Resolution and super‐resolution

Many papers have claimed the attainment of super‐resolution, i.e. resolution beyond that achieved classically, by measurement of the profile of a feature in the image. We argue that measurement of the contrast of the image of a dark bar on a bright background does not give a measure of resolution, but of detection sensitivity. The width of a bar that gives an intensity at the center of the bar of 0.735 that in the bright region (the same ratio as in the Rayleigh resolution criterion) is for the coherent case with central illumination. This figure, which compares with for the Abbe resolution limit with central illumination, holds for the classical case, and so is no indication of super‐resolution. Theoretical images for two points, two lines, arrays of lines, arrays of bars, and grating objects are compared. These results can be used a reference for experimental results, to determine if super‐resolution has indeed been attained. The history of the development of the theory of microscope resolution is outlined.     

Reduction of coherent artefacts in super‐resolution fluorescence localisation microscopy

Super‐resolution localisation microscopy techniques depend on uniform illumination across the field of view, otherwise the resolution is degraded, resulting in imaging artefacts such as fringes. Lasers are currently the light source of choice for switching fluorophores in PALM/STORM methods due to their high power and narrow bandwidth. However, the high coherence of these sources often creates interference phenomena in the microscopes, with associated fringes/speckle artefacts in the images. We quantitatively demonstrate the use of a polymer membrane speckle scrambler to reduce the effect of the coherence phenomena. The effects of speckle in the illumination plane, at the camera and after software localisation of the fluorophores, were characterised. Speckle phenomena degrade the resolution of the microscope at large length scales in reconstructed images, effects that were suppressed by the speckle scrambler, but the small length scale resolution is unchanged at ～30 nm.     

Strain mapping accuracy improvement using super‐resolution techniques

Super‐resolution (SR) software‐based techniques aim at generating a final image by combining several noisy frames with lower resolution from the same scene. A comparative study on high‐resolution high‐angle annular dark field images of InAs/GaAs QDs has been carried out in order to evaluate the performance of the SR technique. The obtained SR images present enhanced resolution and higher signal‐to‐noise (SNR) ratio and sharpness regarding the experimental images. In addition, SR is also applied in the field of strain analysis using digital image processing applications such as geometrical phase analysis and peak pairs analysis. The precision of the strain mappings can be improved when SR methodologies are applied to experimental images.     

Two‐dimensional structured illumination microscopy

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern‐shift and ‐rotation. Moreover, our two‐dimensional approach also yields a better signal‐to‐noise ratio in the evaluated image.     

Advances in three‐dimensional super‐resolution nanoscopy

Three‐dimensional optical super‐resolution imaging is capable of providing 3D visualization of cellular structures in nanoscale detail. The past decade has witnessed the blossoming of 3D super‐resolution imaging technologies. In this review, we comprehensively discuss and compare the imaging depth, resolution enhancement, and imaging speed of the existing 3D super‐resolution imaging techniques.     

Simultaneous multiplane imaging‐based localization encoded (SMILE) microscopy for super‐resolution volume imaging

We propose a widefield‐based rapid super‐resolution volume imaging technique. This technique requires encoding single molecules to their respective planes and subsequent identification of the locus of individual molecule (both in the focal plane and off‐focal planes). Experimentally, this is achieved by precise calibration of system PSF size and its natural spread in the off‐focal planes using sub‐diffraction fluorescent beads. The specimen plane touching the coverslip is chosen as the focal plane whereas planes far from coverslip (situated at large penetration depths) represent off‐focal planes. The identification and sorting of single molecules are carried out by setting multiple cut‐offs to the respective PSFs and a 3D super‐resolved volume image is reconstructed. SMILE microscopy technique eliminates the need for multiple z‐plane scanning, minimizes radiation‐dose and enables rapid super‐resolution volume imaging.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Nano‐level position resolution for particle tracking in digital in‐line holographic microscopy

Three‐dimensional particle tracking in biological systems is a quickly growing field, many techniques have been developed providing tracking characters. Digital in‐line holographic microscopy is a valuable technique for particle tracking. However, the speckle noise, out‐of‐focus signals and twin image influenced the particle tracking. Here an adaptive noise reduction method based on bidimensional ensemble empirical mode decomposition is introduced into digital in‐line holographic microscopy. It can eliminate the speckle noise and background of the hologram adaptively. Combined with the three‐dimensional deconvolution approach in the reconstruction, the particle feature would be identified effectively. Tracking the fixed beads on the cover‐glass with piezoelectric stage through multiple holographic images demonstrate the tracking resolution, which approaches 2 nm in axial direction and 1 nm in transverse direction. This would facilitate the development and use in the biological area such as living cells and single‐molecule approaches.     

MRT letter: A unified accelerated maximum likelihood technique for widefield,confocal, and super‐resolution 4Pi microscopy

We propose an algorithmic technique for accelerating maximum likelihood (ML) algorithm for image reconstruction in fluorescence microscopy. This is made possible by integrating Biggs–Andrews (BA) method with ML approach. The results on widefield, confocal, and super‐resolution 4Pi microscopy reveal substantial improvement in the speed of 3D image reconstruction (the number of iterations has reduced by approximately one‐half). Moreover, the quality of reconstruction obtained using accelerated ML closely resembles with nonaccelerated ML method. The proposed technique is a step closer to realize real‐time reconstruction in 3D fluorescence microscopy. Microsc. Res. Tech. 78:331–335, 2015. © 2015 Wiley Periodicals, Inc.     

吹扫捕集-GC/MS/SIM法测定水体中挥发性有机污染物

建立了吹扫捕集-GC/MS/SIM法测定水体中挥发性有机污染物的方法.在进行质谱分析时,选择了离子跳变扫描方式（SIM）.方法的检出限达到了10-12水平.     

A simple,straightforward correlative live‐cell‐imaging‐structured‐illumination‐microscopy approach for studying organelle dynamics

Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two‐fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high‐speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high‐speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells. Microsc. Res. Tech. 78:777–783, 2015. © 2015 Wiley Periodicals, Inc.     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

Sub‐100‐nanometre resolution in total internal reflection fluorescence microscopy

Combining total internal reflection fluorescence microscopy with structured illumination allows optical wide‐field imaging with sub‐100‐nanometre resolution. We present a novel objective‐launch set‐up for standing wave illumination that takes advantage of a tunable transmission diffraction grating and transparent phase shifters actuated by electro‐active polymers to control the excitation pattern in three dimensions. Image acquisition is completed in less than 1 s. To reconstruct the extended image spectrum, we apply a new apodization function that results in a lateral resolution of 89 nm for green emission wavelength.     

A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities

Multi‐emitter fitting algorithms have been developed to improve the temporal resolution of single‐molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm^–2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high‐density super‐resolution microscopy), at densities up to 20 molecules μm^–2, and better than DAOSTORM at higher molecular densities. The application of WTM to a high‐density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high‐density reconstruction. WTM algorithm is a computationally fast, multi‐emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

High-resolution confocal microscopy using synchrotron radiation

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF₂ optics allows for confocal imaging over the wavelength range 200–700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofuro[3,2-c][1]benzopyran-6-one ( Chem. Abstr. Reg. No . 479-13-0). The trivial name will be used throughout this paper.)     

Automatic particle detection in microscopy using temporal correlations

One of the fundamental problems in the analysis of single particle tracking data is the detection of individual particle positions from microscopy images. Distinguishing true particles from noise with a minimum of false positives and false negatives is an important step that will have substantial impact on all further analysis of the data. A common approach is to obtain a plausible set of particles from a larger set of candidate particles by filtering using manually selected threshold values for intensity, size, shape, and other parameters describing a particle. This introduces subjectivity into the analysis and hinders reproducibility. In this paper, we introduce a method for automatic selection of these threshold values based on maximizing temporal correlations in particle count time series. We use Markov Chain Monte Carlo to find the threshold values corresponding to the maximum correlation, and we study several experimental data sets to assess the performance of the method in practice by comparing manually selected threshold values from several independent experts with automatically selected threshold values. We conclude that the method produces useful results, reducing subjectivity and the need for manual intervention, a great benefit being its easy integratability into many already existing particle detection algorithms. Microsc. Res. Tech., 76:997–1006, 2013. © 2013 Wiley Periodicals, Inc.     

Stable and super‐low friction of amorphous carbon nitride coatings in nitrogen gas by using two‐step ball‐on‐disk friction test

Effect of running‐in process on friction behaviour of carbon nitride (CNx) coating in N₂ gas stream was investigated with a newly introduced two‐step ball‐on‐disk friction test, where the rubbed Si₃N₄ ball in the pre‐sliding (step 1) was replaced by a new CNx‐coated Si₃N₄ ball in the subsequent sliding stage under N₂ gas (step 2). The two‐step friction test is clarified to be a simple but effective technique for obtaining contact material combination of self‐mated CNx coatings and for achieving stable and low frictions of CNx coatings. Friction coefficients of CNx/CNx in N₂ gas stream decrease greatly from 0.07 without pre‐sliding to less than 0.025 in two‐step friction tests. The minimum friction coefficient of 0.004 was obtained by introducing 500 cycles of pre‐sliding in ambient air. These stable and low frictions are attributed to the generation of self‐mated CNx coatings and the formation of a lubricious layer on the disk surface. Copyright © 2014 John Wiley & Sons, Ltd.