Mapping birefringence in three dimensions using polarized light field microscopy: the case of the juvenile clamshell.

We report methods to generate three-dimensional maps of birefringence, its position and orientation in juvenile shells of the Atlantic hard clamshell (Mercenaria mercenaria). For measuring the retardance and optic axis orientation of curved shell surfaces in three dimensions, we developed enhanced acquisition and processing algorithms and combined results from conventional and light field imaging approaches to reconstruct the three-dimensional shell shape and its anisotropic optical properties. Our work represents the first successful attempt to generate such maps at a spatial resolution of about 2 µm and angular steps of about 9° in terms of the inclination angles of the optic axis. The maps of clamshell birefringence provide structural insights into the early mineralization during juvenile clamshell development.

Polarized light field microscopy: an analytical method using a microlens array to simultaneously capture both conoscopic and orthoscopic views of birefringent objects.

For the comprehensive analysis of anisotropic materials, a new approach, called 'polarized light field microscopy' is introduced. It uses an LC-PolScope to which a microlens array was added at the image plane of the objective lens. The system is patterned after the 'light field microscope' that achieves both lateral and axial resolution in thick specimens in a single camera exposure. In polarized light field microscopy, the microlens array generates a hybrid image consisting of an array of small conoscopic images, each sampling a different object area. Analysis of the conoscopic images reveals the birefringence of each object area as a function of the propagation direction of transmitted light rays. The principles and utility of the instrument that we are calling 'light field LC-PolScope' are demonstrated with images of a thin, polycrystalline calcite film, revealing the azimuth and inclination angle of the optic axis for many crystals simultaneously, including crystals with diameters as small as 2 microm. Compared to traditional conoscopy and related methods, the vastly improved throughput and quantitative analysis afforded by the light field LC-PolScope make it the instrument of choice for measuring 3D birefringence parameters of complex structures.

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes.

Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.

Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones.

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.

Microtubule flux mediates poleward motion of acentric chromosome fragments during meiosis in insect spermatocytes.

We applied a combination of laser microsurgery and quantitative polarization microscopy to study kinetochore-independent forces that act on chromosome arms during meiosis in crane fly spermatocytes. When chromosome arms located within one of the half-spindles during prometa- or metaphase were cut with the laser, the acentric fragments (lacking kinetochores) that were generated moved poleward with velocities similar to those of anaphase chromosomes (approximately 0.5 microm/min). To determine the mechanism underlying this poleward motion of detached arms, we treated spermatocytes with the microtubule-stabilizing drug taxol. Spindles in taxol-treated cells were noticeably short, yet with polarized light, the distribution and densities of microtubules in domains where fragment movement occurred were not different from those in control cells. When acentric fragments were generated in taxol-treated spermatocytes, 22 of 24 fragments failed to exhibit poleward motion, and the two that did move had velocities attenuated by 80% (to approximately 0.1 microm/min). In these cells, taxol did not inhibit the disjunction of chromosomes nor prevent their poleward segregation during anaphase, but the velocity of anaphase was also decreased 80% (approximately 0.1 microm/min) relative to untreated controls. Together, these data reveal that microtubule flux exerts pole-directed forces on chromosome arms during meiosis in crane fly spermatocytes and strongly suggest that the mechanism underlying microtubule flux also is used in the anaphase motion of kinetochores in these cells.

The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes.

OBJECTIVE: To evaluate how well polar body location predicts spindle localization and to examine spindle morphology. DESIGN: Randomized, controlled animal study. SETTING: University-affiliated research laboratory. ANIMAL(S): Mature, female golden hamsters. INTERVENTION(S): After superovulation with pregnant mare serum gonadotropin and hCG, metaphase II oocytes were obtained and imaged under digital polarization microscopy. MAIN OUTCOME MEASURE(S): Identify the meiotic spindle in living, unfixed hamster oocytes and determine spindle location relative to the polar body. RESULT(S): Spindles were imaged in 30 oocytes and only in 5 of them could the polar body predict the spindle localization. In the remaining oocytes, the spindles presented a random distribution within the cytoplasm. CONCLUSION(S): These data show that the polar body location is not an accurate predictor for meiotic spindle location in mammalian oocytes.

Analysis of edge birefringence.

We present an experimental and theoretical study of the phenomenon of edge birefringence that appears near boundaries of transparent objects which are observed with high extinction and high resolution polarized light microscopy. As test objects, thin flakes of isotropic KCl crystals were immersed in media of various refractive indices. The measured retardation near crystal edges increased linearly with both the crystal thickness (tested between 0.3 and 1 micron), and the difference in refractive indices n between crystal (n = 1.49) and immersion liquids (n between 1.36 and 1.62). The specific edge birefringence, i.e., the retardation per thickness and per refractive index difference, is 0.029 on the high refractive index side of the boundary and -0.015 on the low refractive index side. The transition through zero birefringence specifies the position of a boundary at a much higher precision than predicted by the diffraction limit of the optical setup. The theoretical study employs a ray tracing procedure modeling the change in phase and polarization of rays passing through the specimen. We find good agreement between the model calculations and the experimental results indicating that edge birefringence can be attributed to the change in polarization of light that is refracted and reflected by dielectric interfaces.

Birefringence of tropomyosin crystals.

The birefringence of tropomyosin crystals was measured in the temperature range 5 degrees-35 degrees C. The experimental results are compared with a simple model calculation based on the theory developed by Wiener for the optical properties of colloidal systems. The difference between experimental and theoretical values is less than 15%, which denotes a good agreement given the simplicity of the model. A value of 0.011 was obtained for the intrinsic birefringence of the tropomyosin molecule. The temperature dependence of the crystal birefringence could be accounted for in part by a change of the unit cell parameters; this change was experimentally observed by others in x-ray diffraction experiments.

Probing f-actin flow by tracking shape fluctuations of radial bundles in lamellipodia of motile cells.

We examined the dynamics of radial actin bundles based on time-lapse movies of polarized light images of living neuronal growth cones. Using a highly sensitive computer vision algorithm for tracking, we analyzed the small shape fluctuations of radial actin bundles that otherwise remained stationary in their positions in the growth cone lamellipodium. Using the tracking software, we selected target points on radial bundles and measured both the local bundle orientations and the lateral displacements between consecutive movie frames. We found that the local orientation and the lateral displacement of a target point are correlated. The correlation can be explained using a simple geometric relationship between the lateral travel of tilted actin bundles and the retrograde flow of f-actin structures. Once this relationship has been established, we have turned the table and used the radial bundles as probes to measure the velocity field of f-actin flow. We have generated a detailed map of the complex retrograde flow pattern throughout the lamellipodium. Such two-dimensional flow maps will give new insights into the mechanisms responsible for f-actin-mediated cell motility and growth.

New polarized light microscope with precision universal compensator.

A new type of polarized light microscope ('new pol-scope') for fast and orientation-independent measurement of birefringent fine structure has been developed. The design of the new pol-scope incorporates a precision universal compensator made from two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at all points of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen. The sensitivity of the current instrument is 0.1 nm of specimen retardance, measured with data gathered in 0.43 s at all 640 x 480 image points. Examples of birefringence measurements in biological (microtubule arrays) and industrial (magneto-optical disc substrate) specimens are presented.

Point-spread functions of a polarizing microscope equipped with high-numerical-aperture lenses.

In an effort to establish the imaging properties of a new type of polarized-light microscope, we recorded images of small, uniaxial, birefringent crystals. We show that the sequence of in-focus and out-of-focus images, the so-called point-spread function, of a submicroscopic crystal can be used to measure the orientation of its optic axis in three-dimensional space. By analogy to conoscopic images out-of-focus images reveal the changes in relative phase shift between the extraordinary and the ordinary rays that propagate at different directions through the crystal. We also present simulated images of a pointlike anisotropic scattering particle and compare these with our experimental findings. The theoretical model is based on a complete vectorial theory for partial coherent imaging by use of polarized light and high-numerical-aperture lenses.

Birefringence of macromolecules. Wiener's theory revisited, with applications to DNA and tobacco mosaic virus.

We summarize Wiener's theory of the dielectric constant of heterogeneous systems and extend its application to suspensions of particles with corrugated surfaces and interstitial solvent. We retain a simple geometrical shape for the particles and account specifically for the solvent associated with the particles. We calculate the birefringence of the rodshaped Tobacco Mosaic Virus (TMV) particle and of DNA and find excellent agreement between our numerical results and experimental values from the literature.

Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones.

We investigated the motion of filopodia and actin bundles in lamellipodia of motile cells, using time-lapse sequences of polarized light images. We measured the velocity of retrograde flow of the actin network and the lateral motion of filopodia and actin bundles of the lamellipodium. Upon noting that laterally moving filopodia and actin bundles are always tilted with respect to the direction of retrograde flow, we propose a simple geometric model for the mechanism of lateral motion. The model establishes a relationship between the speed of lateral motion of actin bundles, their tilt angle with respect to the direction of retrograde flow, and the speed of retrograde flow in the lamellipodium. Our experimental results verify the quantitative predictions of the model. Furthermore, our observations support the hypothesis that lateral movement of filopodia is caused by retrograde flow of tilted actin bundles and by their growth through actin polymerization at the tip of the bundles inside the filopodia. Therefore we conclude that the lateral motion of tilted filopodia and actin bundles does not require a separate motile mechanism but is the result of retrograde flow and the assembly of actin filaments and bundles near the leading edge of the lamellipodium.