3D Super-resolution Imaging Research Articles

Isotropic 3D Super Resolution Imaging with Self-Bending Point Spread Function

We introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional (3D) super-resolution fluorescence imaging. We designed a side-lobe-free SB-PSF for the incoherent fluorescence emission and implemented a two-channel detection scheme for the SB-PSF to enable unambiguous 3D localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF ideal for precise 3D localization of molecules over a large imaging depth. Using this SB-PSF, we demonstrated super-resolution imaging with isotropic localization precisions of 10-15 nm in all three dimensions over a 3 μm imaging depth without sample scanning.

Super Resolution Imaging of Thick Samples by Means of Selective Plane Illumination Microscopy

Recently, super-resolution techniques based on single molecule localization became a popular tool to understand biological processes and image structures below the diffraction limit. In the last few years a fast development in the field allowed multicolor and 3D super-resolution imaging of biological samples. However, super-resolution techniques are currently well established at the cellular level but their application to more complicate samples, such as tissues and embryos, still remain a challenging task. within this context, recently, the coupling of localization based techniques and selective plane illumination microscopy [1], allowed to extend the application range to thicker tissues ( 50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM) both in the linear and non linear regime. Particular attention is addressed to the advantages provided by two photon photo-activation in scattering environments.IML-SPIM allows to image cellular spheroids with sub-diffraction resolution in depth and can be applied to image cellular substructures in Zebrafish early developmental stages as well.(1) Cella Zanacchi F. et al., “Live-cell 3D super-resolution imaging in thick biological samples.,” Nat Methods, 8, no. 12, pp. 1047-1049, (2011)(2) Truong T. V. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.,” Nat Methods, 8, no. 9, pp. 757-760, (2011). Huisken, J., et al. Science 305, 1007-1009 (2004).

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE)

Super-resolution imaging with photo-activatable or photo-switchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field. We demonstrate for the first time a method integrating a Fisher information efficient DH-PSF design, a surface relief optical phase mask, and an optimal 3D localization estimator. 3D super-resolution imaging using photo-switchable dyes reveals the 3D microtubule network in mammalian cells with localization precision approaching the information theoretical limit over a depth of 1.2 µm.

Adaptive optics enables 3D STED microscopy in aberrating specimens

Stimulated emission depletion (STED) microscopy allows fluorescence far-field imaging with diffraction-unlimited resolution. Unfortunately, extending this technique to three-dimensional (3D) imaging of thick specimens has been inhibited by sample-induced aberrations. Here we present the first implementation of adaptive optics in STED microscopy to allow 3D super-resolution imaging in strongly aberrated imaging conditions, such as those introduced by thick biological tissue.

3D Multicolor Super-Resolution Imaging Offers Improved Accuracy in Neuron Tracing

The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.

Live-Cell Super-Resolution Fluorescence Imaging at High Spatiotemporal Resolutions

Super-resolution fluorescence microscopy accomplished nanometer resolutions in optical imaging along with multi-color and three-dimensional capability in fixed specimens. For live cells, time and spatial resolutions has been limited by the intrinsic tradeoff between the two resolutions. Using bright, fast switching cyanine dyes, we achieved high spatiotemporal resolutions with stochastic reconstruction microscopy (STORM). We obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of clathrin-coated pits and their transferrin cargo labeled with photoswitchable dyes either directly or via SNAP tags in living cells. The high photon output and fast switching rate of the cyanine dye enabled us to demonstrate 2D imaging at spatial resolutions of ∼25 nm and temporal resolutions as fast as 0.5 sec, which represent a 2-3 fold improvement in spatial resolution and 50-100 fold improvement in temporal resolution compared to previously reported resolutions obtained using single-molecule-localization based super-resolution approaches. Furthermore, we demonstrate live-cell 3D volumetric super-resolution imaging for the first time, with a spatial resolution of ∼30 nm in the lateral directions and ∼50 nm in the axial direction at time resolutions down to ∼1 sec. Two-color 3D super-resolution imaging in live cells was also achieved using photoswitchable probes with distinct emission spectra. In addition, we obtained similarly high spatial and temporal resolutions imaging other important cellular structures. These imaging capabilities open a new window for resolving ultrastructures in living cells.

2D/3D Super-Resolution Microscopy by Means of Individual Molecule Localization (IML) and Selective Plane Illumination Microscopy

In the last few years localization based techniques, which exploit photoactivation, photoconversion or ground state depletion of fluorescent molecules, became a popular tool for super-resolution imaging of biological samples. Recently, approaches based on two photon excitation have been implemented in order to get axial confinement of the photoactivation process allowing for 3D super-resolution imaging of biological samples [1]. However, a topic of interest is still represented by the widening of super-resolution applications to thick samples (>15 μm). Within this scenario, light sheet based fluorescence microscopy techniques provide optical sectioning since illumination is confined to a thin planar region perpendicular to the detection axis and represent a suitable tool to confine the photoactivation process along the optical axis. In particular, single plane illumination microscopy (SPIM), has been proved to be a useful tool for biological investigations of thick living samples [2].Here we demonstrate three-dimensional super-resolution live cell imaging through thick biological specimen (>50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM).The confined excitation provided by SPIM and the improved signal-to-noise ratio allows for nanometric localization of single molecules in thick scattering samples. A PALM approach [3] and elliptical stretching of the point spread function allow to perform 3D super-resolution imaging of biological live samples in depth (up to 100 μm). IML-SPIM allowed to image cellular spheroids with < 35 nm lateral precision and sub-diffraction resolution in depth [4].(1) York, A.G. et al. Nat Methods, 8, 327–333 (2011).(2) Huisken, J., et al. Science305, 1007–1009 (2004).(3) Hess, S.T., Girirajan,T.P.K. and Mason, M.D. Biophys J., 91(11), 4258–4272 (2006).(4) Cella Zanacchi et al. Nature Methods (accepted).

SOFI-based 3D superresolution sectioning with a widefield microscope

Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial. Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope. SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (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 superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40nm in 3D) over a large field of view (1212μm).

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

BackgroundOptical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.Methodology/Principal FindingsWe show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.Conclusions/SignificanceOur approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Fast, three-dimensional super-resolution imaging of live cells

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast switching probes enabled us to achieve 2D imaging at spatial resolutions of ~25 nm and temporal resolutions as fast as 0.5 sec. We also demonstrated live-cell 3D volumetric super-resolution imaging. A 3D spatial resolution of ~30 nm in the lateral directions and ~50 nm in the axial direction was obtained at time resolutions down to 1 – 2 sec with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we further demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.

Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores

We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.

Minimizing detection errors in single molecule localization microscopy

Fluorescence microscopy using single molecule imaging and localization (PALM, STORM, and similar approaches) has quickly been adopted as a convenient method for obtaining multicolor, 3D superresolution images of biological samples. Using an approach based on extensive Monte Carlo simulations, we examined the performance of various noise reducing filters required for the detection of candidate molecules. We determined a suitable noise reduction method and derived an optimal, nonlinear threshold which minimizes detection errors introduced by conventional algorithms. We also present a new technique for visualization of single molecule localization microscopy data based on adaptively jittered 2D histograms. We have used our new methods to image both Atto565-phalloidin labeled actin in fibroblast cells, and mCitrine-erbB3 expressed in A431 cells. The enhanced methods developed here were crucial in processing the data we obtained from these samples, as the overall signal to noise ratio was quite low.

QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ

To the Editor: Although conventional microscopes have a reso-lution limited by diffraction to about half the wavelength of light, several recent advances have led to microscopy methods that achieve roughly tenfold improvements in resolution. Among them, photoactivated light microscopy (PALM) and stochastic optical resolution microscopy (STORM) have become particularly popular, as they only require relatively simple and affordable modifications to a standard total internal reflection fluorescence (TIRF) microscope and have been extended to three-dimensional (3D) super-resolution and multicolor imaging.

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.

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

Polarization sensitive, three-dimensional, single-molecule imaging of cells with a double-helix system

Double-helix point spread function photoactivation-localization microscopy allows three-dimensional (3D) superresolution imaging of objects smaller than the optical diffraction-limit. We demonstrate polarization sensitive detection with 3D super-localization of single-molecules and unveil 3D polarization specific characteristics of single-molecules within the intracellular structure of PtK1 cells expressing photoactivatable green fluorescent protein. The system modulates orthogonal polarization components of single-molecule emissions with a single spatial light modulator and detects them separately with a single detector. Information obtained from the two polarization channels demonstrates polarization based contrast in 3D superresolution imaging. Further, we show that the 3D information from the two channels can be optimally combined to yield up to 30% improvement in localization precision relative to a single polarization channel system.

Stereo Photoactivated Localization Microscopy for Super-Resolution 3D Bioimaging

Serial localization of single photoactivated fluorescent molecules allows imaging of cells and tissues with theoretically unlimited high resolution. Recently significant progresses have been made towards the extension of this technique to three dimensions. However, to obtain axial localization of single molecular emitters, most of current techniques rely on extracting information from the out-of-focus region. Moreover, the axial resolution is generally lower than the lateral resolution.Here, we demonstrate a 3D super-resolution imaging technique by stereo photoactivated localization microscopy (Stereo PALM), in which a mirror is placed at 45 degree with respect to the microscope stage, at the sample region. The mirror creates a side-view image of the activated molecules and thereby the axial localization turns into the lateral localization in the mirrored image. In this fashion, a 3D high resolution image can be reconstructed with an equal resolution in lateral and axial directions. This technique is very simple to implement and can be readily combined with other imaging techniques. Stereo PALM imaging of micron-size beads coated with photoswitchable fluorophores is demonstrated, and its application to super-resolution 3D imaging of mitochondria and other biological samples is discussed.