A High-Resolution Combined Scanning Laser- and Widefield Polarizing Microscope for Imaging at Temperatures from 4 K to 300 K

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as for example birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a $^4$He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via Faraday effect or imaging of structural features, such as twin-walls in tetragonal SrTiO$_3$. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism and superconductivity.Comment: 14 pages, 11 figures. The following article has been accepted by Review of Scientific Instruments. After it is published, it will be found at http://aip.scitation.org/journal/rs

Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

The functions and consequences of force at kinetochores

Chromosome segregation requires the generation of force at the kinetochore—the multiprotein structure that facilitates attachment of chromosomes to spindle microtubules. This force is required both to move chromosomes and to signal the formation of proper bioriented attachments. To understand the role of force in these processes, it is critical to define how force is generated at kinetochores, the contributions of this force to chromosome movement, and how the kinetochore is structured and organized to withstand and respond to force. Classical studies and recent work provide a framework to dissect the mechanisms, functions, and consequences of force at kinetochores.National Institute of General Medical Sciences (U.S.) (Grant GM088313

Visualization and Analysis of 3D Microscopic Images

In a wide range of biological studies, it is highly desirable to visualize and analyze three-dimensional (3D) microscopic images. In this primer, we first introduce several major methods for visualizing typical 3D images and related multi-scale, multi-time-point, multi-color data sets. Then, we discuss three key categories of image analysis tasks, namely segmentation, registration, and annotation. We demonstrate how to pipeline these visualization and analysis modules using examples of profiling the single-cell gene-expression of C. elegans and constructing a map of stereotyped neurite tracts in a fruit fly brain

Developments in the Photonic Theory of Fluorescence

Conventional fluorescence commonly arises when excited molecules relax to their ground electronic state, and most of the surplus energy dissipates in the form of photon emission. The consolidation and full development of theory based on this concept has paved the way for the discovery of several mechanistic variants that can come into play with the involvement of laser input – most notably the phenomenon of multiphoton-induced fluorescence. However, other effects can become apparent when off-resonant laser input is applied during the lifetime of the initial excited state. Examples include a recently identified scheme for laser-controlled fluorescence. Other systems of interest are those in which fluorescence is emitted from a set of two or more coupled nanoemitters. This chapter develops a quantum theoretical outlook to identify and describe these processes, leading to a discussion of potential applications ranging from all-optical switching to the generation of optical vortices