Axial Phase-Darkfield-Contrast (APDC), a new technique for variable optical contrasting in light microscopy

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.     

Improved Zernike‐type phase contrast for transmission electron microscopy

Zernike phase contrast has been recognized as a means of recording high‐resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of π/2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of ‐π/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only π /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.     

Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field

Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark‐ or bright‐field illumination. Thus, the particular visual information associated with interference and phase contrast, dark‐ and bright‐field illumination is joined in real‐time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark‐field illumination. In particular, haloing and shade‐off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators.     

High‐contrast optical microscopy of graphene sheets

In the way of making graphene an industry‐friendly material, it must be mass‐produced with high‐quality and reduced cost over large areas. Assisted by machine‐learning techniques, rapid, nondestructive and accurate determination of large graphene sheets on SiO₂/Si substrates has been made possible in recent years by the optical microscopy method. Optimization of the substrate to achieve the maximum contrast can further extend the application of the optical microscopy method for quality control of the mass‐produced graphene. Graphene/n₂/n₃three‐layer structures, where n₂ and n₃ are refractive indices, are routinely used for identifying the number of graphene layers by optical reflection microscopy. In this paper, two analytical equations are derived that can be easily used for high‐contrast optical imaging of graphene sheets without any need to resort to the cumbersome numerical methods. One of the equations is derived for choosing the best material with refractive index n₂ that when coated on a substrate with refractive index n₃, maximizes the optical contrast. The other equation is derived for finding the best thickness of the SiO₂ layer in graphene/SiO₂/Si structures, which are in common use for fabrication of graphene‐based devices. The results are implemented in a MATLAB GUI, see Supporting Information, to assist the users in using the equations.     

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

Using the optical methods described, phase specimens can be observed with a modified light microscope in enhanced clarity, purified from typical artifacts which are apparent in standard phase contrast illumination. In particular, haloing and shade‐off are absent, lateral and vertical resolution are maximized and the image quality remains constant even in problematic preparations which cannot be well examined in normal phase contrast, such as specimens beyond a critical thickness or covered by obliquely situated cover slips. The background brightness and thus the range of contrast can be continuously modulated and specimens can be illuminated in concentric‐peripheral, axial or paraxial light. Additional contrast effects can be achieved by spectral color separation. Normal glass or mirror lenses can be used; they do not need to be fitted with a phase plate or a phase ring. The methods described should be of general interest for all disciplines using phase microscopy. Microsc. Res. Tech., 76:1050–1056, 2013. © 2013 Wiley Periodicals, Inc.     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Advantages of embedment‐free section transmission electron microscopy

The usefulness of embedment‐free section transmission electron microscopy (TEM) is stressed for present and future morphological analyses, and several examples are demonstrated which are revealed in sections for the first time by this method: en‐face views of slit diaphragm of renal glomerulus and fenestrated diaphragm of capillary endothelium, transparency of neural myelin, attenuated endothelium and some basement laminae, labyrinth architecture of vacuoles within lipid droplets, and enhanced 3D effect of ultrastructures, the latter of which is the case in electron tomography. In addition, the biological significance of structured appearance (microtrabecular lattices) of the cytoplasmic matrix, which is disclosed by this method, are briefly reviewed in relation to the sol–gel transition of cytoplasmic heterogenous proteins. Since the ultrastructures of various cells and tissues in this method are confirmed to be well correspondent to those in conventional epoxy section TEM except for isotropic dimensional changes, and because there is no necessity for any special expensive equipments other than those for the conventional TEM, the embedment‐free section TEM method with these advantages, deserves much more wide application to the morphological research including electron tomography. Microsc. Res. Tech. 76:1257–1265, 2013. © 2013 Wiley Periodicals, Inc.     

Phase‐contrast hard X‐ray microscopy using synchrotron radiation for the properties of skeletal muscle in mouse hind limbs

Radiation beam interface contrast X‐ray microscopy provides resolution of a few dozen nanometers from fixed whole muscle biopsies, allowing better reconstruction of the microstructure of the muscle than is currently possible with classic histological techniques. Fixed soleus muscle biopsies have been evaluated from the walk‐in mouse model using phase‐contrast X‐ray microscopy, and results presented that corroborate the accuracy of the method used, and its potential for application in physiotherapy and occupational therapy studies. We believe that this method will enhance existing morphometric methods of analysis, leading to accurate reconstruction of other thick specimens that would otherwise require thin sectioning and reconstruction through deconvolution algorithms.     

Image scanning microscopy: an overview

For almost a century, the resolution of optical microscopy was thought to be limited by Abbé’s law describing the diffraction limit of light. At the turn of the millennium, aided by new technologies and fluorophores, the field of optical microscopy finally surpassed the diffraction barrier: a milestone achievement that has been recognized by the 2014 Nobel Prize in Chemistry. Many super‐resolution methods rely on the unique photophysical properties of the fluorophores to improve resolution, posing significant limitations on biological imaging, such as multicoloured staining, live‐cell imaging and imaging thick specimens. Structured Illumination Microscopy (SIM) is one branch of super‐resolution microscopy that requires no such special properties of the applied fluorophores, making it more versatile than other techniques. Since its introduction in biological imaging, SIM has proven to be a popular tool in the biologist's arsenal for following biological interaction and probing structures of nanometre scale. SIM continues to see much advancement in design and implementation, including the development of Image Scanning Microscopy (ISM), which uses patterned excitation via either predefined arrays or raster‐scanned single point‐spread functions (PSF). This review aims to give a brief overview of the SIM and ISM processes and subsequent developments in the image reconstruction process. Drawing from this, and incorporating more recent achievements in light shaping (i.e. pattern scanning and super‐resolution beam shaping), this study also intends to suggest potential future directions for this ever‐expanding field.     

Pipeline for illumination correction of images for high‐throughput microscopy

The presence of systematic noise in images in high‐throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non‐homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high‐content screen readouts due to software‐based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real‐world high‐throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z′‐factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high‐content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post‐hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open‐source image analysis pipelines publicly available. This software‐based solution has the potential to improve outcomes for a wide‐variety of image‐based HTS experiments.     

A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three‐dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.     

Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.     

An anomalous contrast in scanning electron microscopy of insulators: the pseudo-mirror effect

In a scanning electron microscope, electron-beam irradiation of insulators may induce a strong electric field due to the trapping of charges within the specimen interaction volume. On one hand, this field modifies the trajectories of the beam of electrons subsequently entering the specimen, resulting in reduced penetration depth into the bulk specimen. On the other hand, it leads to the acceleration in the vacuum of the emitted secondary electrons (SE) and also to a strong distortion of their angular distribution. Among others, the consequences concern an anomalous contrast in the SE image. This contrast is due to the so-called pseudo-mirror effect. The aim of this work is first to report the observation of this anomalous contrast, then to give an explanation of this effect, and finally to discuss the factors affecting it. Practical consequences such as contrast interpretations will be highlighted.     

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.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.