BB43CH14-Lippincott-Schwartz ARI 19 May 2014 15:54 Photocontrollable Fluorescent Proteins for Superresolution Imaging Daria M. Shcherbakova,1,2 Prabuddha Sengupta,3 Jennifer Lippincott-Schwartz,3 and Vladislav V. Verkhusha1,2 1Department of Anatomy and Structural Biology, and 2Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461; email: [email protected] 3Section on Organelle Biology, Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; email: [email protected] Annu. Rev. Biophys. 2014. 43:303–29 Keywords The Annual Review of Biophysics is online at PALM, RESOLFT, PAGFP, PAmCherry, EosFP biophys.annualreviews.org This article’s doi: Abstract 10.1146/annurev-biophys-051013-022836 Superresolution fluorescence microscopy permits the study of biological Copyright c 2014 by Annual Reviews. processes at scales small enough to visualize fine subcellular structures All rights reserved that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. Annu. Rev. Biophys. 2014.43:303-329. Downloaded from www.annualreviews.org The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysi- by Yeshiva University - Albert Einstein College of Medicine on 06/06/14. For personal use only. cal properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitch- able fluorescent proteins, and we detail their particular usefulness in single- molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable pro- teins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are rel- evant to cell and developmental biology, neuroscience, cancer biology and biomedicine. 303 BB43CH14-Lippincott-Schwartz ARI 19 May 2014 15:54 Contents INTRODUCTION............................................................... 304 OPERATIONAL PRINCIPLES OF PHOTOCONTROLLABLE PROTEIN-BASED SUPERRESOLUTION TECHNIQUES . 305 Conceptual Basis of Photoactivated Localization Microscopy (PALM) and Related Single-Molecule Techniques . 305 Resolution of Photoactivated Localization Microscopy (PALM) and Related Single-Molecule Techniques . 306 Conceptual Basis and Resolution of Reversible Saturatable Optical Fluorescence Transition (RESOLFT) and Related Ensemble Techniques . 308 PHOTOCONTROLLABLE FLUORESCENT PROTEINS AS TOOLS FORSUPERRESOLUTIONMICROSCOPY.................................. 309 Photophysical and Biochemical Properties of Photocontrollable Fluorescent Proteins . 309 Comparison of Superresolution Techniques. 312 MODERN PHOTOCONTROLLABLE FLUORESCENT PROTEINS . 313 Photoactivatable Fluorescent Proteins (PAFPs). 313 Photoswitchable Fluorescent Proteins (PSFPs) . 317 Reversibly Photoswitchable FPs (rsFPs). 318 APPLICATIONS OF SUPERRESOLUTION MICROSCOPY USING PHOTOCONTROLLABLEFLUORESCENTPROTEINS................... 321 Spatial Relationship Between Single Molecules . 321 StructuralDetailsofMolecularAssemblyandNanowriting....................... 322 DynamicsofSingleProteinMolecules........................................... 324 ImagingofLivingCells......................................................... 324 INTRODUCTION Optical imaging using genetically encoded fluorescent proteins (FPs) is arguably the most widely used approach for noninvasive studies of biological processes at different scales (from molecules to organisms). Because of light’s diffraction limit, however, the inherent nature of light microscopy limits its ability to visualize objects with sizes smaller than 200 nm in the lateral direction and Annu. Rev. Biophys. 2014.43:303-329. Downloaded from www.annualreviews.org 500 nm in the axial direction. This poses a problem for imaging small, abundant subcellular structures that have sizes in the tens of nanometer range. To overcome this obstacle, several by Yeshiva University - Albert Einstein College of Medicine on 06/06/14. For personal use only. superresolution imaging techniques have been invented (6, 29, 35, 36, 63). These techniques make FP: fluorescent it possible to obtain images with exquisite spatial resolution and therefore are now widely used by protein researchers. Diffraction limit: the FPs enable straightforward and specific labeling of cells without a need to deliver any additional minimal distance at substances. Modern FPs are available in various forms. In this review, we focus on light-controllable which objects can be FPs whose fluorescence is regulated by light irradiation of specific wavelengths. These FPs can distinguished in light microscopy that is half be classified into three forms: photoactivatable FPs (PAFPs), photoswitchable (also called photo- of the wavelength of convertible) FPs (PSFPs), and reversibly photoswitchable FPs (rsFPs). PAFPs undergo activation light according to from a nonfluorescent (dark) state to a fluorescent state, whereas PSFPs can be converted from one Abbe’s law fluorescent state (or “color”) to another. In contrast to PAFPs and PSFPs, which can be photocon- verted only once, rsFPs can be photoswitched repeatedly between fluorescent and nonfluorescent 304 Shcherbakova et al. BB43CH14-Lippincott-Schwartz ARI 19 May 2014 15:54 states. We refer to these light-controllable FPs as photocontrollable FPs. Although the brightness, photostability, and photoactivation characteristics of organic dyes and other fluorophores can be superior to those of photocontrollable FPs, these substances require sophisticated approaches to Photocontrollable increase labeling specificity and to deliver them to biological cells. The simplicity and specificity FP: any fluorescence of labeling proteins of interest with photocontollable FPs have made these proteins increasingly protein whose popular tools for superresolution imaging. excitation and/or In recent years, the engineering of new photocontrollable FPs has been spurred by their ap- emission can be plicability to superresolution microscopy. As a result, researchers may now choose among many modulated with light photocontrollable FPs. The optimal choice depends on the particular imaging technique. All PALM: photocontollable FP-based superresolution methods image fluorophores separately over time by photoactivated localization controllably turning them on and off. Two strategies have been employed to construct a super- microscopy resolution image (Figure 1). One strategy is based on localizing individual fluorophore molecules RESOLFT: through cycles of stochastic activation of a small subset of photocontrollable FPs in a densely la- reversible saturatable beled structure (6, 36). This approach was first demonstrated in the similar techniques called pho- optical fluorescence toactivated localization microscopy (PALM) (6), fluorescence PALM (36) and stochastic optical transition reconstruction microscopy (63), and has been expanded to include different single molecule–based Point-spread PALM variations. The second strategy uses nonlinear optical approaches (29, 35) to reduce the function (PSF): the spot size of an ensemble of excited photocontrollable FPs (38). This technique is called reversible spatial intensity profile saturatable optical fluorescence transition (RESOLFT) imaging. of light emitted by a point light source or In this review, we survey photocontrollable FPs and their uses in PALM and RESOLFT super- object observed resolution imaging techniques. For an in-depth description of other superresolution techniques through an optical we refer readers to the recent reviews (33, 43, 57). We first discuss the conceptual bases of the two instrument such as a superresolution imaging strategies, highlighting critical parameters important for each strategy microscope to work. We then review modern photocontrollable FPs, along with their advantages and limi- tations for use in superresolution microscopy. Finally, we consider how PALM and RESOLFT superresolution imaging have helped clarify different biological questions. OPERATIONAL PRINCIPLES OF PHOTOCONTROLLABLE PROTEIN-BASED SUPERRESOLUTION TECHNIQUES Conceptual Basis of Photoactivated Localization Microscopy (PALM) and Related Single-Molecule Techniques The development of advanced microscopy instrumentation has now made detection of a single fluorescent protein possible (46). The focused emission from a single protein molecule produces a Annu. Rev. Biophys. 2014.43:303-329. Downloaded from www.annualreviews.org diffracted, blurred spot with an intensity profile represented by the point-spread function (PSF) of the microscope. For high numerical aperture (NA) lenses and visible light, the size of this spot is by Yeshiva University - Albert Einstein College of Medicine on 06/06/14. For personal use only. approximately 200 × 500 nm in the lateral and axial dimensions, respectively. The intensity profile of this spot (i.e., the PSF) can be fitted with a two-dimensional Gaussian, and its center can be localized to within tens of nanometers (82). Because the image produced by a single molecule is distended (i.e., a blurred spot), fluoresc- ing protein