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 of image series in which single molecules can be revealed by high molecules that are irreversibly photobleaching or blinking off, and magnification streaming acquisition of fluorescent samples fol- the appearance of molecules that are blinking on. lowed by image subtraction. This technique, which we call It has been analytically shown that maximum precision of bleaching/blinking assisted localization microscopy (BaLM), is single molecules imaged with a low background is obtained with based on the detection of fluorescent molecules that are either lost a pixel size equal in dimension to the PSF of the imaging system to irreversible photobleaching or lost/gained by blinking. BaLM (22). This calculation is why PALM, which has an inherently low does not require prebleaching before single molecules are detec- background (8), uses pixel dimensions close to the PSF of the ted, distinguishing it from prior approaches that require pre- system (typically ≈150 nm diameter-sized pixel) in acquiring bleaching to detect single molecules (18, 19). BaLM works with data. In BaLM, where single molecules are identified from dif- all commonly used synthetic fluorescent dyes and genetically ference images, there is much higher background. The higher expressed fluorescent proteins. Thus, BaLM can be readily used in background comes from a variety of sources, including fluctua- multicolor superresolution experiments, mapping out the molec- tions in ensemble fluorescent intensities from frame to frame ular distribution of up to four different proteins in a sample, as and from bleaching/blinking of nearby molecules. If one sub- shown here. Furthermore, BaLM allows for molecules to be lo- tracts images acquired with a pixel size similar to the PSF of the calized by similar algorithms already used for the analysis of PALM peak intensity under these conditions, single maxima get aver- data. As such, BaLM offers a practical and versatile approach for aged out because of the higher background. It thus becomes obtaining superresolution images that can stand alone or be used in difficult to detect single peaks by using the 150-nm pixel size used conjunction with other superresolution approaches. in PALM, as schematically illustrated in Fig. S1A. To overcome

Fig. 1. Detection of molecules and construction of BaLM image. (A) Diffraction limited TIRF image of microtubules localized with an Alexa Fluor 488 secondary antibody. Arrowheads denote ﬂuorescent beads used for image alignment. (B) Magniﬁed view of the yellow box in A.(C) Three representative consecutive frames of a subtraction image series showing before and after two molecules bleached/blinked off. (D) The distribution of photon counts of single molecules (n = 15 microtubules). (E) The localization of the two molecules in C Middle.(F) The distribution of localization precisions of BaLM localized molecules (n = 15 microtubules). (G) Schematic demonstrating how localized molecules from different frames are summed to create a BaLM image reconstruction. (H) A BaLM image reconstruction of the microtubules shown in B.(I) Image intensity linescans (normalized to 1) through the dotted line from the diffraction limited image in B (purple line in graph) and the dotted line from the BaLM reconstruction in H (blue line in graph). (Scale bars: A,5μm; B, 1 μm; C and E,0.6μm.)

21082 | www.pnas.org/cgi/doi/10.1073/pnas.1117430109 Burnette et al. Downloaded by guest on September 27, 2021 this problem, BaLM uses higher magnification to obtain smaller pixel size (≈60 nm). This magnification allows much better detection of single maxima in the difference images arising from bleaching/blinking of molecules (see illustration in Fig. S1B). Additionally, by spreading out the PSF of the molecules over more pixels, it is possible to image more molecules in a diffraction limited spot before saturating each individual pixel. To demonstrate that BaLM works using a standard EMCCD camera, we used a secondary magnification element to create an effective pixel size of 61 nm and imaged microtubules in COS-7 cells labeled with a primary antibody to α-tubulin and an Alexa Fluor-488 labeled secondary. Microtubules are biopolymers with a 25-nm diameter tubular structure, making them useful test objects for superresolution techniques (10). The diffraction- limited image from the first frame of a time-lapse acquisition of microtubules from the periphery of a fixed cell is shown in Fig. 1 A and B. Frames from this series were subsequently subtracted to detect signal intensities that disappeared/appeared. Three representative frames from a subtraction series is shown in Fig. 1C. Single local maxima were then identified in subtracted images as having a higher signal than the background (Fig. 1C Middle). These local maxima were then defined as single molecules if, (i) their radius was <2.5× the SD of the effective PSF, and, (ii) they were emitting between 2,000 and 12,000 photons over a 1-s integration time (Materials and Methods). This range was determined based on previously reported measurements of photon counts of Alexa dyes (23), as well as examination of the photon counts of single molecules at the end of bleaching acquisitions in our experiments (Materials and Methods). The distribution of the photon counts of molecules satisfying the criteria described above is shown in Fig. 1D. Single molecules were then fit with a theoretical Gaussian to obtain localization precisions (Fig. 1 E and F; σ = 48.0 ± 26.1 nm, see Materials and Fig. 2. BaLM reconstructions created with the ImageJ plugin, QuickPALM. Methods). The rendered superresolution localizations of the (A–C) Diffraction limited image (A), BaLM image (B), and overlay (C)of molecules in Fig. 1C Middle are shown in Fig. 1E. a microtubule labeled with Alexa Fluor 488. Graph in C is a line scan through To create the BaLM reconstruction, all superresolution the yellow line in C.(D) Overlay of the diffraction limited image (purple) and CELL BIOLOGY BaLM image (light blue) of myosin IIC-GFP. (Scale bars: 2 μm.) localizations were combined into one single probability map (see schematic in Fig. 1G and reconstruction in Fig. 1H). Note that the microtubule-based structures in the BaLM reconstruction with Alexa Fluor 488 from a bleaching data set and the sub- appear to be thinner than in the diffraction-limited image (Fig. sequent BaLM image reconstructed with QuickPALM are shown H B fi 1 compared with Fig. 1 ). To con rm this difference in width, in Fig. 2 A–C. Line scans drawn across an area of overlapping we examined line scans through the same region in the diffrac- C Inset I microtubules (Fig. 2 ) reveal no separation of the mi- tion limited data and the BaLM reconstruction (Fig. 1 ). Note crotubules in the diffraction limited image but clear separation in that the two microtubules in this region are resolved in the the reconstructed BaLM image. BaLM data, but not in the diffraction-limited data (Fig. 1I). fl BaLM reconstructions also can be acquired from samples When doing immuno uorescence labeling of proteins, there expressing GFP-tagged proteins. To demonstrate this possibility, is always background fluorescence from nonspecific interactions we exogenously expressed the molecular motor, myosin IIC with either the growth substrate or cell (Fig. S1). These fluo- fused to EGFP, in COS-7 cells (Fig. 2D). Myosin II has a ste- rophores will show up in the BaLM analysis and in any other point reotypical periodic localization along actin filament-based stress localization-based superresolution technique (Fig. S2). Although fi fi bers that can be detected with diffraction limited imaging (Fig. spatial ltering of the data could eliminate these molecules, as has D been recently shown (24), we present nonfiltered images to em- 2 , see periodic purple signal from myosin IIC-GFP that dis- tributes along elongated actin filaments). BaLM imaging of this phasize the quality and sensitivity of BaLM imaging. fi It can take a few hours to perform BaLM analysis with our sample provides signi cantly higher resolution of myosin IIC- custom Matlab-based software to get an estimate of the locali- EGFP, showing new substructure associated with each diffrac- D zation precision of each molecule within a typical dataset of tion-limited puncta (Fig. 2 , see BaLM localizations in blue). σ 1,000–3,000 images. However, identifying molecules and creating The mean localization precision ( ) of individual EGFP mole- ± a BaLM reconstruction from the center of mass of these molecules imaged with BaLM was 57.2 22.9 nm. ’ cules can be done quickly with a freely available ImageJ plugin To compare BaLM s performance with another superres- software package, QuickPALM (Fig. 2). This plugin also makes olution technique, we compared microtubule structures localized it possible to process BaLM data exclusively by using ImageJ with BaLM to those localized with PALM (Fig. 3). PALM im- plugins in <5 min (Materials and Methods). QuickPALM defines aging and processing was performed as reported (8) on COS-7 single molecules based on the size, signal/noise ratio, and shape cells expressing α-tubulin fused to photoactivatable GFP (PA- of their PSF but does not calculate a precision. However, once GFP) (Fig. 3 A and B). Our localization precision was found to be the user knows the average precision of single molecules for ≈20–30 nm, which is similar to previous reports of PALM using a certain set of imaging conditions, then QuickPALM becomes PA-GFP (8). Examples of diffraction-limited images and PALM a quick and useful tool that can be used to produce BaLM or BaLM reconstructions are shown in Fig. 3 A–D. To quantify images. The diffraction limited image of microtubules labeled the improvement in the resolution obtained with PALM and

Burnette et al. PNAS | December 27, 2011 | vol. 108 | no. 52 | 21083 Downloaded by guest on September 27, 2021 Fig. 3. Structural resolution of BaLM compared with PALM. (A and B) Total raw diffraction limited image (A) and PALM image (B) from a cell expressing α-tubulin-PAGFP. (C and D) Diffrac- tion limited image (C) and BaLM image (D)of microtubules labeled with Alexa Fluor 488. (E) Linescans (lines in A and B) through a single microtubule in both the diffraction limited image (purple) and the PALM image (blue) showing the decreased width of the microtubule as visualized with PALM. (F) Linescans (lines in C and D) through a single microtubule in both the diffraction limited image (purple) and the PALM image (blue) showing the decreased width of the microtubule as visualized with BaLM. (G) Quantiﬁcation of the widths of microtubules measured as the width of signal distribution from diffraction limited images (301.8 ± 45.7 nm, n = 10 microtubules, 5 linescans per microtubule), PALM images (46.6 ± 6.3 nm, n =10 microtubules, 5 linescans per microtubule), and BaLM images (64.9 ± 11.7 nm, n = 10 microtubules, 5 linescans per microtubule). Error bars denote SD. *P value from Student’s t test <0.001 when distribution are compared with diffraction limited microtubule widths. (Scale bars: 1 μm.)

BaLM to that of the diffraction-limited images, we created line microtubules with the immunogold particles, we measured the scans of the intensity distribution across the width of microtubules distance from the molecule or gold particle’s centroid to the and measured the width of the distribution at half of the maxi- middle of the microtubule (see schematic in Fig. 4D). The average mum signal (Fig. 3 E and F, dotted lines). As expected for the distance from the center of microtubules for the immunogold diffraction-limited image, the half-width was ≈300 nm (301.8 ± particle was 17.0 ± 13.7 nm, whereas the average distance for 45.7 nm). We found that the half-width of microtubules in both BaLM-localized molecules was 32.7 ± 22.8 nm. The better per- PALM and BaLM images was significantly narrower than those formance of electron microscopy stems from the subnanometer from the diffraction-limited images (PALM, 46.6 ± 6.3 nm; localization precision with which the gold particles can be local- BaLM, 64.5 ± 11.7 nm; Fig. 3G). PALM localization of micro- ized. However, BaLM produces a higher density of labeling and tubules is slightly more precise because of the better precision can be more practical for the simultaneous localization of differ- obtained from PALM data compared with BaLM. This difference ent proteins. is primarily due to the lower background signal in PALM data. The number of proteins that can be localized with BaLM To further validate the performance of BaLM, we next com- should only be limited by the ability to separate the emission of pared BaLM images of microtubules to that obtained with con- different fluorophores while still being able to detect single ventional immunogold electron microscopy (Fig. 4). The locali- molecules above camera noise. To explore this possibility, we zation resolution of the immunogold technique is mainly limited investigated the feasibility of doing BaLM analysis on multiple by the size of the immunogold particle and the antibodies. types of fluorescent dyes in a single sample. We first labeled three Therefore, we performed electron microscopy on rotary shad- different proteins in cells using immunofluorescence. Myosin IIA, owed cells that were fixed by using the same procedure as for the clathrin light chain, and microtubules were labeled with Alexa BaLM samples. Microtubules were localized with a secondary Fluor 647, Alexa Fluor 561, and Alexa Fluor 488, respectively antibody conjugated to 15-nm gold particles (Fig. 4C). To com- (Fig. 5A). A dataset in which all molecules were irreversibly pare the distribution of BaLM-localized fluorophores along bleached was acquired for each channel in the following order:

Fig. 4. Structural resolution of BaLM compared with electron microscopy. (A and B) Diffraction limited image (A) and locations of the center of molecules localized with BaLM (B) from a single microtubule. (C) Electron micrograph of a rotary-shadowed microtubule labeled with gold particles. (D) Schematic shows how the centers of the molecules or gold beads were measured from the center line of a microtubule and graph shows the averages and SDs of these distances. BaLM molecules deviated an average of 32.7 ± 22.8 nm (n = 258 molecules from three microtubules) from the center of microtubules, whereas immunogold particles deviated 17.0 ± 13.7 nm (n = 139 gold particles from six microtubules). #P < 0.05. (Scale bars: 100 nm.)

21084 | www.pnas.org/cgi/doi/10.1073/pnas.1117430109 Burnette et al. Downloaded by guest on September 27, 2021 localize more than two different proteins by using PALM because of the limited variety of photoactivatable probes that can be used in combination. By contrast, BaLM can use the same probes used in conventional fluorescence microcopy. An example of this versatility is illustrated in Fig. 5, where a microtubule labeled with secondary antibodies having one of four different Alexa dyes was imaged and processed with BaLM analysis. Previous multicolor superresolution imaging typically uses two (25) or three fluorophores (10). With BaLM, the number of different fluorophores that can be combined in the same sample is limited only by the user’s ability to separate their fluorescence emission. Using BaLM, researchers can design experiments using several different imaging channels with a combination of genetically expressed or synthetic fluorescent molecules. The simplicity and practicality of BaLM can be a critical advantage in relation to other superresolution methods when localization is combined with additional procedures such as array tomography (26, 27) and correlative microscopy (28, 29). Array tomography uses repeated cycles of immunofluorescence labeling, imaging, and stain elution (26, 27). Thus, BaLM could extend the versatility of array tomography by allowing the use of a broad range of readily available fluorescent immunoprobes. BaLM could also be used in correlative electron and fluorescence microscopy for the combined localization of molecules and the ultrastructural visualization of their microenvironment (29–31). The general method in which molecules are localized with Fig. 5. Multicolor BaLM. (A) BaLM reconstruction showing the localization BaLM, PALM, and STORM are very similar and, in fact, these of microtubules (purple), clathrin pits (yellow), and myosin IIA filaments techniques can be used in various combinations. For example, (blue) in the same cell. Immunofluorescent labeling of myosin IIA, clathrin a PALM sample that already contains a photoactivatable protein light chain, and microtubules was performed with Alexa Fluor 647, Alexa such as PAGFP can be counterstained with other fluorescence Fluor 488, and Alexa Fluor 561, respectively. The channels were imaged in the following order: Alexa Fluor 647, Alexa Fluor 488, and Alexa Fluor 561. dyes and imaged with both PALM and BaLM. Furthermore, (B) The diffraction limited images (Left) and the BaLM images (Right)of BaLM in principle can be adapted to produce 3D localization by a single microtubule that has been labeled with a mixture of Alexa Fluor 405 using a similar approach to that developed for 3D STORM (12, (purple), Alexa Fluor 488 (green), Alexa Fluor 561 (orange), and Alexa Fluor 32). This 3D localization can be achieved by using astigmatism in 647 (red) secondary antibodies is shown. The channels were imaged in the the fluorescence image by simply adding a cylindrical lens in the following order: Alexa Fluor 647, Alexa Fluor 405, Alexa Fluor 488, and Alexa imaging path to cause fluorescent molecules to appear elliptical CELL BIOLOGY Fluor 561. (Scale bars: 2 μm.) (33). The ellipticity of the single molecule image can be used to calculate the molecule’s axial position with high precision. The localization precision in PALM/STORM-type super- Alexa Fluor 647, followed by Alexa Fluor 488 and then Alexa resolution techniques depends on several factors including Fluor 561. BaLM localization of these three proteins revealed camera performance, fluorophore properties, and synchroniza- discrete nonoverlapping structures, indicating that there was in- fl fi A tion of image acquisition to uorophore activation/emission signi cant bleed-through between channels (Fig. 5 ). Addition- cycles. Properties of fluorophores such as brightness, quantum ally, we labeled microtubules with a mixture of competing yield, and photostability, vary widely (10, 34, 35). The number of fl uorescently conjugated secondary antibodies: Alexa Fluor 405, photons obtained per activation cycle significantly influences the Alexa Fluor 488, Alexa Fluor 561, and Alexa Fluor 647. Although localization precision (34). Furthermore, the measurement and fi the secondary antibodies showed different labeling ef ciencies calculation of the background is essential to the precision cal- fi (Alexa Fluor 647 showed particularly low labeling af nity when culation. The overall background signal of a BaLM image is mixed with competing antibodies), BaLM worked in all four increased compared with PALM/STORM because it is produced channels. The diffraction limited and BaLM images of a single by the subtraction of two images, thus combining the background microtubule from the four separate channels is shown in Fig. 5B. noise of the two images. Our measurements for the precision of This difference demonstrates that one can use BaLM to produce an Alexa dye with BaLM is ≈50 nm, which is significantly less similar structural information in multiple channels. Finally, it than the precision reported for PALM/STORM (≈10–20 nm) should be possible to expand the use of BaLM to more than (8, 9). Although this lower precision may be an inherent disad- four channels. vantage of BaLM, we feel that its simplicity and accessibility make BaLM a viable alternative to more technically difficult Discussion point-localization techniques. Additionally, with the development In this article, we describe a single molecule superresolution of fluorescent probes with higher photostability and improve- imaging technique, BaLM, which relies on the intrinsic bleaching ments in the overall performance of EMCCD cameras, the res- and blinking behavior of fluorescent molecules. BaLM is a prac- olution of these two techniques will be more comparable. tical procedure for generating superresolution images of samples Bleaching and blinking behaviors are intrinsic to virtually all containing any commonly used fluorophore. BaLM does not re- fluorescent probes (36). Although bleaching is often regarded as quire the use of engineered photoactivatable proteins or photo- a hindrance, and blinking is often ignored, we show here that switchable synthetic dye pairs, although it should work with these both characteristics can be used to produce superresolution as well. images. Fluorescence bleaching has been used to count mole- A common need in fluorescence microscopy is the ability to cules in a molecular complex (20, 21, 37, 38). BaLM could also image multiple fluorophores and to compare localization of be potentially extended to provide information about the actual multiple proteins. Currently, it is technically challenging to number of fluorescent molecules in a multilabeled complex

Burnette et al. PNAS | December 27, 2011 | vol. 108 | no. 52 | 21085 Downloaded by guest on September 27, 2021 sample. During the logarithmic decay of fluorescence, the blinking Bleaching/blinking off events and blinking on events were identified with events are relatively rare in comparison with the bleaching events the delta F down setting or the delta F up setting, respectively, of T func- (37); thus an algorithm could be developed to separately account tions. BaLM molecules were localized and image reconstructions were ren- for the bleaching events and derive approximate quantitative dered by using QuickPALM (43). See SI Materials and Methods for details. information on the fluorophores being localized, analogous to what has been recently developed for PALM (39). Data Analyses Using BaLM Software. Custom BaLM programs were written in MatLab (Mathworks). Single molecule localization and estimation of pre- Materials and Methods cision of localization were performed in a similar manner as described (8, 22). Cell Culture and Sample Preparation. COS-7 or PtK1 cells were cultured, See SI Materials and Methods for details. extracted, and fixed as described (40). An α-tubulin antibody (Sigma) was used to localize microtubules. For expression experiments, cells were trans- Note Added in Proof. While this paper was in review, another paper published fected with myosin IIC-EGFP or α-tubulin-PAGFP. Replicas of critical point a similar approach to BaLM using bleaching of a single synthetic dye (44). As dried samples for electron microscopy were prepared as described (41, 42). shown here, BaLM utilizes both the bleaching and blinking behaviors of See SI Materials and Methods for details. synthetic dyes or fluorescent proteins and can be used in multi-spectral imaging. Image Acquisition. Four hundred to 3,000 images were acquired by using variable angle TIRF with a Nikon Eclipse Ti microscope with either an Evolve ACKNOWLEDGMENTS. We thank Uri Manor, Ronald Petralia, Stephan (Photometrics) or an iXon 847 (Andor) EMCCD camera. Total magnification Brenowitz, and the members of the B.K. and J.L.-S. laboratories for was 40 or 61 nm per pixel. This high magnification allowed the use of more of comments on the manuscript; and Robert Adelstein for the generous the dynamic range of the camera while avoiding pixel saturation. See SI gift of the myosin IIC-EGFP construct. This research was supported by Materials and Methods for details. the Intramural Programs of the National Institute of Deafness and Other Communication Disorders and the National Institute of Child Health and Human Development. D.T.B. was supported by a Pharmacology Practical Image Analyses Using ImageJ. We used freely available ImageJ Research Associate Fellowship from the National Institute of General plugins (http://rsbweb.nih.gov/ij/plugins) to analyze BaLM datasets. To cor- Medical Sciences, National Institutes of Health during the course of rect drift in the images, we used the translational setting of StackReg. these studies.

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