Histochem Cell Biol (2014) 141:577–585 DOI 10.1007/s00418-014-1186-1 REVIEW The changing point‑spread function: single‑molecule‑based super‑resolution imaging Mathew H. Horrocks · Matthieu Palayret · David Klenerman · Steven F. Lee Accepted: 20 January 2014 / Published online: 11 February 2014 © Springer-Verlag Berlin Heidelberg 2014 Abstract Over the past decade, many techniques for limit, gaining over two orders of magnitude in precision imaging systems at a resolution greater than the diffraction (Szymborska et al. 2013), allowing direct observation of limit have been developed. These methods have allowed processes at spatial scales much more compatible with systems previously inaccessible to fluorescence micros- the regime that biomolecular interactions take place on. copy to be studied and biological problems to be solved in Radically, different approaches have so far been proposed, the condensed phase. This brief review explains the basic including limiting the illumination of the sample to regions principles of super-resolution imaging in both two and smaller than the diffraction limit (targeted switching and three dimensions, summarizes recent developments, and readout) or stochastically separating single fluorophores in gives examples of how these techniques have been used to time to gain resolution in space (stochastic switching and study complex biological systems. readout). The latter also described as follows: Single-mol- ecule active control microscopy (SMACM), or single-mol- Keywords Single-molecule microscopy · Super- ecule localization microscopy (SMLM), allows imaging of resolution imaging · PALM/(d)STORM imaging · single molecules which cannot only be precisely localized, Localization microscopy but also followed through time and quantified. This brief review will focus on this “pointillism-based” SR imaging and its application to biological imaging in both two and Fluorescence microscopy allows users to dynamically three dimensions. observe phenomena in living cells; however, the wave When observed through any optical imaging system, a nature of light and its associated diffraction restrict the single-point emitter is blurred due to the diffractive prop- resolution of light microscopy: When light of wavelength λ erties of light. This blur, or point-spread function (PSF), is focused by a lens with a numerical aperture NA, objects of the point emitter is not due to an imperfect focus of the that are closer together than d λ/(2NA) cannot be eas- optics, but is an inherent property of the wave-like nature of = ily discerned. This is the Abbe diffraction limit (Abbe light. The PSF can be well approximated by a 2D-Gauss- 1873), and it has until recently restricted the resolution of ian function, the width of which is proportional to the emit- fluorescence microscopy to ~250 nm. New methods, col- ted wavelength (Santos and Young 2000). The PSF can lectively grouped under the term super-resolution (SR) be thought of as a probability distribution function of the microscopy, have been developed to overcome this physical position of the point emitter; therefore, through determina- tion of the center of an individual PSF, the Abbe diffrac- tion limit can be overcome and the emitter can be localized Mathew H. Horrocks and Matthieu Palayret have contributed at SR. The calculation of the PSF center and consequently equally. the precision of the localization are dependent on two main properties: the number of photons detected N, via 1/√N, * M. H. Horrocks · M. Palayret · D. Klenerman · S. F. Lee ( ) and the pixel size of the camera (Thompson et al. 2002). Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK The essential step leading to pointillism-based SR e-mail: [email protected] microscopy was the ability to control the activation of point 1 3 578 Histochem Cell Biol (2014) 141:577–585 emitters, and the techniques now distinguish themselves “off,” and many solutions to this exist. In this review, we based on how emitters are spatially isolated. The first poin- highlight three popular classes used in biological imaging: tillism-based SR techniques were all demonstrated in 2006, exploiting the innate photo-physics of fluorophores, chemi- and these were photo-activated localization microscopy cal/spectral structural modification, and finally diffusion. (PALM) (Betzig et al. 2006), stochastic optical reconstruc- tion microscopy (STORM) (Rust et al. 2006), and fluores- cence photo-activation localization microscopy (fPALM) Photo‑physical manipulation (Hess et al. 2006). One requirement for accurate super-localization is that When pumped to their excited states, organic dyes can relax the point emitters are well separated in space so that their via a number of mechanisms (Fig. 1a). Fluorescence occurs PSFs do not overlap. This is rarely possible with biological when the fluorophore relaxes from its first excited singlet structures, meaning that innovative methods for decreasing state (S1) via emission of a photon, with a temporal proba- the concentration of active fluorophores and separating dense bility well modeled by an exponential decay and character- or agglomerated emitters have had to be developed. Gener- istic fluorescence lifetime (τ) on the order of nanoseconds. ally, this involves stochastically switching a subset of fluoro- However, inter-system crossing to non-fluorescent states, phores on at any given time, imaging them, determining their such as triplet states (Tn), may also occur, and when in such PSF centers, and then switching them off. This process is states, the fluorophores are unable to rapidly relax and enter then repeated with other emitters, and multiple cycles allow further excitation-emission cycles, here the fluorescence a SR image to be built up from the fitted centers (Fig. 1b). lifetime is typically on the order of micro- to millisec- This requires methods for switching fluorophores “on” and onds. This leads to the molecules undergoing fluorescence AB S1 ... T1 Dark State S0 Original fluorescently Diffraction-limited Stochastically Super-resolution labeled structure Image selected subsets of reconstruction fluorophores localized C Photoswitching of Cy5: D Photoswitching of oxazines: E Photoswitching of Kaede and Eos: O O3S SO3H N O N N R1 Fully conjugated: Fluorescent N O ON state oxazine: N HN NH N N N O SO3 R2 400nm RS RS Conjugation O disrupted: O3S SO3H Non-fluorescent N O N N R1 OFF state Red-shifted N SR product: chromophore: O HN H N N N N 2 H N O SO3 R2 Fig. 1 Photo-physics of organic fluorophores and basis for SR not be resolved. In the next image, on–off control of the fluorophore imaging. a Modified Jablonski diagram including intersystem cross- between “on” and “off” states allows individual fluorophores to ing to the first triplet state, and transition to a generic dark state. b be localized. Repetition of this procedure gives the final SR image Principles of stochastic SR. From the left, the “Springer Horse” (right). Scale bar is 1 μm in length. c Photo-switching of Cy5 by for- (2.2 μm 2.2 μm) is simulated labeled with fluorophores. In mation of a thiol adduct. d Photo-chemical reduction in the oxazine the next image,× simulated data of all of the dye molecules emit- ATTO655 chromophore. e Photo-chemical switching of the chromo- ting simultaneously, and so the diffraction-limited images overlap phore in the fluorescent proteins Eos and Kaede on the camera, and information about the underlying structure can- 1 3 Histochem Cell Biol (2014) 141:577–585 579 intermittently, so-called blinking, as they cycle into and out oxygen-depleted buffers containing a thiol (Heilemann et al. of the “dark” state. Unfortunately, population of these dark 2005), and it can enter a meta-stable dark state, caused via states can often be part of the photo-bleaching pathway, thiol addition to the excited state of the fluorophore (Demp- and so the number of switching cycles is limited. How- sey et al. 2009) (Fig. 1c). This dark state can be depopu- ever, this blinking can still be used to temporally separate lated through irradiation with a shorter wavelength of light the emitters and therefore allow for SR imaging (Hell and yielding the on-state of the dye once again. Excitation at Kroug 1995). This process is also observed in genetically this shorter wavelength can either be achieved directly (van encodable fluorescent proteins (FPs), for example enhanced de Linde et al. 2011b), or by using a reporter–activator dye yellow fluorescent protein (eYFP), which has been shown pair(Dempsey et al. 2011), for example Cy5–Cy3, where the to undergo power-dependent blinking under irradiation of close proximity of the two dyes leads to the on-switching of 514-nm laser excitation; this has been used to study super- the reporter, Cy5, at lower laser powers. This photo-switch- resolved spatial distributions of proteins in bacterial models ing has also been successfully implemented with other cya- (Ptacin et al. 2010; Lee et al. 2011). nine dyes, such as Alexa Fluor 647 (Heilemann et al. 2005), An alternative strategy to control the photo-physics of Cy5.5 (Heilemann et al. 2008), Cy7 (Bates et al. 2007), fluorophores is to perturb the excited state kinetics. For Alexa Fluor 680 (van de Linde et al. 2011b), Alexa Fluor instance, the fluorescence quantum yield of far-red fluoro- 700 (Baddeley et al. 2011), and Alexa Fluor 750 (Lampe phores is typically low; however, non-radiative decay routes et al. 2012). Rhodamines and oxazines (Fig. 1d) can also can be selectively turned off through the use of deuterated be switched using various buffer conditions, for example, (heavy) water-based buffers, leading to an increase in the flu- switching occurs in the presence of thiols (millimolar con- orescence quantum yield by, in some cases, over 200 %. This centrations) (Heilemann et al. 2009; van de Linde et al. has been shown in multiple common classes of dyes, includ- 2011a; Dempsey et al.