Review pubs.acs.org/ac
Unveiling the Inner Workings of Live Bacteria Using SuperResolution Microscopy Hannah H. Tuson and Julie S. Biteen*
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Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
CONTENTS
Super-Resolution Imaging Methods Extending Single-Molecule Fluorescence Imaging to Three Dimensions Biomolecule Labeling Methods Single-Molecule Data Analysis Sources of Error Applications of Single-Molecule Imaging to Bacteria Cytoskeletal Organization DNA Structure DNA Replication and Repair Transcription and Translation Extensions of Super-Resolution Imaging to Medically Relevant Bacteria New Insights in Established Fields Outlook and Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References
bacterial biology has emerged in parallel with super-resolution imaging.12−16 The recent recognition of super-resolution microscopy by the Nobel Committee for Chemistry17 is a testament to the power of this emerging collection of imaging techniques to transform biology. Here, we review super-resolution microscopy with a specific focus on the use of single-molecule microscopy in living bacteria. To motivate our quest to beat the diffraction limit of light, we first explain the limitations of conventional microscopy. Next, we describe the different types of super-resolution microscopies, and we discuss the more recent extension of single-molecule fluorescence (SMF) imaging into the axial (z) dimension. We then describe various methods of fluorescently labeling molecules of interest. We discuss how to analyze single-molecule data once it is obtained and explain some sources of error. After this overview, we highlight several of the most interesting recent applications of single-molecule microscopy inside living bacteria. Finally, we outline some of the remaining challenges for this important technique and discuss how they are being addressed. In conventional microscopy, the light from an isolated, infinitesimally small point source is diffracted by the optical components of the microscope such that the image on the camera is of a punctate spot significantly larger than the point source itself. The actual size of this spot (d) is a function of the wavelength of the emission light (λ) and the numerical aperture of the objective (NA). This relationship was first described by Ernst Abbe in 1873 and has been formalized as d = λ/2NA.18 The numerical aperture is itself defined as NA = n sin θ, where n is the refractive index of the imaging medium and θ is the half angle of the largest cone of light that can enter the lens. Because n is ≲1.5 for an oil immersion lens and θ has a 90° maximum, NA is limited to ∼1.5, and only decreasing the wavelength can provide a direct means of improving the image resolution. Electron microscopy makes use of the very short wavelength of electrons19 to obtain images with very high resolutions down to a few angstroms; however, this method typically requires extensive sample preparation that is incompatible with live cells. Transmission electron microscopy (TEM) collects the electrons that pass through a sample and thus requires very thin samples, 1 μm at most, and optimally only tens of nanometers.20 Scanning electron microscopy (SEM) collects backscattered or secondary electrons from the surface of a sample and typically requires a conductive surface, a constraint
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ptical microscopy has proven to be one of the most powerful tools in biology, in part because it allows direct, noninvasive visualization of dynamic processes inside live cells. It has enabled a myriad of biological discoveries, ranging from the first observation of cells in cork tissue1 by Robert Hook in 1665 to the highly complex neural mapping allowed by the “Brainbow” method.2 However, until recently, microscopy was limited by the diffraction limit of light, which dictates that features smaller than ∼250 nm cannot be resolved. Excitingly, the past 25 years have seen the emergence of various methods collectively known as super-resolution microscopy.3−10 These modern techniques have allowed scientists to avoid the standard diffraction limit and localize objects in vitro with a resolution as good as 1 nm.11 Even in “messier” live cells, superresolution imaging commonly attains ∼30 nm resolution. Although these improved microscopy techniques have been important throughout biology, super-resolution imaging has been especially critical for studies of molecules inside bacteria. While eukaryotic cells are typically 10−100 μm in diameter (1− 2 orders of magnitude larger than the diffraction-limited image of a single molecule), the prototypical Escherichia coli cells are only 2−3 μm long and 0.8−1 μm wide, i.e., of the same order of magnitude as the diffraction limit of light (Figure 1). As a result, traditional imaging methods were ill-suited for studying the inner workings of bacteria cells and the field of subcellular © XXXX American Chemical Society
Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2015
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The electron microscopy techniques of TEM, SEM, and ECT all require the sample to reside in a vacuum chamber to avoid scattering of the electron beam by impact with gas molecules. More recently, environmental SEM (ESEM) has used a dual chamber such that the electron beam passes primarily through a vacuum before entering the sample chamber, which is filled with gas to atmospheric pressure. This setup allows samples to be hydrated and uncoated and has been successfully used to image live bacteria.26 However, as with conventional SEM, this technique can only be used to visualize the cell surface. Although EM methods continue to evolve, fluorescence microscopy, which uses probes that are excited and emit in the visible wavelength range, remains the preferred way to image inside living cells despite the limited resolution. Referring back to Abbe’s equation and assuming an emission wavelength of 500 nm and a microscope objective NA of 1.4, within cells, we cannot resolve objects that are closer together than ∼180 nm using conventional light microscopy. However, this apparent limitation can be circumvented by several methods, discussed in more detail in the following section. One of these methods, and the primary focus of this review, is to image single molecules one at a time.
Figure 1. Single-molecule imaging provides resolution better than the diffraction limit of light by fitting the image of a fluorescent emitter. (A) Left, a diffraction-limited image of a single fluorescent molecule. Right, the same spot (bottom) is plotted as a matrix of pixel intensities (middle) and then fit to a Gaussian function (top) to determine the emitter position with nm-scale precision. (B) The size of most commonly studied bacteria is in the range of 0.8−1 μm wide and 2−4 μm long. This schematic, which is drawn to scale, shows a 300 nmwide diffraction-limited image.
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SUPER-RESOLUTION IMAGING METHODS Generally, super-resolution methods fall into two categories: those that use nonuniform illumination patterns and nonlinear electronic transitions and those in which only a single molecule is imaged at a time (see Table 1 for a summary of classical schemes for super-resolution imaging). Stimulated emission depletion (STED) microscopy is arguably the most prevalent technique in the first category. In STED, the fluorescence excitation beam is surrounded by a high-powered donut-shaped depletion beam, and the sample is scanned with this dual beam. After excitation, the depletion beam forces fluorophores to relax into a higher energy state via saturated emission depletion. The red-shifted stimulated emission can be filtered out spectrally or temporally, and the result is an excitation spot much smaller than the diffraction limit, with the size of the spot decreasing and the resolution improving as the power of the depletion beam increases.27 Thus, knowledge of the location of the activation spot can be combined with emission intensity to produce a high-resolution map of the sample under study. This scanning microscopy technique is similar to confocal
which in turn necessitates treating biological samples by either coating them in conductive material such as gold or impregnating them with a contrast agent such as osmium.21 The related technique of electron cryotomography (ECT) uses TEM to produce the 3D reconstruction of a sample from a series of 2D images taken at different tilt angles.22 While this technique also requires stationary thin samples, flash-freezing, which produces vitreous ice, keeps the sample in a near-native state. This technique has been used with great success to study the bacterial cell wall, for example, showing that peptidoglycan in Gram positive cells runs around the circumference of the cell rather than parallel to the long axis.23 Additionally, ECT showed that the helical structures observed by fluorescence microscopy when MreB is tagged with YFP24 are in fact an artifact of the tag and that MreB does not form extended filaments in its native state.25 Table 1. Classical Super-Resolution Imaging Schemes
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Figure 2. Schematic of a typical single-molecule fluorescence microscope. Appropriate lasers are selected for the fluorescent probes to be used. In this example, the 488 nm laser can excite green probes such as mCitrine and PAGFP; the 561 nm laser can excite red probes such as PAmCherry and mMaple; and the 406 nm laser can activate photoactivatable or photoswitchable probes such as PAGFP, PAmCherry, and mMaple. Excitation filters clean up the laser light to suppress unwanted long-wavelength signals, and quarter-wave plates circularly polarize the light to excite all probes equally regardless of their orientation. Linked shutters allow coordination of multiple wavelengths. In this example, the 561 nm excitation laser shutter automatically closes when the 406 nm activation laser shutter is opened. A telescope widens the laser beam to fill the back aperture of the microscope objective and provides a larger illuminated imaging area. Mirrors and a periscope direct the laser beam into the microscope, and the dichroic filter sends the laser light through the objective and up to the sample. Fluorescent signals are collected by the objective, and any reflected laser light is removed by a long-pass emission filter and a dichroic filter before detection by the camera sensor. This single-molecule imaging setup has a flexible design that can readily accommodate additional components to enable other imaging modalities, including 3D imaging and two-color imaging.
a series of harmonics that have a higher spatial frequency than the initial illumination pattern and can provide a resolution better than 50 nm.39,40 For the remainder of this Review, we focus on the family of microscopy techniques that achieves super-resolution information by imaging single molecules one at a time. The wide-field microscopy technique of SMF imaging takes advantage of the fact that the center of the diffraction-limited image (or point spread function, PSF) of an isolated infinitesimally small emitter indicates the position of the emitter with subdiffraction limited precision (Figure 1A). Therefore, when only one emitter fluoresces at a time per diffraction-limited area, SMF schemes can localize that emitter. Single-molecule methods have the added advantage that, because of short imaging times, super-resolution information about molecular position can be combined with single-molecule tracking to provide quantitative information about the dynamics of individual molecules.41 Single-molecule techniques have so far been the more commonly used super-resolution method for imaging biomolecules in live cells.14,16,42−47 One important advantage of single-molecule microscopy is that, in contrast to the patterned illumination techniques, single-molecule methods do not require specialized instrumentation to manipulate the light source. A standard microscope can be used for SMF imaging as long as it is equipped with the appropriate filters and an efficient camera (Figure 2).48 Low-power continuous-wave (CW) lasers with appropriate excitation filters are typically used as the excitation source. Good long-pass filters after the sample remove scattered excitation light; these filters are critical because the fluorescence signal intensity is many orders of magnitude smaller than the excitation intensity.48 A high quantum efficiency camera, which will capture as many emitted
microscopy, in which a sample is scanned with an excitation beam; however, in confocal microscopy, the location of the beam is known only with diffraction-limited certainty. The use of the depletion beam provides a more precise location of where excitation is occurring. In vitro resolutions of 2.4 and 16 nm have been reported for STED based on nitrogen vacancy centers28 or dyes,29 respectively, with resolutions in live cells more commonly in the 50−100 nm range.30,31 STED dyes are typically organic molecules, but STED has also been demonstrated with fluorescent protein (FP) tags in eukaryotic cells.32−34 Although one of the first demonstrations of STED was an image of the E. coli cell membrane at a resolution of 33 nm,35 since then this technique has been only infrequently applied to bacteria.36,37 Other super-resolution microscopy methods based on nonuniform excitation have also been developed. Ground state depletion microscopy (GSD; also called GSD followed by individual molecular return [GSDIM]) is a variation on STED in which depletion is realized by “shelving” electrons in the triplet state.38 In structured illumination microscopy (SIM), rather than using a donut-shaped beam, the sample is illuminated with light that has a defined, spatially repeating pattern. The interaction of the light with the sample creates an interference pattern, and high-resolution information about the sample can be extracted from the Moiré fringes of the pattern. SIM can improve imaging resolution by a factor of 2. An extension of this method, saturated SIM (SSIM; also called saturated patterned excitation microscopy [SPEM]) takes advantage of the nonlinear response of fluorescence to increased excitation power near saturation. The SSIM illumination pattern peak excitation intensity is at or above the saturating intensity. The resulting emission pattern contains C
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The first three reports of such single-molecule-based superresolution imaging, achieved by stochastic optical reconstruction microscopy (STORM),8 photoactivated localization microscopy (PALM),9 and fluorescence photoactivation localization microscopy (FPALM),54 are based on the same principle: individual fluorophores are sequentially activated, imaged and localized, and then bleached. Once this cycle is repeated enough times, a very high density of molecular positions is built up, and ultimately, a picture of all of the activated fluorophores in the entire cell can be developed. Historically, PALM made use of photoactivatable or photoswitchable FPs,9,54 while the first realizations of STORM used antibodies conjugated to a pair of dyes (an activator and a reporter).8 Thus, PALM probes usually undergo irreversible bleaching, while STORM probes can stochastically switch back on some time after bleaching. As these single-molecule superresolution methods have been further developed, a wider variety of probes has been used. Direct STORM (dSTORM) uses single dyes that reversibly turn on and off in a reducing buffer that promotes the formation of a long-lived triplet state.55 Caged versions of dyes such as rhodamine and dicyanomethylenedihydrofuran (DCDHF) are nonfluorescent until uncaging and can be used for PALM imaging.56,57 Finally, “standard” FPs, like mCitrine, that are not optimized to be photoactivatable, may still also be activated by violet light after bleaching.52,58 As an alternative to using photoswitching or photoactivation, the points accumulation for imaging in nanoscale topography (PAINT) microscopy59 scheme instead exploits that fact that freely diffusing fluorescent particles are moving too fast to be captured at most camera speeds. However, if a fluorophore temporarily or permanently attaches to a surface, it becomes stationary and can be imaged until it either desorbs and diffuses away or bleaches. By imaging a large number of these events, a high-resolution image of the structure to which the fluorophores bind, for example, DNA, can be obtained.60 Due to the speed limitations of typical cameras, many modern super-resolution techniques make use of this type of motionbased filtering, where faster-moving molecules are not imaged because their PSFs are so blurred as to be undetectable.61,62 In living cells, single-molecule motion provides an additional handle for super-resolution imaging. Fluorescent molecules can be tracked over time, revealing the distribution of dynamics in a heterogeneous system. From single-molecule tracks, one can determine the diffusion coefficient of a labeled protein; whether its motion is confined and if so, what is the radius of confinement; and the number of different protein populations (for example, fast, medium, slow) and the distribution of total proteins into each of those populations.63,64 Indeed, by localizing the same molecule with nm-scale precision in successive frames, even one molecule can give super-resolution information about the cellular environment. A wealth of information can be obtained from SMF imaging. In addition to determining the location of a fluorophore very precisely, in an SMF microscope, even low copy number proteins can be detected.65 The ability to detect single copies of proteins also circumvents the ensemble averaging that is inherent in bulk microscopy. Additionally, the number of labeled proteins can be counted either by measuring the total cellular fluorescence or by watching for the drop in fluorescence each time a molecule bleaches and counting the number of steps.66 While the cellular background fluorescence makes this technique more difficult to achieve in cells, stepwise photo-
photons as possible, is also desired because the resolution depends on the number of detected photons. Furthermore, fluorescent labels must be bright enough that a single molecule produces sufficient photons to be detected by the camera, and background signal must be reduced as much as possible, for example, by using minimal rather than rich medium.49 SMF microscopy depends on the principle that a single point source will produce a diffraction-limited PSF, which can be fit with high precision to determine the true location of that emitter within the larger PSF width.7 Thus, the primary requirement for single-molecule imaging, beyond a fluorescence imaging system capable of detecting single molecules, is that the fluorescent molecules being imaged are well separated to produce this well understood PSF. Because an isolated molecule will be diffracted to an image with a radius of ∼λ/2NA according to the Abbe limit, the Rayleigh criterion dictates that two molecules cannot be resolved if they are closer together than 0.61λ/NA. If this separation is interpreted purely as an upper limit on the spatial density, this restricts SMF microscopy to sparsely labeled samples. Sparse labeling becomes particularly difficult when the labeled molecules are mobile, as even two molecules in a large cell may cross paths. Sparse labeling can be achieved for nonmoving molecules by expressing the labeled protein as a small subset of the total protein population, but this ratio is difficult to control precisely; there is no guarantee that the labeled proteins will be nicely spaced out. Furthermore, when imaging static structures, the Nyquist criterion, which dictates that the sampling must be 2× more dense than the desired resolution, must be satisfied to get an accurate picture of the sample.50 Thus, only rough features can be resolved in a sample labeled with markers that are spaced out well enough to satisfy the Rayleigh criterion, and the traditional diffraction limit persists. Though the position and motion of an isolated single molecule can be very precisely determined, SMF can only achieve super-resolution imaging of fine features in a sample when combined with a secondary scheme to allow detection of only a few molecules at a time within a densely labeled system.5 This secondary discrimination has been achieved in many ways. Indeed, in the first demonstration of single-molecule fluorescence detection in 1989, Moerner and Kador addressed one pentacene molecule at a time within a collection of pentacene molecules, each with a narrow zero-phonon absorption band at a subtly different peak frequency due to inhomogeneous broadening, by scanning the excitation laser frequency.3 It was later realized that the spatial position of molecules in the same imaging volume could also be separated if they were spectrally distinct.51 However, these early experiments had to be performed at extremely low temperatures (∼1.6 K) to maintain the narrow absorption line widths. In the past decade, temporal separation of fluorophores has been widely adopted to enable super-resolution imaging at room temperature. By using labels whose fluorescent emission can be driven to turn on and off such that only one is on at a time in each diffraction-limited volume,52,53 a higher density of single molecules can be imaged to build up a super-resolution image over time. Using this concept, SMF-based superresolution microscopy was extended to live cells in 2006, when several groups simultaneously demonstrated methods of temporally separating fluorophores in cells.8,9,54 Following that discovery, single-molecule imaging has rapidly gained popularity throughout the biology community. D
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Figure 3. Double-helix point spread function has better precision than astigmatism or biplane imaging over a wider range of axial positions. (Top row) 6000 detected photons. (Bottom row) 1000 detected photons. In all cases, noise is Poissonian with a mean of 2 photons per pixel and the localization precision is plotted as a function of axial position, z. (A) and (D), localization precision in the x dimension, σx; (B) and (E), localization precision in the y dimension, σy; (C) and (F), localization precision in the z dimension, σz. Reprinted with permission from Badieirostami, M.; Lew, M. D.; Thompson, M. A.; Moerner, W. E. Appl. Phys. Lett. 2010, 97, 161103 (ref 106). Copyright 2010, AIP Publishing LLC.
the light collected by the objective into two paths that are focused onto two different detectors. The difference in the path lengths of the two light beams allows simultaneous imaging of two different focal planes, producing resolutions of 30 nm in the x−y dimension and 75 nm in the z dimension.102,103 Another more recent 3D method is double-helix point spread function (DH-PSF) microscopy. In this approach, emission light is manipulated in phase space rather than in real space. By manipulating the signal with a spatial-light modulator or phase mask at the Fourier plane of the microscope, the regular PSF is transformed into a bilobed PSF, where the angular orientation of the lobes is a function of the emitter z position. This method has provided in vitro resolutions of 10 nm in the x−y plane and 20 nm in the z direction, with a 2 μm depth of field.104 In live cells, this technique has been used to track single mRNA molecules in Saccharomyces cerevisiae with a resolution of 25 nm in the x−y dimension and 50 nm in the z dimension,105 as well as to determine the position of the cytoskeletal protein crescentin in relation to the Caulobacter crescentus cell membrane.92 A theoretical comparison of the best possible localization precision of the astigmatism, multifocal plane, and DH-PSF microscopy techniques shows that the DH-PSF provides relatively consistent localization precision over a large depth of field. Astigmatism and multifocal plane imaging have much greater variability, with the best localization precision closest to the focal plane. At a high signal-to-noise ratio, DH-PSF is consistently superior across the whole examined depth of field, while at low signal-to-noise ratio, astigmatism and multifocal plane imaging provide better precision than DH-PSF in a narrow window close to the focal plane (Figure 3).106 However, astigmatism and multifocal plane imaging have thus far been more widely adopted, perhaps because they are more straightforward extensions of 2D SMF microscopy. 3D imaging continues to expand as new methods are developed. Astigmatism and multifocal plane microscopy have recently been combined for high-speed, high-resolution imaging of microtubules in mammalian cells.107 Beyond single-molecule schemes, several tools based on patterned illumination have been developed to perform noninvasive 3D super-resolution imaging of thick samples such as tissue and large cells. SIM can be extended to three dimensions to double
bleaching has been successfully used on a few occasions, for instance, to determine the cooperativity of starch utilization system proteins in human gut bacteria.64
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EXTENDING SINGLE-MOLECULE FLUORESCENCE IMAGING TO THREE DIMENSIONS The methods discussed so far improve localization precision and thus resolution in the imaging plane. However, resolution in the axial (z) direction is also important. Although bacteria are very thin, without z information, it is impossible to determine whether a signal is coming from the membrane or the nucleoid, for example. Projecting 3D motion onto a 2D plane produces several artifacts: in the most extreme case, a protein that is moving purely in the z direction will appear to be stationary without 3D information, but any motion with a z component will appear slower than it actually is. Additionally, in purely two-dimensional super-resolution reconstructions, the density of localizations will appear artificially high near the cell membrane due to the effect of projecting data from a threedimensional object onto a plane.94 The z resolution in conventional microscopy is restricted to d = 2λ/NA2, so even in confocal microscopy, which optically sections a sample, the axial resolution is worse than the in-plane resolution by a factor of 4/NA. However, several single-molecule methods have been developed to provide super-resolution information about the position of a molecule in the z dimension alongside subdiffraction-limit information in the x−y plane.95 A number of these methods have primarily been used for in vitro imaging;72,96−100 those that have seen the most use in live cells are described below. The astigmatism approach is currently the most commonly used 3D method in bacterial imaging. In this technique, a cylindrical lens in the imaging pathway after the sample produces a PSF that is elongated along the x or y axis such that the PSF aspect ratio and the direction of elongation identify the position of the emitter above or below the focal plane.101 This technique was used to image the bacterial cytoskeletal protein FtsZ in E. coli with resolutions of
