High-resolution Phase-contrast Microscopy Research Articles

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Synthesis of anisotropic Au–Cu alloy nanostructures and its application in SERS for detection of methylene blue

Au–Cu alloy nanostructures have been synthesized in aqueous phase through co-reduction of HAuCl4.3H2O and CuCl2.2H2O by glucose in presence of hexadecylamine at ∼80 °C. By changing the synthesis conditions, nanostructures of various shapes such as nanowires, multiply twinned tripod, tetrapod, etc were observed. Systematic variation of the synthesis condition not only leads to change in size and particle morphology but also develops various other crystallographic characters in the nanoparticles. Alloying behavior of Au–Cu has been examined through transmission electron microscope operating in its conventional and analytical modes coupled with high resolution phase contrast microscopy. These results suggested that nanostructures are composed of homogeneous Au–Cu alloy. Preferential attachment along {111} and {100} crystallographic facets of Au–Cu alloy nanoparticles led to the formation of nanowires. Multiply twinned branched shape Au–Cu (width of branch ∼30 nm) nanostructures exhibit localized surface plasmon resonance maxima in the near-infrared region. The branched shape Au–Cu alloy nanostructures display better surface enhanced Raman scattering response in the detection of methylene blue as compared to spherical Au nanoparticles.

Step velocity in tetragonal lysozyme growth as a function of impurity concentration and mass transport conditions

The step velocity on {1 1 0} faces of tetragonal lysozyme crystals has been measured in situ by high-resolution phase contrast microscopy. The movement of the steps shows a distinct anisotropy with respect to the growth velocity and the interaction with impurities. Under high-purity conditions (using lysozyme of 99.99% purity), the step velocity v step in the 〈1 1 0〉 direction exceeds the one in the 〈0 0 1〉 direction by a factor of 6. Spiral hillocks as well as 2D islands show an elongated, lens shaped morphology with pronounced tips in the 〈1 1 0〉 direction. The ratio of v step〈1 1 0〉/ v step〈0 0 1〉 is reduced to ≈2–4 when commercial grade lysozyme (purity: 98.5%) is used. The morphology of the hillocks/islands changes from the lens-shaped one to a rounded one. The step velocity into the fast growing 〈1 1 0〉 direction is significantly more affected by impurities than the growth into the 〈0 0 1〉 direction. The step velocity of monolayer islands (5.6 nm in height), of multilayer islands and of growth spirals is similar within the accuracy of the measurements. On the topside, the crystal is exposed to protein solution of ≈0.5 mm width. This results in convective mass transport conditions. On the backside of the crystal, there is a small gap of a few hundred nanometers. Within this gap, the crystal is growing under diffusive transport conditions. Changing the focus position from the topside of the crystal to the backside, the growth can be observed on the same crystal under convective as well as under diffusive conditions. Using commercial grade lysozyme, the growth velocity is higher under diffusive conditions than under convectively dominated ones. The morphology of the islands grown from a commercial grade solution under diffusive mass transport conditions is similar to the island morphology obtained otherwise only from high-purity solutions. Using high-purity solution, the step velocity and the island morphology does not differ as a function of the mass transport conditions. These results prove the extension of the depletion zone of the impurities and the reduced transport of impurities toward the growing interface under diffusive transport conditions.

The Ultrafine Structure of Carbon Fibers

High resolution phase contrast microscopy has been used to study the ultrafine structure of Morganite I and Thornel high modulus carbon fibers. Both are commercial fibers processed at 2800° to 3000°C, the former made from a polyacrylonitrile and the latter from an cellulose (rayon) precursor. Graphitizing is combined with stretching in order to align the basal planes parallel to the fiber axis to secure the high modulus. The ultrafine structures of these fibers have previously proved elusive.The fibers have been looked at in two ways. Since they are irregular in shape it was found possible to direct the electron beam normal to the fiber axis and image the basal planes viewed edge-on at the periphery of a fiber. The image disappears as one moves into the thicker region, so that only the structure near the surface is accessible. The second approach was to fracture the fiber by applying local bending stresses.

Intimate Structure of High Modulus Carbon Fibres

No evidence of transverse grain boundaries has been found in high modulus graphite fibres by high resolution phase contrast microscopy. Instead, the graphite planes have been found to flow over long distances in the direction of the fibre axis coalescing to graphite “crystallite” regions. The latter have the size and shape already suggested by X-ray diffraction techniques. The planes literally “flow” around intruding material, and evidence for elongated voids is occasionally found.

Modification of plastic culture dishes for high-resolution microscopy of living cells.

The plastic Cooper-type of dish has been adapted to high power, high resolution phase-contrast microscopy by drilling a 3/4 inch hole in the center of the bottom of the dish and cementing a 30 × 30 mm coverslip over this hole. Once the trypsinized cell suspension has been added and the dish closed, the top of the dish is sealed to the bottom by means of a roll of plasticene to prevent loss of CO2 and water. The preparation is maintained in a stage incubator. The same field can be observed with an inverted phase contrast microscope for 3 to 4 days. Either high dry (40-45x) or oil immersion objectives may be used to study the cells. The high resolution obtainable is especially useful in evaluating the effects of viruses or chemical agents on living cells in monolayer cultures.