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.     

Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimens

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity.     

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.     

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.     

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.     

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.     

Automatic detection and analysis of cell motility in phase‐contrast time‐lapse images using a combination of maximally stable extremal regions and Kalman filter approaches

Phase‐contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase‐contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase‐contrast images in time‐lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time‐lapse movies, the MSER‐based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase‐contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time‐consuming large‐scale dynamical analysis of cultured cells.     

Mirror lenses in light microscopy—Theoretical considerations and practical implications

Various types of mirror lenses were developed some decades ago designed for several special tasks in microscopy. When compared with high‐end glass lenses, mirror lenses lead to an extraordinary image quality because of their supraapochromatic color fidelity and their high planarity; the working distances are significantly longer, and in most cases, the depth of field and resolution are higher than in concurrent glass lenses. In this respect, the microscopic advantages of mirror lenses seem to be comparable with the advantages of mirror telescopes in astronomy; for in observations of celestial bodies, mirror telescopes lead to better results than refractors based on glass lenses. When mirror lenses are available in light microscopy, not only common illumination modes can be carried out in transmitted or incident light, but also new and specific axial illumination techniques can be utilized, which cannot be achieved with normal glass lenses. This axial illumination, called “luminance contrast,” can be carried out in various modes, so that the resulting images can be compared with dark‐field, phase or interference‐contrast images. In all variants, especially, fine details within transparent specimens can be visible in maximized contrast and resolution, and blooming or haloing artifacts are significantly reduced or absent. These findings are based on theoretical consideration, and intensive practical tests carried out with several mirror lenses constructed in various optical designs. All in all, supramicroscopic image qualities could be expected if mirror lenses were produced based on the optimized manufacturing techniques available nowadays. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

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.     

An easily built diffuse illumination system effective at both very low and moderate magnifications, for observing in situ stained slides

Effective study of in situ stained sections often requires illumination that is difficult to achieve with commonly used research microscopes. One must be able to switch quickly and conveniently from the very lowest to moderate magnifications. At all magnifications contrast due to light scatter must be minimized, so that the weak staining that signifies low gene expression can be observed reliably. For the lowest power objectives (e.g., 1.25× or 2×) many microscopes require that the condenser be removed to illuminate the full field of view. This is not only very inconvenient when switching magnifications, but without a condenser the low numerical aperture of the illuminating light beam results in unwanted contrast due to light scatter. We have devised a simple system that diffusely illuminates the full field of view of the lowest power objective (1.25×) and has high enough numerical aperture for use with the 25× and 40× objectives. A key feature is the use of a large diameter ring light (internal diameter 5.8 cm), placed on the microscope base, to illuminate a large diameter diffuser placed just below the slide.     

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.     

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.     

Selectable light‐sheet uniformity using tuned axial scanning

Light‐sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three‐dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light‐sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade‐off between light‐sheet thickness and area over which this thickness is preserved. Recently, an increase in light‐sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z‐scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light‐sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.     

Quantitative X-ray projection microscopy: phase-contrast and multi-spectral imaging

We outline a new approach to X‐ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high‐brightness field‐emission gun (FEG)‐based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 µm and have demonstrated novel features such as: (i) phase‐contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy‐resolved imaging to simultaneously produce multiple quasi‐monochromatic images using broad‐band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude‐retrieval algorithms to enable new real‐time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object–plane information from intermediate‐field images, unlocking the potentially greater contrast and resolution of the intermediate‐field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.     

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.     

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.     

Third‐harmonic generation imaging of breast tissue biopsies

We demonstrate for the first time the imaging of unstained breast tissue biopsies using third‐harmonic generation (THG) microscopy. As a label‐free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour‐associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright‐field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.     

Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object

We demonstrate simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object. Subject to the assumptions explicitly stated in the derivation, the algorithm solves the twin‐image problem of in‐line holography and is capable of analysing data obtained using X‐ray microscopy, electron microscopy, neutron microscopy or visible‐light microscopy, especially as they relate to defocus and point projection methods. Our simple, robust, non‐iterative and computationally efficient method is applied to data obtained using an X‐ray phase contrast ultramicroscope.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation‐independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation‐independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation‐independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.