MetaMorph Microscopy Automation and Image Analysis Software

Catalytic nanoparticles represent a potential clinical approach to replace or correct aberrant enzymatic activities in patients. Several diseases, including many blinding eye diseases, are promoted by excessive oxidant stress due to reactive oxygen species (ROS). Cerium oxide and platinum nanoparticles represent two potentially therapeutic nanoparticles that de-toxify ROS. In the present study, we directly compare these two classes of catalytic nanoparticles. Cerium oxide and platinum nanoparticles were found to be 16 ± 2.4 and 1.9 ± 0.2 nm in diameter, respectively. Using surface plasmon-enhanced microscopy, we find that these nanoparticles associate with cells. Furthermore, cerium oxide and platinum nanoparticles demonstrated superoxide dismutase catalytic activity, but did not promote hemolytic or cytolytic pathways in living cells. Importantly, both cerium oxide and platinum nanoparticles reduce oxidant-mediated apoptosis in target cells as judged by the activation of caspase 3. The ability to diminish apoptosis may contribute to maintaining healthy tissues.

Automated image-based and biochemical assays have greatly increased throughput for quantifying cell numbers in in vitro studies. However, it has been more difficult to automate the counting of specific cell types with complex morphologies in mixed cell cultures. We have developed a fully automated, fast, accurate and objective method for the quantification of primary human GFAP-positive astrocytes and CD45-positive microglia from images of mixed cell populations. This method, called the complex cell count (CCC) assay, utilizes a combination of image processing and analysis operations from MetaMorph™ (Version 6.2.6, Molecular Devices). The CCC assay consists of four main aspects: image processing with a unique combination of morphology filters; digital thresholding; integrated morphometry analysis; and a configuration of object standards. The time needed to analyze each image is 1.82 s. Significant correlations have been consistently achieved between the data obtained from CCC analysis and manual cell counts. This assay can quickly and accurately quantify the number of human astrocytes and microglia in mixed cell culture and can be applied to quantifying a range of other cells/objects with complex morphology in neuroscience research.

How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na⁺ and K⁺ currents, and fire mature action potentials by 7 weeks of differentiation.