Single-Molecule Projects:

Development of Novel Microscopies for Superresolution In Three Dimensions Using Photoswitching/Photoactivation and Single-Molecule Imaging

Current Subgroup Members: Dr. Steffen Sahl, Adam Backer, Mikael Backlund, Alex Diezmann, Matthew D. Lew

PUBLICLY AVAILABLE CODE

RECENT ADVANCES-see below

OVERVIEW: Three-Dimensional Super-resolution Imaging of Single Molecules using a Double-Helix Point Spread Function (DH-PSF)

We have developed a unique method for 3D super-resolution with single fluorescent molecules where the PSF of the microscope has been engineered to have 2 rotating lobes where the angle of rotation depends on the axial position of the emitting molecule. In other words, the PSF appears as a double-helix along the z axis of the microscope, so it is called the double-helix PSF (DH-PSF) for convenience. This method is based on earlier work of our collaborator Rafael Piestun at University of Colorado who showed that a rotating DH-PSF could be formed by a superposition of Gauss-Laguerre (GL) modes that form a line in the GL modal plane (1). His student, Prasanna Pavani, modified the PSFdesign to increase efficiency, and used it for both photon-unlimited scatterers and very bright moving fluorescent microspheres(2). The figure at the right shows the image of a single fluorescent sphere at different z-positions relative to the usual focal position of the microscope. You are not seeing double, but, rather, the actual behavior of the DH-PSF sampled by the fluorescent bead! Various z-slices of the PSF appear as pairs of two spots. The angle of the line between the two spots can be used to read out the z-position of the object; the lower part of the figure shows a calibration curve extracted from the bead images. The DH-PSF can be generated by inserting a phase mask in the Fourier transform plane of the microscope.

We have recently shown(3) that a particularly useful photon-limited source, a single fluorescent molecule, can be imaged far beyond the diffraction limit by using a DH-PSF. In thick samples, we have demonstrated super-localization of single fluorescent molecules with precisions as low as 10 nm laterally and 20 nm axially over axial ranges >2 µm. The DH-PSF imaging system can be used to identify the 3D position of many molecules in a single image as long as the PSFs from the different emitters do not appreciably overlap. We have demonstrated this capability by using a sample containing a low concentration of the fluorophore DCDHF-P embedded in a ≈2 µm-thick PMMA film. The figure at right, left side, compares the standard (upper) and the DH-PSF(lower) images of 2 single molecules at different 3D positions selected to be fairly close to the focal plane for purposes of illustration only. In general, molecules away from the focal plane appear quite blurry in the standard PSF image. In contrast, the DH-PSF image encodes the axial position of the molecules in the angular orientation of the molecules’ DH-PSF lobes, which are distinctly above the background with approximately the same intensity through the entire z range of interest. This increased depth-of-field is illustrated directly in the right side of the figure, which shows a representative DH-PSF image of multiple molecules in a volume. Each molecule is seen to exhibit 2 lobes oriented at an angle that is uniquely related to its axial position, and the x,y,z, positions of these molecules are shown in Ref. 3..

Finally, to demonstrate true superresolution, we used single-molecule photoactivated localization microscopy (PALM) to determine the 3D location of many single molecules in a polymer sample, where many pairs of molecules were much closer than the standard diffraction limit. Our method may thus be called DH-PALM, for Double-Helix PALM. The photoactivatable molecule is from the new class of aryl azide fluorogens we have recently developed(4). The resulting image is shown at the right below, and the inset illustrates localizations of two molecules only 36 nm apart. For full details, see Ref. (3). Our work illustrates a new and powerful method for 3D superresolution imaging, because the DH-PSF has far more Fisher information (changes more rapidly with z) than is the case in other approaches for extracting 3D position information.

(1) R. Piestun, Y. Y. Schechner, and J. Shamir, Journal of the Optical Society of America A 17, 294-303 (2000).

(2) S. R. P. Pavani and R. Piestun, Optics Express 16, 3484-3489 and 22048-22057 (2008).

(3) S. R. P. Pavani*, M. A. Thompson*, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995-2999 (2009) [Journal Link]

(4) S. J. Lord, N. R. Conley, H.-l. D. Lee, R. Samuel, N. Liu, R. J. Twieg, W. E. Moerner, JACS 130, 9204 (2008) [Slide] [journal link: JACS]

 

Recent Reviews:

Mikael P. Backlund, Matthew D. Lew, Adam S. Backer, Steffen J. Sahl, and W. E. Moerner, “The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging,” Minireview, ChemPhysChem 15, 587-599 (2014), published online December 30, 2013. DOI

Steffen J. Sahl and W. E. Moerner, "Super-resolution Fluorescence Imaging with Single Molecules,” Curr. Opin. Struct. Biol. 23, 778-787 (2013), published online 8 August 2013. DOI

W. E. Moerner, “Microscopy beyond the diffraction limit using actively controlled single molecules,” J. Microsc. 246, 213-220 (2012), published online 12 April 2012. DOI

Michael A. Thompson, Matthew D. Lew, and W. E. Moerner, “Extending Microscopic Resolution with Single-Molecule Imaging and Active Control,” Annual Reviews of Biophysics 41, 321-342 (published online 9 Jun 2012). DOI

 

RECENT ADVANCES:

Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements

FtsZ SRSingle-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the sta tistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e. goodness-of- t tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the tted model with the observed trajectory so that extracted parameters are not over-interpreted (the tools are applicable to linear or nonlinear SDEs calibrated from non-stationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT tracking pplications.  

Christopher P. Calderon, Michael A. Thompson, Jason M. Casolari, Randy C. Paffenroth, and W. E. Moerner, “Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements,” Michael D. Fayer Festschrift, J. Phys. Chem. B 117, 15701-15713 (2013) published online September 9, 2013. DOI[Slide]

 

Single-molecule orientation measurements with a quadrated pupil

FtsZ SRThis paper presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single  molecule (SM), which uses a specially designed phase mask, termed a “quadrated pupil,” conjugate to the back focal plane (BFP) of a conventional widefield microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter, and makes this anisotropy amenable to quantitative analysis at the image plane.  In comparison to older image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies and optical aberrations. Precision on the order of 1-5 degrees is achieved in proof-of-concept experiments for both azimuthal (φ) and polar (θ) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator (SLM) that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies.            

Adam S. Backer, Mikael P. Backlund, Matthew D. Lew, and W. E. Moerner, “Single-molecule orientation measurements with a quadrated pupil,” Optics Lett. 38, 1521-1523 (2013), published online March 15, 2013. DOI[Slide]

Rotational Mobility of Single Molecules Affects Localization Accuracy in Super-Resolution Fluorescence Microscopy

FtsZ SRThe asymmetric nature of single-molecule (SM) dipole emission patterns limits the accuracy of position determination in localization-based super-resolution fluorescence microscopy. This effect depends upon axial position of the molecule with respect to the focal plane. More importantly, in actual experiments, the degree of mislocalization depends highly on the rotational mobility of the SM; only for SMs rotating within a cone half angle α > 60° can mislocalization errors be bounded to ≤ 10 nm. Simulations demonstrate how low or high rotational (orientational) mobility can cause resolution degradation or distortion in super-resolution reconstructions. The design of optimal fluorescent label attachments may be altered to affect the local orientational mobility of the fluorophore.

Matthew D. Lew*, Mikael P. Backlund*, and W. E. Moerner (*equal contributions), “Rotational Mobility of Single Molecules Affects Localization Accuracy in Super-Resolution Fluorescence Microscopy,” Nano Lett. 13, 3967- 3972 (2013), published online January 29, 2013. DOI[Slide]

 

Simultaneous, Accurate Subdiffraction Measurement of the 3D Position and Orientation of Single Molecules Enabled by the Double-Helix Point Spread Function Microscope

FtsZ SRRecently, single-molecule-based super-resolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically employ image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole  when it is rotationally immobile, depending highly on the molecule’s three-dimensional (3D) orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ~50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation of its lateral position to appear larger than that expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve standard deviations in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths we are able to improve from a lateral standard deviation of 116 nm (~4x worse than the photon-limited precision, 28 nm) to 34 nm (within 6 nm of the photon limit).

Mikael P. Backlund*, Matthew D. Lew*, Adam S. Backer, Steffen J. Sahl, Ginni Grover, Anurag Agrawal, Rafael Piestun, and W. E. Moerner (equal contributions), “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Nat. Acad. Sci. (USA) 109, 19087-19092 (2012), published online 5 November 2012. DOI[Slide]

 

Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism

FtsZ SRSingle-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells.  Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway.  We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring.  We quantify these dynamics and determine the FtsZ depolymerization time to be < 100 ms.  We image the Z-ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre-divisional stage.

Julie Biteen, Erin D. Goley, Lucy Shapiro, and W. E. Moerner, “Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism, ChemPhysChem 13, 1007-1012 (2012), published online January 20, 2012. DOI [Slide]

 

The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision

The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision σ of this approach using single emitters in a cell under typical background conditions, demonstrating σ < 20 nm laterally and <30 nm axially for N ≈ 1180 photons per localization. Combined with light-induced blinking of single-molecule labels, we present proof-of-concept imaging beyond the optical diffraction limit of microtubule network structures in fixed mammalian cells over a large axial range in three dimensions.

 

Hsiao-lu D. Lee*, Steffen J. Sahl*, Matthew D. Lew, and W. E. Moerner, “The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision,” Appl. Phys. Lett. 100, 153701 (2012), published online 9 April 2012. DOI [Slide]

 

Three-dimensional super-resolution co-localization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled super-resolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, opening a new frontier in noninvasive imaging of structures within living cells.  However, live cell super-resolution imaging has been challenged by the need to image 3D structures relative to their biological context, such as the cellular membrane.  We have developed a technique, termed Super-resolution by PoweR-dependent Active Intermittency and Points Accumulation for Imaging in Nanoscale Topography (SPRAIPAINT) that combines imaging of intracellular eYFP fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser.  Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile Red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required.  Here we demonstrate live cell 3D super-resolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, co-localized with the surface of the bacterium Caulobacter crescentus using a double helix point spread function microscope.  Three-dimensional co-localization of intracellular protein structures and the cell surface with super-resolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-30 nm in 3D) over a large field of view (12×12 μm).

Matthew D. Lew*, Steven F. Lee*, Jerod L. Ptacin, Marissa K. Lee, Robert J. Twieg, Lucy Shapiro, and W. E. Moerner, “Three-dimensional super-resolution co-localization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus,” Proc. Nat. Acad. Sci. (USA) 108, E1102-E1110 (2011) and 108, 18577-18578 (2011), published online 26 October 2011. [Slide] [Journal Link] [MOVIE 7.7MB .wmv-see SI Videos for more]

 

Corkscrew point spread function for far-field three-dimensional nanoscale localization of point-like objects

We have developed a new point spread function (PSF), termed the corkscrew PSF, which can localize objects in three dimensions throughout a 3.2 µm depth of field with nanometer precision using a wide-field imaging microscope. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging fluorescent beads on the surface of a triangular PDMS grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z. This new PSF should provide a useful complement to the DH-PSF for 3D imaging with wide-field microscopy.

Matthew D. Lew, Steven F. Lee, Majid Badieirostami, and W. E. Moerner, “Corkscrew point spread function for far-field three-dimensional nanoscale localization of point-like objects," Optics Letters 36, 202-204 (2011), published online December 14, 2010. [Slide] [Journal Link]

 

Three-Dimensional Localization Precision of the Double-Helix Point Spread Function versus Astigmatism and Biplane

Wide-field microscopy with a double-helix point spread function (DH-PSF) provides three-dimensional (3D) position information beyond the optical diffraction limit. We compare the theoretical localization precision for an unbiased estimator of the DH-PSF to that for 3D localization by astigmatic and biplane imaging using Fisher information analysis including pixelation and varying levels of background. The DH-PSF results in almost constant localization precision in all three dimensions for a 2 μm thick depth of field, while astigmatism and biplane improve the axial localization precision over smaller axial ranges. For high signal-to-background ratio, the DH-PSF on average achieves better localization precision.

Majid Badieirostami, Matthew D. Lew, Michael A. Thompson,, and W. E. Moerner, “Three-Dimensional Localization Precision of the Double-Helix Point Spread Function versus Astigmatism and Biplane," Applied Physics Letters 97, 161103 (2010), published online October 18, 2010. [Journal Link]

 

Tracking mRNA in Live Yeast Cells Beyond the Diffraction Limit in 3D

Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25 nm precision in the x and y dimensions and 50 nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types.

Michael A. Thompson, Jason Casolari, Majid Badieirostami, Patrick O. Brown, and W. E. Moerner, “Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function," Proc. Nat. Acad. Sci. (USA) 107, 17864-17871 (2010), published online October 4, 2010. [Slide] [Journal Link][Free access pdf]

 

Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function - Quantifying Localization Precision with Measurement and Fisher Information

Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). We describe in detail how the localization precision quantitatively depends upon the number of photons detected and the z position of the nanoscale emitter, thereby showing a ~10 nm localization capability along x, y, and z in the limit of weak emitters. Experimental measurements are compared to Fisher information calculations of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions.

M. A. Thompson*, M. D. Lew*, M. Badieirostami, and W. E. Moerner, (*equal contributions), “Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatio-Temporal Resolution Using a Double-Helix Point Spread Function,” Nano Letters 10, 211 (2010), published online December 15, 2009. [Slide] Journal Link] [Movie - see SM movies page]

 

In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function

The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells.

M. D. Lew, M. A. Thompson, M. Badieirostami, and W. E. Moerner, “In-vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function,” Proc. SPIE 7571, 75710Z-1-75710Z-13 (2010).[Journal Link]

PRESS HIGHLIGHTS :

 

This work was supported in part by The National Institutes of General Medical Sciences Grant No. R01GM085437. The DH-PSF was originally developed by R. Piestun at the University of Colorado.