As is well known, biological fluorescence microscopy can light up different structures in cells with a wide array of labels, sufficient signal-to-background ratio, and high specificity. However, the price often paid for using visible light is the relatively poor spatial resolution compared to X-ray or electron microscopy. The basic problem is that fundamental diffraction effects limit the resolution to roughly the optical wavelength divided by twice the numerical aperture of the imaging system, λ
/(2NA). Since the largest values of NA for state-of-the-art, highly corrected microscope objectives are in the range of 1.3–1.6, the spatial resolution of optical imaging has, until recently, been limited to ~180 nm for visible light. We describe here a way to provide “super-resolution,” or imaging far better than the optical diffraction limit, while still using a far-field optical microscope. Various techniques for increasing resolution with far-field imaging have been suggested and realized, including stimulated emission depletion (Hell and Wichmann, 1994
; Klar and Hell, 1999
), structured illumination (Gustafsson, 2005
; Heintzmann et al., 2002
), and single-molecule spectral separation (Ambrose et al., 1991
; Betzig, 1995
; van Oijen et al., 1998
) (for comparison of the various methods see Moerner, 2006
; Hell, 2007
). Alternatively, this chapter concentrates on a complementary set of methods based on single-molecule wide-field imaging and the ability to turn single emitters on and/or off.
Single fluorescent molecular labels 1–2 nm in size provide a way around the resolution limit. How can single molecules help? A molecule absorbs light with a probability proportional to the square of the dot product between the local optical electric field and the molecule’s transition dipole moment. The image formed by emission from a single molecule essentially maps out the point-spread function (PSF) of the microscope because the molecule is a nanoscale light absorber approaching a point source. This was realized in the early days of single-molecule spectroscopy, where the fluorescence excitation signal from one molecule was used to map the size of the focused pumping laser beam (Ambrose et al., 1991
). By measuring the shape of the PSF, the position of its center can easily be determined much more accurately than its width by numerically fitting the observed PSF with a model function, such as a Gaussian or Airy shape. This idea, characterizing and fitting the PSF to achieve “super-localization,” or position information far below the diffraction limit, is well known in many areas of science, and was applied early on to single nanoscale fluorescent beads with many emitters (Gelles et al., 1988
). Later, the super-localization approach was applied to low-temperature single-molecule images (Güttler et al., 1994
; van Oijen et al.,1999
), where both spatial information and a secondary variable, detuning the wavelength of the excitation laser over time, were used to separately detect and localize molecules within a diffraction-limited spot.
At room temperature, spectral broadening limits the ability to separate many similar emitters by pumping wavelength. To keep the images of adjacent molecules from overlapping, very low concentrations of the emitting molecule are usually required. If a single molecule moves through a relatively static structure, such as a filament for example, then the super-localized positions of the molecule from each microscope image can be summed to yield a super-resolution image of the structure (Kim et al., 2006
). The accuracy with which a single molecule can be located depends fundamentally upon the Poisson process of photon detection, so the most important variable is the total number of photons detected above background, with a weaker dependence on the size of the detector pixels and background noise (Michalet and Weiss, 2006
; Ober et al., 2004
; Thompson et al., 2002
). However, to qualify for improved resolution, it is necessary according to the Nyquist–Shannon theorem to obtain position information for at least two emitters within each resolution element (Nyquist, 1928
; Shannon, 1949
). In order to achieve super-resolution imaging in a more general fashion, it was necessary to develop a way to work with high concentrations of labels, where the PSFs would otherwise overlap. In 2006, three research groups applied photoactivation or photoswitching of single emitters directly to generate super-resolution images in biologically relevant systems. Photoactivated GFP fusions were used in the method of Betzig et al. (2006)
, PALM (photoactivated localization microscopy), and in the method of Hess et al. (2006)
, FPALM (fluorescence photoactivation localization microscopy). Photoswitching produced by proximal Cy3 and Cy5 molecules in the presence of thiols was the key element in the method of Rust et al. (2006)
, STORM (stochastic optical reconstruction microscopy).
The basic technique for achieving super-resolution in all of these reports involves turning on only a sparse subset of all the labels present at any given time (see ). (Active, intentional control of the concentration of emitting single molecules is essential, thus these methods may generally be termed “single-molecule active-control microscopy,” or SMACM, but another acronym may not be needed.) After imaging, superlocalizing, and then photobleaching a sparse group of single molecules, a new subset is turned on and the process is repeated to build up a full image of the labeled structure. The “turning on” process is random in that the members of the small subset of labels that turn-on are not known in advance, but this is inherent in the method. Over the course of the data acquisition cycles, the activated labels should sample as much of the underlying labeled structure as possible. Final resolutions down to 10–20 nm have been achieved, and it is this impressive improvement of resolution that has caused much excitement in the field.
Schematic showing the key idea of super-resolution imaging of a structure by PALM
In this chapter, molecules and methods for super-resolution microscopy by imaging of switchable single molecules will be reviewed, focusing on the authors’ research. The molecules include two classes of small-molecule labels that can be photoactivated or photoswitched between emissive and dark states, as well as the first-reported photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The methods described include protocols for super-resolution studies in live bacteria and a novel method for obtaining three-dimensional super-resolution image information using a microscope with a double-helix PSF.