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We investigate rotational diffusion of fluorescent molecules in angular potential wells, the excitation and subsequent emissions from these diffusing molecules, and the imaging of these emissions with high-NA aplanatic optical microscopes. Although dipole emissions only transmit six low-frequency angular components, we show that angular structured illumination can alias higher-frequency angular components into the passband of the imaging system. We show that the number of measurable angular components is limited by the relationships between three time scales: the rotational diffusion time, the fluorescence decay time, and the acquisition time. We demonstrate our model by simulating a numerical phantom in the limits of fast angular diffusion, slow angular diffusion, and weak potentials.

Intrinsic optical signal (IOS) imaging has been widely used to map the patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, reflectance, and scattering of the brain tissue. Here, we use polarized light for IOS imaging to monitor structural changes of cellular and subcellular architectures due to their neuronal activity in isolated brain slices. To reveal fast spatiotemporal changes of subcellular structures associated with neuronal activity, we developed the instantaneous polarized light microscope (PolScope), which allows us to observe birefringence changes in neuronal cells and tissues while stimulating neuronal activity. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at a high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatiotemporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by electrical stimulation at Schaffer collaterals. Upon stimulation, changes in traditional IOS signals were broadly uniform across the area, whereas birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those produced at low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced an increase in both transmittance and birefringence signals. Blocking synaptic transmission with inhibitors CNQX (for AMPA-type glutamate receptor) and D-APV (for NMDA-type glutamate receptor) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that the detection of activity-induced structural changes of the subcellular architecture in dendrites is possible in a label-free manner.

We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics—a form we call the dipole spatio-angular transfer function. We discuss the implications of our analysis for all quantitative fluorescence microscopy studies and lay out a path toward a complete theory.

We investigate the properties of a single-view fluorescence microscope in a 4f geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are small when the sample contains either many randomly oriented fluorophores in each resolvable volume or unconstrained rotating fluorophores.

A number of histopathology studies have utilized the label free microscopy method of Second Harmonic Generation (SHG) to investigate collagen organization in disease onset and progression. Here we explored an alternative label free imaging approach, LC-PolScope that is based on liquid crystal based polarized light imaging. We demonstrated that this more accessible technology has the ability to visualize all fibers of interest and has a good to excellent correlation between SHG and LC-PolScope measurements in fibrillar collagen orientation and alignment. This study supports that LC-PolScope is a viable alternative to SHG for label free collagen organization measurements in thin histology sections.

We propose a network of national imaging centers that provide collaborative, interdisciplinary spaces needed for the development, application, and teaching of advanced biological imaging techniques. Our proposal is based on recommendations from a National Science Foundation (NSF)-sponsored workshop on realizing the promise of innovations in imaging and computation for biological discovery.

The consequences of drug-based Arp2/3 inhibition in sea urchin coelomocytes include dramatic changes in lamellipodial architecture, a lamellipodial-to-filopodial shape change in suspended cells, and a novel actin structural organization in spreading cells. The generation of actin arcs induced by Arp2/3 inhibition is arrested by formin inhibition.

During meiosis I in crane-fly spermatocytes, the increase in microtubule density in K-fibers clearly originates at the kinetochore. The increased density first appears at the kinetochore, then elongates toward the pole, probably by addition of tubulin at microtubule plus ends anchored in the kinetochore.

We report capillary-force-assisted self-assembly (CAS) as a method for preparation of thin films of chemically reduced graphene oxide (rGO) with unidirectional organization of rGO sheets. The films were initiated at the contact line of the air–liquid–solid interface and form directly on solid substrates dipped in an isotropic colloidal suspension of rGO. Assisted by capillary forces at the contact line, the suspension undergoes an isotropic-to-anisotropic phase transition and becomes aligned with the film growth direction as the contact line moves across the substrate surface. We determined the degree of order in rGO films and assemblies by birefringence and diattenuation imaging. The slow axis of the rGO platelets within the CAS films displayed a narrow angular distribution (±3°) within a film area of 1 mm2, resulting in the highest possible order parameter (S) of ∼1 with 8-fold enhancement of electrical conductivity compared to films formed by traditional techniques such as filtration. Our straightforward film fabrication technique is scalable to produce large areas of films, and by controlling the rates of convective to diffusive mass transport, films with varying degree of order can be produced.

Experiments reveal pac-man motility in kinetochores of X-Y chromosomes, even though their normal behavior is dominated by traction fiber mechanics. A laser microbeam is used to release kinetochores in anaphase from tension. There is a poleward motion of released kinetochores twice as fast as normal and faster than tubulin flux.

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is "park," in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery and fluorescent speckle microscopy on kinetochores and associated microtubules during anaphase in crane fly spermatocytes. Kinetochores detached from their chromosomes moved at twice their normal speed, entering a motile state identified as “park.”

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n – 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.

This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n − 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.

To test the “traction fiber” model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are “smart,” meaning they embody a position-sensitive mechanism that controls their activity.

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.

We present a theoretical and experimental study of radially symmetric aberration caused by the differential transmission and phase shift of p- and s-polarized components of an axial beam passing through spherical lenses and plane parallel plates. We give a general description of the aberrations for an axial beam. The extinction is calculated as a function of the numerical aperture for uncoated lenses and for plane parallel plates. In our theoretical analysis, the polarization of output rays is described 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, optimal lens shape factors that minimize the rays’ polarization aberrations are found. Techniques for measurement of radially symmetric birefringence in a lens system are described. Finally, we discuss strategies for polarization rectification and introduce new designs including meniscus rectifiers and a liquid crystal rectifier that can actively compensate a wide variety of polarization aberrations. Good correlations between theory and experimental results for microscope optical systems with coated and uncoated optical elements are found. Our results enable us to suppress depolarization and remove anomalous diffraction in a modern microscope equipped with high-numerical-aperture lenses. © 2002 Society of Photo-Optical Instrumentation Engineers.

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 (∼0.5 μm/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 ∼0.1 μm/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% (∼0.1 μm/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.

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.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

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.

The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP‐tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open‐access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user‐friendly image acquisition to measure and analyze polarized fluorescence. © 2015 by John Wiley & Sons, Inc.