Far-field Fluorescence Microscopy Research Articles

Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences

Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted by the Abbe diffraction limit. We detail system integration and demonstrate system performance using sub-resolution fluorescent beads and applied to a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1) tagged with an enhanced green fluorescent protein (EGFP) at the N-terminal.

Quantum-limited estimation of the axial separation of two incoherent point sources

Improving axial resolution is crucial for three-dimensional optical imaging systems. Here we present a scheme of axial superresolution for two incoherent point sources based on spatial mode demultiplexing. A radial mode sorter is used to losslessly decompose the optical fields into a radial mode basis set to extract the phase information associated with the axial positions of the point sources. We show theoretically and experimentally that, in the limit of a zero axial separation, our scheme allows for reaching the quantum Cram\'er-Rao lower bound and thus can be considered as one of the optimal measurement methods. Unlike other superresolution schemes, this scheme does not require neither activation of fluorophores nor sophisticated stabilization control. Moreover, it is applicable to the localization of a single point source in the axial direction. Our demonstration can be useful to a variety of applications such as far-field fluorescence microscopy.

Super-resolution microscopy approaches to nuclear nanostructure imaging.

The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Nanoscopy of bacterial cells immobilized by holographic optical tweezers.

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscope's image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.

Effect of light polariztion on pattern illumination super-resolution imaging

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.

A software tool for tomographic axial superresolution in STED microscopy.

A method for generating three-dimensional tomograms from multiple three-dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three-dimensional projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with three-to-four-fold improved apparent axial resolution. Registration of the different projections is based on the use of a mutual-information histogram similarity metric. Fusion of the projections into a single view is based on Richardson-Lucy iterative deconvolution algorithm, modified to work with multiple projections. Our tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a limited signal-to-noise ratio (SNR); the reconstruction software (SuperTomo) and its source code will be released under BSD open-source license.

Application of STED microscopy to cell biology questions.

The increasing interest in "seeing" the molecular environment in biological systems has led to the recent quest for breaking the diffraction barrier in far-field fluorescence microscopy. The first nanoscopy method successfully applied to conventional biological probes was stimulated emission depletion microscopy (STED). It is based on a physical principle that instantly delivers diffraction-unlimited images, with no need for further computational processing: the excitation laser beam is overlaid with a doughnut-shaped depleting beam that switches off previously excited fluorophores, thereby resulting in what is effectively a smaller imaging volume. In this chapter we give an overview of several applications of STED microscopy to biological questions. We explain technical aspects of sample preparation and image acquisition that will help in obtaining good diffraction-unlimited pictures. We also present embedding techniques adapted for ultrathin sectioning, which allow optimal 3D resolutions in virtually all biological preparations.

4Pi microscopy of the nuclear pore complex.

4Pi microscopy is a far-field fluorescence microscopy technique, in which the wave fronts of two opposing illuminating beams are adjusted to constructively interfere in a common focus. This yields a diffraction pattern in the direction of the optical axis, which essentially consists of a main focal spot accompanied by two smaller side lobes. At optimal conditions, the main peak of this so-called point spread function has a full width at half maximum: fixed phrase of 100 nm in the direction of the optical axis, and thus is 6-7-fold smaller than that of a confocal microscope. In this chapter, we describe the basic features of 4Pi microscopy and its application to cell biology using the example of the nuclear pore complex, a large protein assembly spanning the nuclear envelope.

Sub-nanometer drift correction for super-resolution imaging.

Spatial resolution of conventional far-field fluorescence microscopy is limited by diffraction of light. Single-molecule localization microscopy (SMLM), such as (direct) stochastic optical reconstruction microscopy (dSTORM/STORM), and (fluorescence) photoactivation localization microscopy (fPALM/PALM), can break this barrier by localizing single emitters and reconstructing super-resolution image with much higher precision. Nevertheless, a SMLM measurement needs to record a large number of image frames and takes considerable recording time. In this process, sample drift becomes a critical problem and cannot be neglected. In this Letter, we present a sub-nanometer precision, low-cost sample drift correction method based on minimizing normalized root-mean-square error (NRMSE) between bright field images. Two optical configurations are suggested for recording bright field and fluorescence images simultaneously or alternately. The method was demonstrated on simulated data, and better than 0.3 nm drift correction precision was achieved. It was also applied on dSTORM imaging of F-actins of 3T3 cell, and the quality of reconstructed super-resolution image was improved observably. This method does not require special hardware, extra labelling or markers, and no precision decline due to photobleaching. It can be applied as an add-on for SMLM setups and achieves sub-nanometer precision drift correction for post-measurement or real time drift compensation.

Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy

The recently proposed fluorescence emission difference (FED) microscopy has been demonstrated to be capable of breaking the diffraction barrier, which restricts the spatial resolution of far-field fluorescence microscopy. In this paper, we report a novel high-speed FED system that can realize real-time super-resolution imaging. By replacing the conventional nanopositioning stage with a galvo mirror, the temporal resolution of FED is improved to nearly one frame per second, which is 100 times faster than that of best existing FED, while the system maintains a super-high spatial resolution of 150 nm, which is far beyond the diffraction barrier. Therefore, the high-speed FED is suitable for large-area observations while avoiding photobleaching. Detailed theoretical analysis, simulations, and experimental real-time resolution tests on 100 nm nanoparticles and biological cells are reported.

Super-resolution microscopy heads towards 3D dynamics

Abstract Resolving fine details of subcellular structures is key to understanding the organization and function of cellular networks. Recent advances in far-field fluorescence microscopy provide the necessary tools to analyze these structures with resolutions well below the classical diffraction limit in all three dimensions. Technical improvements go hand-in-hand with new versions of switchable fluorophores that allow nonlinear optical effects to be more efficiently used to push the resolution limit down further. High contrast combined with the wide spectrum of available colors currently endow these fluorescence-based super-resolution techniques with the power to study the complexity of subcellular organelles and the relation of their constituting components down to the molecular level and under physiological conditions. In this way, they give us a far better understanding of the assembly of macromolecular complexes and their functions within a cell than has been possible before employing conventional imaging methods. In this review, we give an overview of the technical state-of-the art of these technologies, their fundamental and technical trade-offs, and provide typical application examples in this exciting field.

3D STED Microscopy in Scattering Specimens

By breaking the classical diffraction limit, Stimulated Emission Depletion (STED) Microscopy has revolutionized far-field fluorescence microscopy. 25 nm resolution and better have been achieved in two dimensions imaging cultured cells and even neurons in the brain of living mice. 3D super-resolution has also been demonstrated utilizing two opposing objectives or phase filters with a top-hat profile (see Figure), its application to tissue has however been hampered by aberrations introduced by refractive index inhomogeneities.Here we present our latest results in 3D STED microscopy of scattering specimens enabled by the integration of adaptive optics into a custom STED microscope. We will present our current research about the physical and technical concepts of adaptive optics STED microscopy as well as the latest biological applications of adaptive optics STED microscopy.FigureSchematic of 3D STED Microscopy with aberration correction. A top-hat phase filter (a) is modified to compensate for sample-induced aberrations (b) to achieve an optimized 3D depletion profile (c).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Sub-100 nm 3D Detection Volumes by Total Internal Reflection STED Microscopy

Stimulated Emission Depletion (STED) Microscopy (1–2) has revolutionized far-field fluorescence microscopy by breaking the classical diffraction limit: 25 nm resolution and better are routinely achieved in the focal plane.Fig. 1. 20 nm fluorescent bead imaged in confocal and STED mode. The profile across the white box demonstrates <26 nm resolution.View Large Image | View Hi-Res Image | Download PowerPoint SlideWhile comparable axial resolution values have been obtained using two opposing objectives, single-objective STED microscopy can usually not realize values on this size scale.Here we present results obtained with a recently realized STED variant that combines total internal reflection excitation for ∼70 nm axial sectioning capabilities with stimulated depletion for ∼50 nm lateral super-resolution.TIRF STED microscopy represents an attractive super-resolution alternative for live cell microscopy featuring fast scanning with sub-100 nm 3D detection volumes and reduced photo-damage through TIRF excitation.

Sample drift correction in 3D fluorescence photoactivation localization microscopy

The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.

Total internal reflection STED microscopy

Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

Simultaneous near-field and far-field fluorescence microscopy of single molecules

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

Compact Multi-Color STED Microscopy

Stimulated emission depletion (STED) microscopy enables fluorescence imaging on the nanoscale with resolutions of 20 nm and beyond. As samples prepared for far-field fluorescence microscopy are in general also suitable for STED imaging, it has been applied advantageously in biosciences, in particular investigations of subcellular structures and assemblies.In order to expand the regime of applications to high-resolution colocalization studies we designed a setup which allows for STED imaging of up to three different labels (quasi-)simultaneously. This enables us to accurately investigate the interplay of at least three differently labeled cellular components [1]. We distinguish the labels by using different spectral properties of the dyes and, in addition, by separating dyes with similar spectral properties but different fluorescence lifetimes. The design intrinsically provides high spatial resolution for all markers simultaneously, since in STED, the diffraction barrier is overcome by reducing the effective volume in which fluorescence can occur spontaneously to a nanosized region at the very center of the STED beam. Our approach is less prone to drift artifacts than e.g. stochastic imaging concepts where the recording of even a small region of the object extends over the whole image acquisition period, and it is not subject to systematic errors due to the fluorophore orientations which have to be considered in stochastically based high-resolution techniques.[1] D. Neumann, J. Buckers, L. Kastrup, S. W. Hell, S. Jakobs: “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophysics, 3:4 (2010).

Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications

A growing trend in far-field super resolution fluorescence microscopy based on single molecule photoswitching involves the replacement of photoactivatable fluorophores by common organic dyes in which photoswitching reactions or blinking can be induced. This alternative strategy can greatly simplify the sample preparation and imaging scheme in some cases, and enables its application to a wider range of biological systems. This methodology has been applied successfully to unveil the nanoscale organisation of proteins, but little progress has been seen to date in DNA super-resolution imaging. Previous results have shown that blinking can be induced in the DNA-intercalating dimeric dye YOYO-1 in combination with a reducing buffer, and in turn super-resolution images of DNA can be reconstructed. However, monomeric intercalating dyes like YO-PRO-1 are more advantageous for biological applications. This paper shows that both YO-PRO-1 and YOYO-1 can be used in super-resolution imaging, and different sample preparation strategies are compared in terms of spatial resolution and homogeneity of the reconstructed super-resolution images. Moreover, ensemble and single-molecule experiments provide insight into the switching mechanism. The dyes YOYO-1 and YO-PRO-1 hold great potential for their use in nanoscale imaging of DNA topology in biology and nanoscience.