Alignment with sub-pixel accuracy for images of multi-modality microscopes using automatic calibration.

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in-depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x-y detector plane using a custom chrome-on-glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter-modal and 30 intra-modal image pairs, and the success rates are 95 and 100%, respectively, with sub-pixel accuracy. This methodology is widely applicable to any multi-modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.

In vitro imaging of embryonic stem cells using multiphoton luminescence of gold nanoparticles.

Recent advances in nonlinear optical techniques and materials such as quantum wells, nanowires and noble-metal nanoparticles have led to advances in cellular imaging wherein various nanoparticles have been shown to improve both in vitro and in vivo visualization. In this paper, we demonstrate in vitro imaging using multi-photon photoluminescence of gold nanoparticles from two different cell types Dictyostelium discoideum and mouse embryonic stem cells. By observing nanoparticles we show that embryonic stem cells maintained their ability to proliferate for several passages while grown in the presence of gold nanoparticles. The advantages of multi-photon luminescence using gold nanoparticles have important implications for use in stem cell proliferation experiments and in vitro experiments to monitor differentiation.

Automated real-time measurement of chemotactic cell motility.

We have developed a novel method, (ECIS/taxis), for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment, and their positions are monitored using the electric cell-substrate impedance sensor technology to measure the impedance change at a target electrode, that is lithographed onto the substrate, as the cells arrive at the target. In the studies reported here, Dictyostelium discoideum was used as a prototypical, motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose offolate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to the delayed arrival of cells at the target electrode. The multi-well assay configuration allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement.

Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and mechanically inhibited conditions.

Under-agarose chemotaxis has been used previously to assess the ability of neutrophils to respond to gradients of chemoattractant. We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid. Troughs are used instead of wells to increase the area along which the cells can be visualized and to create a uniform front of moving cells. Imaging the transition zone where the cells first encounter the agarose, we find that the cells move perpendicular to the gradient and periodically manage to squeeze under the agarose and move up the gradient. As cells exit the troughs, their cross-sectional area increases as the cells become flattened. Three-dimensional reconstruction of confocal optical sections through GFP-labeled cells demonstrates that the increase in cross-sectional area is due to the flattening of the cells. Since the cells locally deform the agarose and become deformed by it, the concentration of the agarose, and therefore its stiffness, should affect the ability of the cells to migrate. Consistent with this hypothesis, cells in 0.5% agarose move faster and are less flat than cells under 2% agarose. Cells do not exit the troughs and move under 3% agarose at all. Therefore, this assay can be used to compare and quantify the ability of different cell types or mutant cell lines to move in a restrictive environment.

Butyrate suppression of colonocyte NF-kappa B activation and cellular proteasome activity.

Butyrate is derived from the microbial metabolism of dietary fiber in the colon where it plays an important role in linking colonocyte turnover and differentiation to luminal content. In addition, butyrate appears to have both anti-inflammatory and cancer chemopreventive activities. Using confocal microscopy and cell fractionation studies, butyrate pretreatment of a human colon cell line (HT-29 cells) inhibited the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the proinflammatory transcription factor NF-kappaB. Butyrate inhibited NF-kappaB DNA binding within 30 min of TNF-alpha stimulation, consistent with an inhibition of nuclear translocation. IkappaB.NF-kappaB complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular IkappaB composition. Butyrate treatment increased p100 expression, an IkappaB that was not degraded upon TNF-alpha treatment. Butyrate also reduced the extent of TNF-alpha-induced IkappaB-alpha degradation and enhanced the presence of ubiquitin-conjugated IkappaB-alpha. The suppression of IkappaB-alpha degradation corresponded with a reduction in cellular proteasome activity as determined by in vitro proteasome assays and the increased presence of ubiquitin-conjugated proteins. The butyrate suppression of IkappaB-alpha degradation and proteasome activity may derive from its ability to inhibit histone deacetylases since the specific deacetylase inhibitor trichostatin A had similar effects. These results suggest a potential mechanism for the anti-inflammatory activity of butyrate and demonstrate the interplay between short chain fatty acids and cellular proteasome activity.