We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100×/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ∼0.5 nm and lateral resolution if ∼300 nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems.
Conventional DIC microscope shows the two-dimensional distribution of optical path length gradient encountered along the shear direction between two interfering beams. It is therefore necessary to rotate unknown objects in order to examine them at several orientations. We built new DIC beam shearing assembly, which allows the bias to be modulated and shear directions to be switched rapidly without any mechanically rotating the specimen or the prisms. The assembly consists of two standard DIC prisms with liquid crystal cell in between. Another liquid crystal cell is employed for modulating a bias. All components do not require a special design and are available on the market. We describe techniques for measuring parameters of DIC prisms and calibrating liquid crystal cells. One beam-shearing assembly is added to the illumination path and another one to the imaging path of standard microscope. Two sets of raw DIC images at the orthogonal shear directions and two or three different biases are captured and processed within a second. Then the quantitative image of optical path gradient distribution within a thin optical section is displayed on a computer screen. The obtained data are also used to compute the quantitative distribution of optical phase, which represents refractive index gradient or height distribution. It is possible to generate back the enhanced regular DIC images with any desired shear direction. New DIC microscope can be combined with other techniques, such as fluorescence and polarization microscopy.
We describe a combined orientation-independent differential interference contrast OI-DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture (NA) 1.4, thus achieving a level of resolution not previously achieved with phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes, while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.
We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their
orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path
difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow
illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined
with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry
mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to
structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly,
Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization
image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for
staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both
orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those
images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live
cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics
having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference
microscope.
We have been developing a polarized light microscope with liquid crystal universal compensator and circular polarizer (the LC-PolScope) for recording images, which are independent of the orientation of birefringent objects. Separate images show the retardance and the slow axis azimuth distributions of the in-plane birefringence of the focused region in the specimen. However the measured (apparent) retardance still depends on the angle between the crystal optic axis and the axis of the illuminating beam of light. If the illuminating beam is close to parallel to the optic axis the measured retardance value decreases dramatically and becomes zero when the two axes are parallel. The description of birefringent objects oriented in 3-dimensional space requires the introduction of two additional parameters: the principal retardance and the inclination angle. Together with the azimuth angle they completely characterize the birefringence properties of a specimen, assuming the specimen has a uniaxial optical indicatrix. We devised a new technique for measuring the three birefringence parameters without moving the specimen. For exploring the out-of-plane birefringence the new instrument which is based on the LC-PolScope technique contains an additional spatial light modulator, implemented here as a liquid crystal mask. The mask is located in the aperture plane of the condenser lens. Partial occlusion of the condenser aperture changes the direction of the central ray of the cone of light converging on the specimen. So we can obtain the retardance and azimuth images using different sets of illumination rays. For experimental verification we used a biological object called an aster. An aster consists of nearly parallel arrays of microtubules, a stiff biopolymer, radiating from a common organizing center called a centrosome. The object is spherically symmetric, and its 3 dimensional distribution of birefringence orientation can be predicted. Experimental results have shown the developed polarizing microscope can successfully be used for imaging and measuring three-dimensional orientation of birefringent objects
Image processing algorithms for measuring two-dimensional distributions of linear birefringence using a pair of variable retarders are proposed. Several algorithms using from 2 to 5 recorded frames allow to make fast or real time measurements, increase sensitivity and suppress measurement errors. Moreover, the described algorithms can be applied for proposed birefringence imaging systems with fixed retarders and/or Faraday rotators, including systems with images acquired in time sequences or as sets of parallel images.
The article presents a new design of a polarization microscope with a scanning liquid crystal aperture. The scanning device is based on the earlier reported Pol-Scope technique and includes a liquid crystal universal compensator. It is mounted in the front focal plane of the high numerical aperture condenser lens on the microsccope. By occluding different portions of the aperture, an oblique beam of variable tilt angle and azimuth is created for illuminating the specimen. Birefringence measurements are recorded for different mask configurations and results are evaluated to determine the retardance magnitude, azimuth and direction of optic axis of the specimen. We report measurements using small calcite crystals that confirm our theoretical predictions.
We report on the theoretical analysis, measurement and active rectification of depolarization caused by the differential transmission and phase shift of p- and s- polarized components of an axial beam passing through lens surfaces. Our theoretical analysis finds the polarization of output rays as a function of the input ray parameters, the shape factor and refractive index of the lenses used. For rays that are inclined to the optical axis we find optimal lens shape factors that minimize the rays' polarization aberrations. We report measurements of polarization aberrations that were detected in the back-focal plane of a modern microscope equipped with high numerical aperture lenses. Finally, we discuss strategies for polarization rectification and introduce new designs including a liquid crystal rectifier that can actively compensate a wide variety of polarization aberrations.
The paper covers an autocollimating fiber optic sensor for measuring the birefringence of bulk material based on the use of a pulsed laser and multimode optical fiber with a fiber delay line. The specimen birefringence is defined by measurement of the pulse intensities. The basic expressions that describe the operation of the sensor are presented. The suggested scheme can be used for studying ht inner stress in transparent solid bodies, birefringence of crystals, or for measuring temperature, pressure, electrical and magnetic field strength, etc.
We used a new type of polarized light microscope (Pol-Scope) equipped with a precision universal compensator to record images of small, uniaxial birefringent crystals. Pol-Scope measurements of small calcite crystals represent complete in-focus and out-of-focus retardance maps of the crystals. We relate the 3-D retardance maps to the crystal morphologies and the directions of their optic axes in three dimensional space. In addition to single crystal measurements, we also recorded 3-D retardance maps of small clusters of crystals to study the superposition of overlapping image features. We demonstrate that the retardance of overlapping image features in cluster images can be theoretically predicted by combining separately recorded single crystal images. Our experimental and theoretical analysis indicates that the superposition of retardance features is equivalent to adding incoherently the respective intensity values that were recorded with the Pol-Scope using different compensator settings. This analysis is part of a larger effort towards establishing 3-D image restoration procedures for polarized light imaging.
The paper describes the phase-shifting method for measuring 2D birefringence distributions with return-path polarimeter scheme. Eight or sixteen images in polarized light are processed for determining of specimen's retardance and azimuth distributions. The principal formulas that describe the mathematical processing are presented. The method allows us to find the image of specimen's birefringence with nonuniform distribution of fast axis azimuth and retardance. The method gives the exact solution for any retardance value. Approximation equations for determining of small retardance are presented. The measurement method is highly effective for research of vector or tensor physical fields, which are accompanied by birefringence. For example, inner stresses, electrical and magnetic fields, heat flows, birefringence liquid flows et al.
The paper covers an issue of method and device for measurement of 2D retardance and principal plane azimuth distributions. The system based on the use of a laser return-path polarimeter which consists of a rotating polarizing beam splitter and a quarter wave plate. Two or four sets from four images are used for birefringence analysis. This allows to decrease the errors caused by imperfection of a polarizing beam splitter and a quarter wave plate. The basic expressions that describe the operation of the systems are presented. The main sources of error are discussed. The measured results of the 2D birefringence distribution of a plastic and standard phase plate of retardation are shown.
The paper covers an issue of optical polarimetric method for temperature measurement based on the use of a laser autocollimation polarimeter and a sensitive birefringent plate. The sensor works in the following manner. The linear polarized probe beam of the polarimeter illuminates the sensitive element which reflects the radiation backward. The sensitive element result in a phase shift between the beam polarization components which value is proportional to the element temperature. The autocollimation polarimeter determines this phase shift and enables to calculate the temperature value. The distance between the polarimeter an the sensitive element may change from 0 to 100m. The use precisely of the polarization properties of light makes it possible to decrease substantially the effect of beam attenuation on the measurement errors in case where the radiation passes through smoked or dusty space. Small size of the sensitive crystal plate provides rather fast reaction of the sensor to temperature variation in the medium. The basic expression that describes the operation of the system are presented. It is shown that with the use of a quartz plate 1.912mm thick the device makes possible to determine values of temperature in the range of 0 to + 180 degrees C.
The principal stages in technological chain of liquid crystal displays (LCD) production are presented for case where the measurement of optical polarization properties is required. Experimental set-up is described and result of LCD's substrates research are given. Influence of rubbing of polyimide layer on phase shift is considered. Analysis of five polyimide layers grouped with various structure and dopes is carried out. We have established that optical polarization technique allows to evaluate the degree and homogeneity of rubbing, determine the pre-inclination angle of polyimide molecular chains relatively to the surface of substrate.
The paper describes the device for measuring the normal- reflection birefringence with using of the compensation method. The suggested scheme may be used for research of optical anisotropy in optical recording disks, inner stress in transparent solid body, birefringence of crystals and for the measuring of temperature, pressure, electrical and magnetic field strength.
The paper covers the description of a fiber optic sensor of linear displacement with a single multimode fiber and a movement mirror positioned near the output end of the fiber perpendicularly to its axis. The value of the signal of the considered sensor is determined by the rate of a beam reflected from mirror and introduced back into the fiber. New methods of increase in the band of measured shifts and enhancement of the sensor sensitivity are described in the paper. In particular, we propose to fill a space between the fiber face and the mirror by the light-absorbing liquid and to use polarization splitting of input and output beams. Mathematical descriptions of the sensitive element and the polarization beam splitting scheme are considered in the paper. We used a method of geometrical optics in Hamiltonian formulation for mathematical sensor modeling. Presented experimental results confirm the theoretical conclusion.
The paper describes the devices for measuring the normal- reflection birefringence with using of the photometric, balanced, compensation and interferometric methods. The suggested scheme may be used for research of optical anisotropy in optical recording disks, inner stress in transparent solid body, birefringence of crystals and for the measuring of temperature, pressure, electrical and magnetic field strength.
In many systems with use of laser the incident beam when directed to an object is coincident in space with the reflected beam. This take place, for example, in a laser optical disc player. Separation of the reflected beam from the incident beam can be done by dividing them under of polarization with using of a polarization dividing unit. An example of the unit is shown in figure:
The paper describes the device for measuring the normal- reflection birefringence with using of the compensation method. The suggested scheme may be used for research of optical anisotropy in optical recording disks, inner stress in transparent solid body, birefringence of crystals and for the measuring of temperature, pressure, electrical and magnetic field strength.
The paper describes the means of raising the sensitivity of fiber optical sensors involving single multimode and one-mode light guides. First, this is the use of a polarizational beam splitter which is installed in front of the inlet face of a multimode light guide and which fully transmits the polarized radiation from the source to the light guide and reflects half the power of the depolarized output beam to the photodetector. The scheme allows one to raise the output signal level by more than a factor of two as compared with the traditional scheme. Second, in an ordinary one-mode light guide, it is recommended to place a compensator between a polarizational beam splitter and the light guide face. And third, in the case of the use of a single polarized one-mode light guide the compensator should be replaced by a nonmutual magneto-optical element. It is shown that in the circuits with one-mode light guides one can virtually fully eliminate separation power losses of the output beam.
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