Frits Zernike, Professor of Mathematical and Technical Physics, and Theoretical Mechanics at the University of Groningen, The Netherlands, was a most versatile and gifted scientist. His abilities had an extraordinary breadth: he was a personification of the rare combination of a gifted experimental physicist, a skilled mathematician and an extremely good technician. He worked on many different subjects in the field of statistical mechanics or mathematical statistics, theoretical physics, and physical optics. His correlation functions, van Cittert-Zernike's theorem, Zernike's polynomials, achievements in the theoiy of coherence, and contributions to the correction of lens aberrations are still well known. However, the most fameous his achievement is the phase contrast microscope awarded Nobel's Prize in 1953.

Differential interference contrast (DIC) was introduced to microscopy by George Nomarski over four decades ago and since the late 1960s has become increasingly popular. Today, Nomarski's DIC system is part of the basic equipment of advanced microscopes manufactured by all the leading firms in Europe, Japan and USA. Its principle, specific properties (3-D appearing images, optical sectioning, directional sensitivity), and applications are discussed. Modulation contrast microscopy is also mentioned, and a short biography of G. Nomarski is also given.

Problems of the phase object visualization for various spatial frequency filtration techniques and phase modulation depths are considered. The imaging by the distorter presented in the paper is sufficient to formulate conclusions concerning the visualization problems of the phase objects. Using the vectorial interpretation of the phase object imaging we have shown that the satisfactory choice of the visualization method depends on the phase modulation depth, therefore, for an unknown phase object it is necessary to dispose a few different methods simultaneously.

Properties and new possible applications of the slit phase contrast (SPhC) technique are presented. They have been tested by the use of an advanced computer simulation program, performing a digital 2D spatial filtering process. A composition of phase objects (e.g., circles, rectangles, gratings, fibers) is transformed into its Fourier form, then multiplied by the transmittance of the phase strip and finally retransformed. Several soot filters have been prepared and applied to a modified, commercial microscope according to the simulation results. The experiments have involved mainly biological specimens. Special attention has been given to measurements of the refractive index profile of the optical fibers. A detailed theoretical background has been presented and results of additional computer simulation based on the operation of convolution have been discussed.

The vignetting effects in an optical system are considered by means of wave tools and, specifically, by using the concept of harmonic spatial analysis. The considerations are limited to the periodical objects illuminated by the coherent source of light. The results of numerical computations for different objects are discussed and shown on plots.

The use of holograms without reference beam as spatial and amplitude filters in devices for coherent optical data processing and in systems for studying biological processes is discussed. The possibility of a hologram without reference beam to reconstruct the amplitude and phase of the object field with no object present is demonstrated.

Image formation for a range of alternative methods for obtaining phase contrast in conventional and confocal microscopy is considered. In particular, the phase gradient transfer function is presented.

Nomarski's DIC system for transmitted light consists of two birefringent prisms, one of which is located behind the microscope objective, while the other is placed below the condenser. The latter is used as a compensator; it is similar to the former and oriented in such a manner as to cancel out the optical path differences between lightwave components slightly sheared and brought into mutual interference. Due to the sub- condenser birefringent prism, this system suffers from some limitations when anisotropic objects are examined. These limitations can be removed if a subtractive combination of two birefringent prisms is placed behind the microscope objective and the sub-condenser prism is replaced by a slit diaphragm. If the required wavefront shear is equal to or less than the resolving power of the objective (this is the case of submicron objects), then the slit of the sub-condenser diaphragm is very large or even equal to the diameter of the condenser aperture. Moreover, a variable wavefront can be achieved easily by a suitable combination of two birefringent prisms installed behind the microscope objective.

A factor-of-ten over classical diffraction limit enhancement was achieved with a computer-aided phase microscope 'Airyscan'. It also permits dynamic processes registration at any selected point of the image. The theoretical backgrounds of the new approach to the problem of super-resolution are discussed. 'Airyscan' may be useful in a wide range of scientific and industrial applications. Special attention is paid to biophysics and medicine.

A real-time holographic microscope for phase imaging is described. The image-formation process is based on the aberration-correcting capability of phase-conjugate illumination. After it has passed through a phase object, the light from a laser beam is recorded as a reflection hologram within a crystal of barium titanate by the self-pumping process. Such a reflection hologram, when illuminated, returns the phase conjugate of the incident distorted optical field. The object is then displaced slightly, and the phase conjugate of the field produced by the undisplaced object now passes through the displaced object. This produces in the object plane an intensity pattern that is an image of gradients in phase retardation. A microscope (objective and ocular) creates a magnified image of the pattern. A digital processor grabs video frames, subtracting from the gradient image the initial optical field acquired before the shift. The subtractive processing results in a final image free of coherent noise and artifacts. We describe the microscope and its operation and show representative images.

In the present paper, an optical arrangement for precision measurement of the amount of translation and rotation of different objects is proposed.

Methods for increasing measurement sensitivity by using waves reconstructed from a hologram in higher diffraction orders and from a re-recording of a hologram are considered. A review is given of methods for increasing sensitivity during the multiple passage of a beam through a volume being investigated or a hologram. A review of the use of holographic interferometry with increasing sensitivity for transparent media diagnostics is given.

The interferometer fringe contrast ratio C is the most important figure of merit for interferometer performance. Low values of contrast drastically limit the use of double beam interferometers. The large values of contrast present in common path interferometers indicate that this deterioration is due to beam separation produced by nonideal beam splitters. Minimizing the negative effects of such beam splitters by the use of Double Beam Circular Interferometers (DBCI) and the use of plane- parallel beam splitters as well as collimating optics is suggested. Several new types of interferometers are described with improved contrast and stability.

A new class of interferometers, Double Beam Circular Interferometers (DBCI) is introduced. Since these interferometers are in many respects superior to commonly used Mach-Zehnder and Michelson interferometers, DBCIs can be used as their possible replacement.

Nomarski's differential interference contrast (DIC) microscopy is discussed in comparison to Zernike's phase contrast (PhC) microscopy. The possibilities and limits of both are demonstrated by various applications. The high contrast and the use of the full numerical aperture of the DIC optics makes it possible to obtain a series of 'optical sections' through rather thick living specimens (e.g. head of water flea, salivary gland of Drosophila, Xenopus nucleolus, sea urchen egg, mouse embryo). PhC and DIC optics are today available for high resolution light microscopy until N.A. 1.4 Oil as well as for long working distance (LWD) optics, mainly combined with inverted biological microscopes.

Phase contrast microscopy plays a major role in establishing knowledge on in vitro cell behavior in cultured cells. A major focus of these studies has been on the identification of differences between normal and neoplastic cells. Spontaneously metastasizing rat sarcoma was studied to determine the relationship between the rate of appearance of spontaneous metastases and the patterns of in vitro cell behavior. It was found that a slightly acid extracellular milieu (pH 6.5), simulating the natural situation within the tumor, brought about stimulation of directional migration in highly malignant phenotypes. That type of behavior may account for malignant spread of a neoplastic cell from the primary site of the tumor. A set of the in vitro observable properties of the malignant cell phenotype is detailed here and called activated morphotype. A working theory about the origin of the activated morphotype of the neoplastic cell (AMNC) is presented.

Some multicomponent liquid crystal systems show unusual behavior in the phase transition from the isotropic melt to the mesomorphic state. In these systems, the nucleation is performed in the form of filaments, called nematoids, freely suspended in the isotropic melt. The observed aspect ratio (diameter : length) of the nematoids achieves 1:3000. Matured nematoids are rather unstable and undergo rapid shrinkage to droplets. The main features common to essentially all nematoids in the multicomponent (nematic, smectic B, and non-mesogenic chiral dopant) systems are: they can split into two separate threads surrounding homeotropically oriented smectic 'lake' or can undergo the segmentation or double-spiralling before their transformation to droplets. To confirm the supposition that these processes develop the bifilar organization of the nematoids, the microinterferometric analysis was performed by using the Pluta birefracting microinterferometry. This analysis supports the conception that the nematoids represent multiphase systems and are composed of two parallel planar nematic filaments connected with homeotropically oriented smectic wall (gluon). To adjust the mutually perpendicular orientation of molecules, the peripheral filaments and gluon are assumed to be connected with chiral interface. Presumably, the creation of the nematoids is the result of the phase separation in a system with anisotropic surface tensions. It is possible that instability with respect to the segmentation of the nematoid filaments is caused by the anisotropy of the surface tensions at the interfaces within a nematoid whereas the double-spiraling can be the result of a phase transition of the gluon connecting both the filaments or within the chiral interfaces.

The phase stepping technique for data acquisition from transverse interferograms is proposed in the refractive index profiling of optical fibers. The advantages for automatic data extraction are pointed out. Experimental results are presented.

A new method for determining the refractive index profile of GRIN waveguides has been proposed. It uses wedge samples whose preparation is a less time- consuming operation when compared to the known techniques, which use samples with plane parallel faces.

Application possibilities of phase contrast method in investigations of both isotropic and anisotropic optical fibers are discussed. In particular, this method enables the core diameter and other geometrical parameters of GRIN fibers to be determined more accurately when it is combined with scanning microdensitometry. Refractive index profile of some fibers, especially that of monomode fibers, can be determined by using a negative phase contrast device if the difference (Delta) n between refractive indices of the fiber core and cladding is very small (at any rate smaller than 0.03). The result obtained is comparable to that offered by differential microinterferometry. In general, highly sensitive negative phase contrast microscopy, such as that based on the KFA device with soot phase rings (commercially available from the Polish Optical Works, Warsaw), permits us to observe extremely small optical inhomogeneities, which cannot be revealed with the help of conventional microinterferometric techniques.

An effective method of visualization and/or localization of the wave guiding structures is presented. It consists in the two-beam interference. The observation set-up can be related to the Fizeau interferometer but no reference mirror is needed for the observation. Fringe pattern arises from interference of the beams reflected from polished slab-to-air boundaries. The interference order is relatively high (3 10³) thus the well-contrasted fringe pattern arises only with highly monochromatic illumination.

The propagation constants of guided waves were measured by dark mode spectroscopy technique. Series of dark lines known as the m-lines observed on the screen were used to determine propagation constants of guided waves. Theory and experiment on dark spectroscopy in planar waveguides is discussed. On the basis of 4 X 4 matrix algebra is introduced the investigation of plane-wave propagation in absorbing film waveguides.

The role played by optical interference in determining the complex degree of coherence of an electromagnetic field propagating in a fiber waveguide is examined under different conditions of the source spatial and temporal coherence. In particular, the optical interference is examined by evaluating the modulus and the phase of the complex degree of both spatial and temporal coherence at the exit face of two-mode step-index fiber waveguide excited by spatially coherent and quasi-monochromatic source. Moreover, by introducing the averaging operation over a statistical ensemble of fiber waveguides, the statistical quantities of the optical field at the output of the fiber waveguide can be determined.

The phase stepping technique with dia illumination is proposed to evaluate surface relief of dielectric layers. An easy implementation is described in a commercially available polarized light interference microscope and experimental results are presented.

The phase stepping DIC technique is proposed for the surface profiling of highly polished optical substrata. An easy implementation is described in a commercially available polarized light interference microscope for reflected light. Experimental results are presented.

Polarization structure of separate speckles of laser radiation scattered by a rough surface is investigated in different recording zones.

Stray light (glare) in Jamin-Lebedeff interference microscopes, partly due to bubbles in the calcite plates of the objectives, can be reduced by limiting the area of field illuminated. Glare degrades visual image contrast and can cause measuring errors in systems which depend on the measurement or comparison of light intensities, but has no significant effect on measurements in a microinterfereometer which uses phase-modulated light. The apparent OPD of a very thin object mounted in a given medium can be significantly affected by phase changes due to reflections at the interfaces with the preceding and successive media. The error can be reduced or eliminated if the refractive indices of the specimen and the supporting slide are similar, and/or if the reference medium is air and no coverglass is used. In principle, the geometrical thickness and refractive index of a plane object such as a plastic microtome section in a given reference medium can be obtained from microinterferometric measurements using different obliquities of transmitted light. Currently available microinterferometers are not, however, precise and accurate enough for the method to be practical with objects only about a wavelength thick.

An optical correlator as a part of the potential, hybrid system for exact positioning of phase objects in robotics (microrobotics) is proposed. Particularly, a coherent correlator system with image plane in which the required optical correlation is formed in output plane is used. Positive photographic transparency of the object, recorded by phase-contrast method serves as matched filter. The values of light intensity measured in central point of the output plane are related to the actual positions of the input object and allow for searching after required location of the object. The proposed correlator system has been tested experimentally. The obtained results are consistent with the theory.

This paper presents a new and simple technique of phase retardation measurement. It is based on computer aided analysis of diffraction pattern generated by phase object. Application of this method to known intensity distribution in Fourier spectrum allows to find phase distribution in an object. The method is especially aligned for techniques where an unknown phase modulation is gained by exposure of a photo-sensitive media. The described method can help to predict the desired phase shift from exposure and is especially aligned to the problem of Holographic Optical Elements (HOE) manufacturing.