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Bleaching/Blinking Assisted Localization Microscopy for Superresolution Imaging Using Standard fluorescent Molecules

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Bleaching/Blinking Assisted Localization Microscopy for Superresolution Imaging Using Standard fluorescent Molecules Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules Dylan T. Burnettea, Prabuddha Senguptaa, Yuhai Daib, Jennifer Lippincott-Schwartza,1, and Bechara Kacharb,1 aThe Eunice Kennedy Shriver National Institute of Child Health and Human Development and bLaboratory of Cell Structure and Dynamics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892 Contributed by Jennifer Lippincott-Schwartz, November 22, 2011 (sent for review August 11, 2011) Superresolution imaging techniques based on the precise locali- activation of photoactivatable or photoswitchable probes, re- zation of single molecules, such as photoactivated localization spectively, in which only a few molecules in a sample are emitting microscopy (PALM) and stochastic optical reconstruction micros- at any one time (8, 9). This method allows for a cycle in which copy (STORM), achieve high resolution by fitting images of single a sparse field of single fluorescent molecules is imaged, then fluorescent molecules with a theoretical Gaussian to localize them subsequently turned off, and followed by the switching on of with a precision on the order of tens of nanometers. PALM/STORM a sparse set of different molecules. The localization precision rely on photoactivated proteins or photoswitching dyes, respec- of each molecule in each image can then be determined and tively, which makes them technically challenging. We present a superresolution image can be created by combining the local- a simple and practical way of producing point localization-based izations of all of the molecules (8, 9). superresolution images that does not require photoactivatable or The PALM/STORM concept has been expanded to localize photoswitching probes. Called bleaching/blinking assisted locali- up to two different proteins using PALM (14) and three different zation microscopy (BaLM), the technique relies on the intrinsic synthetic dyes using STORM (10). Furthermore, an increased bleaching and blinking behaviors characteristic of all commonly knowledge of the photoswitching characteristics of fluorescent used fluorescent probes. To detect single fluorophores, we simply proteins and synthetic dyes has led to the development of some acquire a stream of fluorescence images. Fluorophore bleach or simplifications of the PALM and/or STORM methods. For ex- blink-off events are detected by subtracting from each image of ample, direct STORM (dSTORM) takes advantage of the ability the series the subsequent image. Similarly, blink-on events are to switch Cy5 or Alexa Fluor 647 with a cycle of green and red detected by subtracting from each frame the previous one. After laser light (11). It was subsequently shown that it is possible to image subtractions, fluorescence emission signals from single switch most Alexa dyes by imaging them in the presence of fluorophores are identified and the localizations are determined a reducing agent (β-mercaptoethanol or DTT) with only excita- by fitting the fluorescence intensity distribution with a theoretical tion laser light (16). Also, intense excitation laser light can be Gaussian. We also show that BaLM works with a spectrum of used to turn off many molecules, and then individual molecules CELL BIOLOGY fluorescent molecules in the same sample. Thus, BaLM extends can be imaged as a subset of the molecules slowly turn back on single molecule-based superresolution localization to samples over time (ground state depletion followed by individual mole- labeled with multiple conventional fluorescent probes. cule return, GSDIM) (17). In another approach, genetically expressed fluorescent proteins are prebleached to a virtual dark nanoscopy | diffraction limit | immunofluorescence | subcellular field of view where only sparse single molecules blink on/off (18, localization | GFP 19). However, the number of blinking fluorescent proteins left after the prebleaching is low relative to the initial pool. n image created from a fluorescently labeled biological After imaging, the above methods use a similar fitting algorithm Asample is composed of the point-spread functions (PSF) of to precisely localize the single molecules and reconstruct a super- many fluorescent molecules. The resolution of a diffraction lim- resolution image. Although the imaging techniques outlined ited fluorescent microscope is ≈300 nm, precluding the fluores- above allow for the creation of superresolution images, they are cent molecules overlapping within this radius from being technically challenging and require a large investment in both time separated from each other (i.e., resolved). Many biological pro- and money to incorporate them into a laboratory’s tool box. Ad- cesses involve changes that occur on spatial scales below this ditionally, neither PALM nor STORM can be performed by simply resolution limit, and, indeed, many cellular organelles are them- imaging of standard fluorescent proteins and/or synthetic dyes. selves entirely diffraction limited. Therefore, the development of When acquiring images of a population of standard fluo- microcopy techniques that produce images that circumvent the rophores, the overall fluorescence intensity decays because of diffraction limit has been the topic of active research in recent fluorescence photobleaching. Two techniques published in 2004 years (1, 2). The result has been the development of several, so- demonstrated the superresolution localization of two to five sin- called superresolution fluorescence techniques, including stimu- gle molecules in a diffraction limited spot using loss of signal as lated emission depletion microscopy (STED) (3, 4), saturated structured illumination microscopy (SSIM) (5–7), and the family of single molecule localization microscopy techniques, founded Author contributions: D.T.B., J.L.-S., and B.K. designed research; D.T.B., Y.D., and B.K. by photoactivated localization microscopy (PALM) and stochas- performed research; D.T.B., P.S., J.L.-S., and B.K. analyzed data; D.T.B., P.S., J.L.-S., and B.K. wrote the paper; and B.K. conceived the technique. tic optical reconstruction microscopy (STORM) (8–15). Conflict of interest statement: The Bleaching/blinking assisted Localization Microscopy Of the superresolution techniques, the single molecule local- (BaLM) technique was developed at the National Institutes of Health by B.K. The authors ization techniques achieve the highest spatial resolution (1). The declare no conflict of interest. fl position of a single uorescent molecule can be determined Freely available online through the PNAS open access option. fi within tens of nanometers by tting its PSF to a theoretical 2D 1To whom correspondence may be addressed. E-mail: [email protected] or kacharb@ Gaussian. This type of single molecule fitting cannot be imple- nidcd.nih.gov. mented when PSFs of neighboring molecules overlap (8). PALM This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and STORM overcome this problem by using stochastic 1073/pnas.1117430109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117430109 PNAS | December 27, 2011 | vol. 108 | no. 52 | 21081–21086 Downloaded by guest on September 27, 2021 individual molecules photobleached (20, 21). The first of the two Results papers reported a technique called SHRImP (single-molecule Fluorescent molecules in a fixed cell can either turn off (i.e., ir- high-resolution imaging with photobleaching) (20). The authors reversibly bleach or reversibly blink-off) or turn on (i.e., blink-on) conjugated two ends of a DNA molecule each with a single from a fluorescent population over time with continuous excita- fluorescent dye. They were able to locate the position of the two tion. In theory, turn-off (i.e., bleach or blink-off) events should be dyes separated by 10–20 nm with 5-nm localization precision (20). detectable simply by subtracting the subsequent image from each The second paper reported NALMS (nanometer-localized mul- image of a stream acquisition. Likewise, blink-on events should be tiple single-molecule) fluorescence microscopy where five mole- detectable by subtracting from each image the previous one. Each cules in a diffraction limited spot were individually detected by subtracted image should then contain the PSF of individual using a similar fitting method (21). Despite this ability, the sole fluorophores that either turned off or on during acquisition of use of photobleaching and photoblinking for mapping out the the two frames. The position of each fluorophore can then be distribution of single molecules at high density, as in PALM or determined by fitting each molecule to a theoretical Gaussian STORM, has not been established. function, similar to how molecules are localized in PALM/ The major technical difficultly in using bleaching and/or blink- STORM (8, 9). BaLM takes advantage of these characteristics of ing of fluorescent molecules to produce superresolution images is fluorescent molecules. That is, it creates a superresolution image the requirement for imaging all of the fluorescing molecules in from single diffraction limited spots generated by subtraction of a sample without saturating the camera, while, at the same time, consecutive images from a stream acquisition of a bleaching flu- being able to detect single molecules above camera noise. We orescently labeled sample. The single diffraction limited spots be- present here a practical experimental technique for producing ing quantified in BaLM are a result of the disappearance
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