Rudolf Oldenbourg

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.

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 × 480 image points. Examples of birefringence measurements in biological (microtubule arrays) and industrial (magneto‐optical disc substrate) specimens are presented.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

Reconfigurable self-assembly through chiral control of interfacial tension

From determining the optical properties of simple molecular crystals to establishing the preferred handedness in highly complex vertebrates, molecular chirality profoundly influences the structural, mechanical and optical properties of both synthetic and biological matter on macroscopic length scales. In soft materials such as amphiphilic lipids and liquid crystals, the competition between local chiral interactions and global constraints imposed by the geometry of the self-assembled structures leads to frustration and the assembly of unique materials. An example of particular interest is smectic liquid crystals, where the two-dimensional layered geometry cannot support twist and chirality is consequently expelled to the edges in a manner analogous to the expulsion of a magnetic field from superconductors. Here we demonstrate a consequence of this geometric frustration that leads to a new design principle for the assembly of chiral molecules. Using a model system of colloidal membranes, we show that molecular chirality can control the interfacial tension, an important property of multi-component mixtures. This suggests an analogy between chiral twist, which is expelled to the edges of two-dimensional membranes, and amphiphilic surfactants, which are expelled to oil–water interfaces. As with surfactants, chiral control of interfacial tension drives the formation of many polymorphic assemblages such as twisted ribbons with linear and circular topologies, starfish membranes, and double and triple helices. Tuning molecular chirality in situ allows dynamical control of line tension, which powers polymorphic transitions between various chiral structures. These findings outline a general strategy for the assembly of reconfigurable chiral materials that can easily be moved, stretched, attached to one another and transformed between multiple conformational states, thus allowing precise assembly and nanosculpting of highly dynamical and designable materials with complex topologies.

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.

POLARIZED LIGHT MICROSCOPY OF SPINDLES

Publisher Summary This chapter presents the technique of polarized light microscopy of spindles starting with the basic setup of the traditional polarized light microscope and ending with state of the art in polarized light microscopy, the Pol-Scope. In addition to the instrumentation, the chapter discusses the ways to improve sensitivity and achieve high signal to noise ratios in recorded images. The chapter then describes the molecular origin of spindle birefringence, including the distinction between form and intrinsic birefringence, and presents some analysis tools that can be applied to find the microtubule density in the spindle based on birefringence measurements. A brief introduction to various cell types, which are amenable to polarized light microscopy is presented and some preparative techniques such as centrifugation to minimize background birefringence contributed by cell components other than the spindle are discussed. The chapter also presents a representative overview of the literature describing polarized light imaging of spindles in living cells.

Increased Birefringence in the Meiotic Spindle Provides a New Marker for the Onset of Activation in Living Oocytes

Abstract 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.

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

Polarized fluorescence microscopy reveals that septins across diverse species assemble into similar higher-order structures consisting of dynamic, paired filaments.

Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates

OBJECTIVE To examine the effects of different thermodynamic control systems on the temperature stability of human eggs during in vitro manipulation, with the integrity of meiotic spindles imaged using the LC-PolScope (Cambridge Research & Instrumentation, Inc., Woburn, MA). DESIGN We performed intracytoplasmic sperm injection (ICSI) and/or imaging of eggs with the temperature regulated by three different systems: thermostated coverslip (system 1), thermostated coverslip combined with objective heater (system 2), and conventional stage warmer (system 3). SETTING Academic in vitro fertilization clinic. PATIENT(S) Oocytes were aspirated from stimulated ovaries of patients undergoing oocyte retrieval for ICSI. INTERVENTION(S) Measurement of temperature regulation in media surrounding eggs during in vitro manipulation and imaging. MAIN OUTCOME MEASURE(S) Rate of oocytes with spindles, fertilization rates, and clinical pregnancy rates after ICSI. RESULT(S) We imaged spindles in more oocytes with system 2 (81.2%) than with system 1 (61.4%). Spindles could not be imaged for system 3 because of technical limitations. Fertilization rates were significantly higher when oocytes were imaged and used for ICSI with system 2 (78.8%) than with system 1 (56.7%) or system 3 (64.0%). Most importantly, a significantly higher clinical pregnancy rate was observed when oocytes were manipulated with system 2 (51.7%) than with system 1 (25.0%) or system 3 (23.1%). No differences were found in average ages, number of previous cycles, number of eggs, or day 3 FSH or E2 levels among groups. CONCLUSION(S) Imaging meiotic spindles with the PolScope provides an intracellular thermostat during ICSI. Rigorous thermal control during ICSI stabilized spindles and increased the fertilization and clinical pregnancy rates achieved after ICSI. The presence of birefringent spindles in living human eggs served as a monitor for in vitro conditions.

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.