Angiogenesis is a general term describing formation of new tube-like microvessel sprouts that are the size of capillary blood vessels. Angiogenesis is fundamental in key stages of embryonic development, organ formation, and wound repair and is also involved in the development and progression of a variety of pathological conditions, including cancer (tumor growth and metastasis), cardiovascular disease, diabetic retinopathy, age-related macular degeneration, atherosclerosis, and rheumatoid arthritis. Because of its diverse roles in key physiological and pathological processes, angiogenesis is an important area of medical research, with a considerable number of angiogenic and anti-angiogenic drugs currently undergoing clinical trials. Cost-effective and efficient screening for potential lead compounds is therefore of prime importance. However, screening methodologies vary in their physiological relevance depending on how faithfully critical aspects of angiogenesis are represented. Cell-based in vitro angiogenesis assays are important tools for screening, which in many cases rely on imaging microscopy to ascertain drug effects. Unfortunately, such screens can be hampered by poorly defined biology, slow image acquisition by manual or semiautomated hardware, and slow data analysis by non-dedicated software. This article describes use of a 96-well microplate in vitro angiogenesis screening system as part of an integrated workflow, comprising (1) setting up the biology in a three-dimensional physiologically relevant system, (2) acquiring a series of image slices ("stacks") using an automated z-stage instrument, (3) collapsing the image stack series into sets of two-dimensional images, (4) segmenting objects of interest, and (5) analyzing the segmentation patterns in order to obtain statistically relevant data.

We report a drug dose-response, end-point study of intracellular filamentous actin (F-actin) by automated fluorescence microscopy, complemented with theoretical kinetic simulation of drug action. We highlight the use of an advanced orientation-sensitive image processing procedure ( transform), specially tailored for the detection of ordered filamentous "patches" in cell images. To examine the extent of stress F-actin disruption caused by the drug, we compare the measured response based on the above transformation with the theoretical data obtained from a quantitative model. We show that the assay data are consistent with the first-order mass action kinetics predicted by a basic reaction model. As a concluding remark, we briefly discuss advantages, perspectives, and challenges of conventional fluorescent microscopy within the context of the quantitative high-throughput screening paradigm.