many basic processes of the cell. In this respect, the recent achievement of video-rate STED imaging of a live neuron with 60 nm lateral resolution represents a major advance [16].
Several advances have been made toward live-cell super-resolution imaging by fluorophore localization techniques.
Image resolution and probe development
The resolution of an image constructed from the localization of many individual fluorescent labels is dependent on several factors, including (i) the accuracy of each localization, (ii) the density of localizations obtained in the image, and (iii) the physical size of the labels themselves. The relationship between resolution and localization precision is fairly straightforward. The ability to resolve two labels as separate entities is limited by the precision with which the position of each
Conclusions
Recent years have witnessed rapid progress in subdiffraction limit fluorescence imaging, facilitated by the development of fluorescent probes with novel properties such as photo-switchable fluorescence emission. In this review we have focused on a method of super-resolution imaging based on the high accuracy localization of individual fluorophores. This method has yielded fluorescence images with spatial resolution an order of magnitude finer than the classical diffraction limit of optical
Acknowledgements
We thank M.V. Bujny and G.T. Dempsey for assistance in the preparation of Figure 1 and Table 1, and for valuable comments and discussions. This work is supported partly by the National Institutes of Health (to XZ). XZ is a Howard Hughes Medical Institute Investigator.
References (66)
- et al.
Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation
Opt Commun
(1992) - et al.
Ultra-high resolution imaging by fluorescence photoactivation localization microscopy
Biophys J
(2006) - et al.
Precise nanometer localization analysis for individual fluorescent probes
Biophys J
(2002) - et al.
Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules
Biophys J
(1994) - et al.
3-Dimensional super-resolution by spectrally selective imaging
Chem Phys Lett
(1998) - et al.
Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters
Biophys J
(2007) - et al.
Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position
Biophys J
(1994) - et al.
The fluorescent toolbox for assessing protein location and function
Science
(2006) - et al.
Electron microscopy in cell biology: integrating structure and function
Nat Rev Mol Cell Biol
(2003) Beitrage zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung
Arch Mikroskop Anat
(1873)
Nonlinear magic: multiphoton microscopy in the biosciences
Nature Biotechnology
Sevenfold improvement of axial resolution in 3D wide-field microscopy using two objective lenses
Proc SPIE
Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy
J Microsc
Optical stethoscopy: image recording with resolution lambda/20
Appl Phys Lett
Breaking the diffraction barrier: optical microscopy on a nanometric scale
Science
Principles of Nano-optics
Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy
Opt Lett
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
Proc Natl Acad Sci U S A
Spherical nanosized focal spot unravels the interior of cells
Nat Methods
Far-field optical nanoscopy
Science
Video-rate far-field optical nanoscopy dissects synaptic vesicle movement
Science
Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy
J Microsc
Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy
Science
I5S: widefield light microscopy with 100-nm-scale resolution in three dimensions
Biophys J
Saturated patterned excitation microscopy—a concept for optical resolution improvement
J Opt Soc Am A
Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution
Proc Natl Acad Sci U S A
Subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
Nat Methods
Imaging intracellular fluorescent proteins at nanometer resolution
Science
Tracking kinesin-driven movements with nanometre-scale precision
Nature
Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization
Science
Ultrahigh-resolution multicolor colocalization of single fluorescent probes
Proc Natl Acad Sci U S A
Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time
Proc Natl Acad Sci U S A
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