Introduction

Pharmaceutical biology deals with drug substances of biogenic origin, their production, chemistry, effects, and uses. Natural products have so far been the most prolific source of drugs, and they continue to provide important inspiration for the development of new drug substances (1). Besides recent developments in separation methods of crude extracts and new and efficient techniques for targeted isolation of lead substances, one major step toward a higher efficiency in pharmaceutical biology is the introduction of predictive high content cell-based assays for the bioactivity-guided isolation of compounds. Here we present a new software tool called AxioVision ASSAYbuilder™ that assists the scientist in performing a variety of high content analyses.

High Content Analysis

High content analysis (HCA) is a combination of cell-based assays, (high resolution) fluorescence imaging, automation, and advanced image analysis (2). It has been widely adopted in the pharmaceutical industries for target identification and validation. Furthermore, in secondary screens HCA detects potential toxicities or elucidates a compound's mechanism of action. In contrast to high-throughput screening (HTS), HCA is able to measure multiple biological pathways simultaneously, or to reveal off-target drug effects (3). However, there is also a growing need to combine research imaging with high content analysis or screening on the same equipment, for example, to reduce necessary investments or to keep learning curves low by having to deal only with one software user interface. Dual-use imaging systems thereby dramatically increase productivity and efficiency, especially in small- to medium-sized labs. A solution that addresses exactly this need consists of the Carl Zeiss Cell Observer^® and the new AxioVision ASSAYbuilder™ high content analysis module.

Materials and Methods

Meaningful biological data can only be extracted from high-quality images, stressing the need for cutting-edge imaging systems as a basis for generation of high content data.

The Carl Zeiss Cell Observer^® is a complete and versatile system based upon the successful Axio Observer. Z1 inverted research microscope. It was developed for observing and documenting living or fixed cells, organisms, and intracellular processes in several dimensions. The Cell Observer^® enables fully automated acquisition of up to 32 fluorescence or brightfield channels, z-stack, time-lapse images, and any number of positions in your specimen, for example, from prestored, editable plate and slide formats. A variety of fluorescence illumination sources, including the new Colibri LED light source, and optional components such as the ApoTome optical sectioning device or a selection of incubators, further expand the possibilities.

AxioVision ASSAYbuilder™ (powered by Cellomics^®) is a work-flow designed tool that makes sophisticated object-based image analysis possible for a wide range of high content screening assays. The module provides intuitive and script-free, workflow-oriented, object-based quantitative analyses of multichannel fluorescence and brightfield images with cross-channel referencing and hierarchical data output (Fig. 1A). Applied to either motorized screening systems, like the Cell Observer^®, or manual optical systems with a digital imaging camera, ASSAYbuilder™ imparts the capability to run measurement protocols on a large number of individual experiments. The images are acquired using AxioVision's advanced image acquisition functions or can also be imported from Zeiss confocal microscopes.

Fig. 1. (Click to enlarge)

According to the desired application and required data output, images are directly analyzed after acquisition with one or more of five different ASSAYbuilder™ Analyst modules: Physiology Analyst, Morphology Analyst, Membrane Analyst, Cell Cycle Analyst, and Motility Analyst. The Analyst modules organize object-based output (Fig. 1B) and analytical processes around common biological assays, and they are widely flexible for basic science research:

• Cell segmentation for the spatial analyses of biological targets follows an interactive display of cell features and cell reference regions.

• Images and relational data are interactively linked and automatically presented, allowing a rapid quality control.

• User-defined demographic tools enable the researcher to target high content measurements of specific subpopulations.

Typical use examples of the AxioVision ASSAYbuilder™module and the Analysts include computation and analysis of:

• Apoptosis and cytotoxicity