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.     

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.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

Phase‐shifting differential interference contrast microscope with Savart shear prism and rotatable analyser

A novel differential interference contrast microscope (DICM) is proposed in this research. It is constituted by inserting a Savart shear prism between the objective and sample of a polarising microscope having a rotatable analyser as the phase‐shifter, and it is with the ability to enhance image contrast using the principle of shearing interferometry. This letter is to introduce the configuration, interpret the interference patterns and present the experimental setup of the DICM. In addition, this letter is to display the experimental results from the uses of the setup; the results demonstrate the validity and ability of the DICM.     

微分相衬干涉显微镜定量测量表面形貌

改进了用于定量测量样品表面形貌的微分相衬干涉显微镜系统,对系统中由Nomarski棱镜引起的相位差β的消零工作提出了一种新的方法,实验证明是有效可靠的,并且对样品表面三维形貌重构、截面轮廓比对及系统的测量精度进行了实验研究.     

Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.     

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.     

A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast

The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.     

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.     

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.     

Measurement-based evaluation of optical pathlength distributions reconstructed from simulated differential interference contrast images

Recently a method was presented for reconstructing optical pathlength distributions (OPDs) from images of weak phase objects obtained by a conventional differential interference contrast (DIC) microscope. A potential application of this technique is the determination of the mass of biological objects: by integrating the optical pathlength over the projected surface of the image of an object, a measure of the dry mass, i.e. the total mass of all solid constituents present in the object, is obtained. To assess the possibilities of DIC microscopy for this application, simulations were performed on computer-generated DIC images of objects of various sizes, shapes and orientation angles. After reconstructing the OPDs from these images, the integrated optical pathlength of each of the test objects was determined, and compared with the expected results. The parameter settings used in the reconstruction algorithm were found to be very important in obtaining a reliable measurement. Using optimal parameter settings, errors in the integrated OPD could be limited to a few per cent for circular objects within the investigated size range. For non-circular objects, however, the orientation angle of the object relative to the lateral shift was found to influence the measured values. Ellipses with their long axes perpendicular to the shift direction had a significantly higher integrated OPD than ellipses orientated parallel to the shift. By adjusting the reconstruction parameters the effect could be limited, but complete elimination of the artefact was not possible within the parameter range investigated.     

Edge detection,three-dimensional cell boundary reconstruction and volume and surface area estimation from differential interference contrast images

Three-dimensional (3-D) cell morphology is important for the understanding of cell function and can by quantified in terms of volume and surface area. Differential interference contrast (DIC, or Nomarski) imaging can enable cell edges to be clearly visualized in unstained tissue due to the slight difference in refractive index between aqueous media and cytoplasm. DIC is affected in only one direction - the direction of the optical shear. A 1-D edge detector was used in that direction with a scale length equal to that of an in-focus edge to highlight cell boundaries. By comparison with the signal from the edge detector on an out-of-focus slice, the in-focus slices could be segmented and, after noise suppression, cell outlines obtained. A voxel paradigm was used to calculate cell volume and differential geometry was used for surface area estimation. We applied this approach to obtain 3-D dimensional information by optical sectioning of motile Amoeba proteus.     

Confocal and differential phase imaging of thin organic films

This paper discusses the imaging of thin organic films in scanning optical microscopes using both differential phase contrast and confocal modes. The Lang-muir-Blodgett technique is used to deposit thin films of controllable thickness. Step structures in these films are considered and theoretical models of the imaging are compared with experimental data. The model provides a measurement of film parameters such as thickness and permittivity. The differential phase contrast mode is also proposed as a simple method of assessment of film quality.     

Contrast enhancement and depth perception in three-dimensional representations of differential interference contrast and confocal scanning laser microscope images

A commonly used three-dimensional reconstruction method in confocal scanning laser microscopy (CSLM) involves the generation of stereo views by stacking optical sections. Under certain conditions the resulting stereo pairs exhibit inferior quality with respect to contrast and depth perception. A method based on digital image processing is described in which the individual images are enhanced prior to reconstruction, thereby increasing the number of usable optical sections by a factor of up to five. Furthermore, we introduce a new contrast enhancement transformation based upon local statistics and a grey-level probability density function that provides improved depth perception. Digital image processing methods map a discrete grey scale onto a continuous, unbounded interval. Inasmuch as a closed, discrete grey scale is required for computer display purposes, we present an appropriate mapping function derived from an entropy criterion.     

Differential contrast imaging of secondary electron signals in cryo-field-emission scanning electron microscopy

Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample.     

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.     

Quantifying cell–matrix adhesion dynamics in living cells using interference reflection microscopy

Focal adhesions and podosomes are integrin‐mediated cell‐substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image‐processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.     

Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.     

基于微分干涉相衬的相位分析法研究

通过对微分干涉相衬显微定量测量方法进行研究,提出了一种更有效的相位分析法。即在不对双光束干涉光路进行改造或处理的前提下,通过对光学成像进行处理而得到理想的结果。即把图像中的光强信号转变成相位信号,并通过维纳滤波对噪声进行了消除,最后获得表面微观形貌定量参数。