An aid to understanding differential interference contrast microscopy: computer simulation

Differential interference contrast (DIC) microscopy has been widely used for many years. However, many biologists employ DIC inefficiently and frequently misinterpret the resulting images. In an effort to improve the standard of use, a method of simulating DIC microscopy on the cathode-ray tube (CRT) and x–y plotter of a computer was therefore developed. This paper describes the program and gives examples of its application, emphasizing some common errors of operation of the DIC microscope and interpretation of the image. Simulations are made for objects of negligible thickness, and in one azimuth of the microscope at a time. The method shows clearly the profiles of the phases and amplitudes of the interfering waves obtained for a variety of objects and demonstrates unequivocally the kinds of images obtained. It is possible to show very quickly how the image changes as instrumental parameters, such as phase-bias, amplitude-ratio, extinction factor, and beam-splitter shear are altered. Simulation was found useful as a tool to learn DICM and as an aid to the interpretation of complicated images obtained with real objects. Examples of common errors in adjusting the microscope and interpreting images are given in this paper. The simulation program was developed for the computer of the Denver Universal Microspectroradiometer (David & Galbraith, 1975; Galbraith et al., 1975), but it can be used on a number of compatible computing systems. The program construction is explained to enable comparable programs to be written for different computers.     

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.     

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated

Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.     

Application of differential interference contrast with inverted microscopes to the in vitro perfused nephron

The study of in vitro perfused individual nephron segments requires a microscope which provides: (1) easy access to the specimen for measurement of cellular solute flux and voltage; (2) an image with high resolution and contrast; (3) optical sectioning of the object at different levels; and (4) rapid recording of the morphological phenomena. This paper describes an example of commercially available apparatus meeting the above requirements, and illustrates its efficiency. The microscope is of the inverted type (Zeiss IM 35) equipped with differential-interference-contrast (DIC) with a long working distance, and an automatically controlled camera system. The microscopic image exhibits cellular and intercellular details in the unstained transporting mammalian nephron segments despite their tubular structure and great thickness and makes obvious function-structure correlations (e.g. cell volume changes); luminal and contraluminal cell borders are well resolved for controlled microelectrode impalement.     

The application of interference contrast microscopy in biology (author's transl)]

Like phase contrast, Nomarski interference contrast microscopy can be used to examine unstained specimens in biology and medicine. The properties of both contrast enhancement techniques are illustrated by various examples. The phase contrast method is especially suited for thin specimens with small differences in refractive index, whereas the interference contrast method supplies good results even of thick specimens. Interference contrast is a valuable supplement to the phase contrast method, and expands the application of microscopy in biology     

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.     

Recording of single-particle hysteresis loops with differential phase contrast microscopy

The magnetic behaviour of laterally patterned structures on a micrometer or nanometer scale in external magnetic fields can be described by their hysteresis loops. However, it is very difficult to record the hysteresis loop of a single (sub-) micron-sized particle. Furthermore, extensive calculations are necessary in order to interpret the shape of the loop and to conclude the micromagnetic domain configuration in the sample from the hysteresis loop only. We have developed a technique which yields both the hysteresis loop and the magnetic domain structure simultaneously. This method uses differential phase contrast microscopy and a microscope with remote control capability for the automatic recording of a single-particle hysteresis loop.     

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.     

Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics

In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long‐established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain‐free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.     

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.     

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.     

Correlation between digital differential voltage contrast and infrared microscopy analysis of latch-up in CMOS

Results of latch-up analysis obtained by scanning electron microscopy voltage contrast and infrared (IR) microscopy are correlated and discussed. Voltage contrast detects surface potential changes due to latch-up firing, while IR microscopy reveals recombination radiation emitted by silicon (Si) regions where current density is maximum. Voltage contrast analysis may be difficult in very large-scale integrated (VLSI) circuits because well and substrate regions can be masked by overlying conductors. On the contrary, thanks to the transparency of Si to IR radiation, IR microscopy can detect latch-up by observing the reverse of the device and is not limited by a high metal density.     

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.     

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 μm within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of ≤10–15 μm into the sample, further compounding the ability to image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology.     

Normarski differential interference microscopy study of the ferroelastic domains in NaCN,RbCN and KCN

Photomicrographs of the ferroelastic domains present in the low temperature, intermediate phase of NaCN, RbCN and KCN are presented with their linear dimensions and angular orientations. The Normarski optical technique used to display the domain surfaces in colour and used to determine the small out-of-plane angular orientations is discussed in detail. Figures and the construction details are presented of the cold stage that allowed microscopic examination of the crystal surfaces by either reflected or transmitted light down to liquid nitrogen temperatures.     

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.     

The direct determination of magnetic domain wall profiles by differential phase contrast electron microscopy.

A new technique for the quantitative investigation of magnetic structures in ferromagnetic thin films is proposed. Unlike previous techniques the detected signal is simply related to the magnetic induction in the film, and as such the direct determination of domain wall profiles is possible. The technique utilizes a differential phase contrast mode of scanning transmission electron microscopy in which the normal bright field detector is replaced by a split-detector lying symmetrically about the optic axis of the system. The difference signal from the two halves of the detector provides the required magnetic information. Analysis of the image formation mechanism shows that, using a commercially available scanning transmission electron microscope equipped with a field emission gun, wall profiles should be obtainable directly from most structures of interest in Lorentz microscopy. Furthermore, signal-to-noise considerations indicate that these results can be obtained in acceptably short recording times. Finally, experimental results using both polycrystalline and single crystal specimens are presented, which confirm the theoretical predictions.     

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.