RECENT ADVANCES:

Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules

  Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments.

Pierre Jouchet, Anish R. Roy, and W.E. Moerner, “Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules,” Opt. Commun. 542, 129589 (2023), (DOI: 10.1016/j.optcom.2023.129589, published online 11 May 2023)                                                                        

Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent a, and a transition to more isotropic dynamics in 3D after such treatment. We think that our methodology in the future will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.l

Anna-Karin Gustavsson, Rajarshi P. Ghosh, Petar N. Petrov, Jan T. Liphardt, and W. E. Moerner, “Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics,” Molec Biol Cell 33:ar47, 1-11 (2022) (DOI: 10.1091/mbc.E21-10-0514, published online 30 March 2022).                                         

Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the “nano-bio interface”). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point-spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

Anish R. Roy, Wei Zhang, Zeinab Jahed, Ching-Ting Tsai, Bianxiao Cui, and W. E. Moerner, “Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy,” ACS Nano 16, 192-210 (DOI: 10.1021/acsnano.1c05313, published online 28 September 2021.                                         

Accurate and rapid structured background estimation in single-molecule localization microscopy using the deep neural network BGnet

Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3D localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, both for simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of super-resolution reconstructions of biological structures.

Leonhard Möckl*, Anish E. Roy*, Petar N. Petrov, and W. E. Moerner, (*equal contributions), “Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet,” Proc. Nat. Acad. Sci. (USA) appearing (DOI: 10.1073/pnas.1916219117, published online 23 December 2019). DOI [Slide]

Accurate phase retrieval of complex 3D point spread functions with deep residual neural networks

Phase retrieval, i.e. the reconstruction of phase information from intensity information, is a central problem in many optical systems. Imaging the emission from a point source such as a single molecule is one example. Here, we demonstrate that a deep residual neural net is able to quickly and accurately extract the hidden phase for general point spread functions (PSFs) formed by Zernike-type phase modulations. Five slices of the 3D PSF at different focal positions within a two micron range around the focus are sufficient to retrieve the first six orders of Zernike coefficients.

Leonhard Möckl, Petar N. Petrov, and W. E. Moerner, “Accurate phase retrieval of complex 3D point spread functions with deep residual neural networks,” Appl. Phys. Lett. 115, 251106 (2019) (DOI: 10.1063/1.5125252, published online 18 December 2019). DOI [Slide]

A new microscope for thick cells: TILT3D: 3D single-molecule super-resolution microscopy with a tilted light sheet

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D superresolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Anna-Karin Gustavsson, Petar N. Petrov, Maurice Y. Lee, Yoav Shechtman, and W.E. Moerner, “3D single-molecule super-resolution microscopy with a tilted light sheet,” Nature Commun. 9, 123 (2018) (DOI: 10.1038/s41467-017-02563-4, published online 9 January 2018). DOI [Slide]

Measurement-based estimation of global pupil functions in 3D localization microscopy

We report the use of a phase retrieval procedure based on maximum likelihood estimation (MLE) to produce an improved, experimentally calibrated model of a point spread function (PSF) for use in three-dimensional (3D) localization microscopy experiments. The method estimates a global pupil phase function (which includes both the PSF and system aberrations) over the full axial range from a simple calibration scan. The pupil function is used to refine the PSF model and hence enable superior localizations from experimental data. To demonstrate the utility of the procedure, we apply it to experimental data acquired with a microscope employing an engineered tetrapod PSF with a 6 µm axial range. The phase-retrieved model demonstrates significant improvements in both accuracy and precision of 3D localizations relative to the uncorrected model based on scalar diffraction theory. The localization precision of the phase-retrieved model is shown to be near the limits imposed by Fisher information theory, and the reproducibility of the procedure is characterized and discussed. Code which performs the phase retrieval algorithm is provided on sourceforge, and this algorithm can be used for other engineered PSFs as well.

Petar Petrov, Yoav Shechtman, and W. E. Moerner, “Measurement-based estimation of global pupil functions in 3D localization microscopy,” Optics Express 25 (7), 7945-7959 (2017) (DOI: 10.1364/OE.25.007945, published online 28 March 2017). DOI [Slide]

Multicolour localization microscopy by point-spread-function engineering

Super-resolution microscopy has revolutionized cellular imaging in recent years. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering – namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging, multiple cameras, or segmented dedicated fields of view. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion and optimization calculations, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path. Using this idea, differently colored emitters in the sample acquire different PSFs which may encode distinct variables.

Yoav Shechtman, Lucien E. Weiss, Adam S. Backer, Maurice Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photonics (in press). (DOI: 10.1038/nphoton.2016.137, published online 8 August 2016). DOI [Slide]

Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask, an experimental demonstration of azimuthal filtering

Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging. Until now, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push towards the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators, especially with modest defocus. We have previously dealt with this problem using (a) polarized imaging with the DH-PSF (see Backlund et al. below), or (b) by noting that if the label is ona floppy tether, the error can be bounded (see Lew et al. below). Here we present the theory and experimental demonstration of an all-optical solution to this problem based on azimuthal filtering in the Fourier plane of the microscope (proposed by M.D. Lew and WEM, Opt. Lett. (2014), see below). To experimentally demonstrate azimuthal filtering, we use a high-efficiency dielectric metasurface polarization/phase device composed of dielectric nanoposts with subwavelength spacing. The removal of lateral position bias is demonstrated both on fluorophores embedded in a polymer matrix, and also for dL5 protein complexes that bind malachite green. The figure at left shows x-y localizations for two fluorophores of different orientation (red bar) moved in z for the standard PSF (upper) and for the Y-phi PSF (lower).

M. P. Backlund, A. Arbabi, P. N. Petrov, E. Arbabi, S. Saurabh, A. Faraon, and W. E. Moerner, "Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask," Nat. Photonics 10, 459-462 (2016) (DOI: 10.1038/nphoton.2016.93, published online 16 May 2016). DOI [Slide]

Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy with a nanohole array

The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope's field of view has not been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50-100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and super-resolution methods demanding high accuracy.

Alex von Diezmann, Maurice Y. Lee, Matthew D. Lew, and W. E. Moerner, “Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy,” Optica 2, 985-993 (2015) (DOI: 10.1364/OPTICA.2.000985, published online November 19, 2015). DOI [Slide]

Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions

We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions (3D) over customizable axial (z) ranges of up to 20 µm, with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel to image flow profiles deep in the liquid. We also demonstrate scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells. The tetrapod PSFs provide an important, non-rotating class of PSFs for flexible and deepimaging in three dimensions.

Yoav Shechtman, Lucien E. Weiss, Adam S. Backer, Steffen J. Sahl, and W. E. Moerner, “Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions,” Nano Lett. 15, 4194-4199 (2015) (DOI: 10.1021/acs.nanolett.5b01396, published online May 5, 2015) DOI [Slide]

Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy

Many single nanoemitters such as single molecules produce dipole radiation which leads to systematic position errors in both particle tracking and super-resolution microscopy.  Via vectorial diffraction equations and simulations, we show that imaging only azimuthally polarized light in the microscope naturally avoids emission from the  component of the transition dipole moment, resulting in negligible localization errors for all emitter orientations and degrees of objective lens misfocus.  Furthermore, localization accuracy is maintained even in the presence of aberrations resulting from imaging in mismatched media.

Matthew D. Lew and W. E. Moerner, “Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy,” Nano Letters 14, 6407-6413 (2014), (DOI:10.1021/nl502914k, published online October 1, 2014) DOI [Slide]

Optimal Point-Spread Function Design for 3D Imaging

Single emitter position determination from a single snapshot is an essential element of particle tracking and single-molecule active control microscopy (SMACM). The precision to which the 3D position of an emitter can be determined is a function of many parameters, such as emitter brightness, background fluorescence, and importantly – the shape of the PSF. To extract from an image of a single nanoscale object maximum physical information about its position, we propose and demonstrate a framework for pupil-plane modulation for 3D imaging applications. The method is based on maximizing the information content of the system, by formulating and solving the appropriate optimization problem – finding the pupil-plane phase pattern that would yield a PSF with optimal Fisher information properties. We use our method to generate and experimentally demonstrate two example PSFs: one optimized for 3D localization precision over a 3 µm depth of field, and another with an unprecedented 5 µm depth of field, both designed to perform under physically common conditions of high background signals.

Yoav Shechtman, Steffen J. Sahl, Adam S. Backer, and W. E. Moerner, “Optimal Point Spread Function Design for 3D Imaging,” Phys. Rev. Lett. 113, 133902 (2014), published online September 26, 2014. DOI [Slide]

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

Single-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

This 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

The 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

Recently, 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

Single-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 :

• Andor Technologies News, 13 May 2010
• Andor Technologies Case Study
• Chemical and Engineering News, 7 September 2009
• Laser Focus World, 1 March 2009

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.