Michael Shribak

Techniques for fast and sensitive measurements of two-dimensional birefringence distributions

We propose image processing algorithms for measuring two-dimensional distributions of linear birefringence using a pair of variable retarders. Several algorithms that use between two and five recorded frames allow us to optimize measurements for speed, sensitivity, and accuracy. We show images of asters, which consist of radial arrays of microtubule polymers recorded with a polarized light microscope equipped with a universal compensator. Our experimental results confirm our theoretical expectations. The lowest noise level of 0.036 nm was obtained when we used the five-frame technique and four-frame algorithm without extinction setting. The two-frame technique allows us to increase the speed of measurement with acceptable image quality.

Entropy-driven formation of a chiral liquid-crystalline phase of helical filaments

We study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.

Orientation-independent differential interference contrast microscopy

We describe a new technique for differential interference contrast (DIC) microscopy, which digitally generates phase gradient images independently of gradient orientation. To prove the principle we investigated specimens recorded at different orientations on a microscope equipped with a precision rotating stage and using regular DIC optics. The digitally generated images successfully displayed and measured phase gradients, independently of gradient orientation. One could also generate images showing distribution of optical path differences or enhanced, regular DIC images with any shear direction. Using special DIC prisms, one can switch the bias and shear directions rapidly without mechanically rotating the specimen or the prisms and orientation-independent DIC images are obtained in a fraction of a second.

Orientation-independent differential interference contrast microscopy and its combination with an orientation-independent polarization system

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.

Polarized light imaging of birefringence and diattenuation at high resolution and high sensitivity.

Polarized light microscopy provides unique opportunities for analyzing the molecular order in man-made and natural materials, including biological structures inside living cells, tissues, and whole organisms. 20 years ago, the LC-PolScope was introduced as a modern version of the traditional polarizing microscope enhanced by liquid crystal devices for the control of polarization, and by electronic imaging and digital image processing for fast and comprehensive image acquisition and analysis. The LCPolScope is commonly used for birefringence imaging, analyzing the spatial and temporal variations of the differential phase delay in ordered and transparent materials. Here we describe an alternative use of the LC-PolScope for imaging the polarization dependent transmittance of dichroic materials. We explain the minor changes needed to convert the instrument between the two imaging modes, discuss the relationship between the quantities measured with either instrument, and touch on the physical connection between refractive index, birefringence, transmittance, diattenuation, and dichroism.

Complete polarization state generator with one variable retarder and its application for fast and sensitive measuring of two-dimensional birefringence distribution.

The complete polarization state generator (PSG), which consists of one rotatable polarizer and one variable retarder with a quarter-wave plate, is introduced. The orientation angle of its output polarization ellipse equals half of the retardance of the variable retarder, and the ellipticity angle corresponds to the polarizer azimuth. The PSG is employed in the quantitative orientation-independent differential polarization microscope, which uses polarized light states with the same ellipticity and different orientation angles. Image processing algorithms using three or four frames are described.

Quantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation

We describe a quantitative orientation-independent differential interference contrast (DIC) microscope, which allows bias retardation to be modulated and shear directions to be switched rapidly without any mechanical movement. The shear direction is switched by a regular liquid-crystal cell sandwiched between two standard DIC prisms. Another liquid-crystal cell modulates the bias. Techniques for measuring parameters of DIC prisms and calibrating the bias are shown. Two sets of raw DIC images with the orthogonal shear directions are captured within 1 s. Then the quantitative image of optical path gradient distribution within a thin optical section is computed. The gradient data are used to obtain a quantitative distribution of the optical path, which represents the refractive index gradient or height distribution. Computing enhanced regular DIC images with any desired shear direction is also possible.

Return-path polarimeter for two-dimensional birefringence distribution measurement

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.

Sensitive measurements of two-dimensional birefringence distributions using near-circularly polarized beam

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.

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

Abstract. 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.