Biomedical Engineering Reference
In-Depth Information
applications, label-free approaches epitomize phenotypic assays, making it possi-
ble to detect major structural reorganizations unaltered primary mammalian cells.
Although many phenotypic assays could be performed in a plate reader format, the
HCS approach described in the next section represents a much more robust and
elegant platform for their execution.
The great advantage of phenotypic assays with respect to characterization of the
biological effects of DOS compounds should not be underestimated. They allow the
ultimate multiplex approach by targeting hundreds (if not thousands) of proteins in
a single test. Although the identification of compounds' intracellular targets may
prove to be challenging, methodological developments within the proteomics and
functional genomics fields should help to streamline the task [14].
12.3.5
Image-Based Screening
A specialized subset of functional cell-based assays relies on quantification of cellular
phenotypic changes made possible by advances in the throughput of fully automated
microscopy systems and image analysis [15]. These imaging assays are often referred
to as
image-based high-content screening assays
, due to their information-rich nature.
The majority of these assays image a defined subregion of each well of a microwell
plate and quantify the morphologic [shape, size, and number of (sub-)cellular struc-
tures], granulometric (texture or spatial distribution of biological markers), and fluo-
rometric (fluorophore intensities) changes at the individual cell (or another object of
interest) level [16]. These cell level data are then analyzed using population statistics
approaches to summarize the response of each well's cell population or assay-specific
cell subpopulation (Table 12.1). Commonly, the cell population data extracted from
image-based assays are multiparametric, since the images acquired provide not just
fluorescent intensity information but also spatial information and an object count
per well. These multiparametric data allow for simultaneous evaluation of multiple
phenotypic parameters at the single cell level and are often used to extract compound
toxicity or cell cycle effects in addition to the specific phenotypic target under inves-
tigation [17,18]. Table 12.1 summarizes phenotypic changes typically investigated
using image-based assays. Most of these make use of the two-dimensional nature of
microscopy images, which allows for the interrogation of spatial changes of fluores-
cent markers such as (sub-)cellular marker location, spatial distribution, morphology
of (sub-)cellular structures, and fluorescent biomarker colocalization.
Another advantage of imaging assays is the ability to multiplex several (routinely,
up to five different fluorescent channels) endogenous markers or fluorescent reporters
in one screening assay. Multiplexing several markers, which investigate different par-
allel pathways or related biological effects, for example, can provide insights into
the potential mechanism of action of compounds or, at a minimum, help validate the
biological response. Although dedicated large-scale image storage (typically, tens of
terabytes) is required, saving the acquired images of the screens for manual review or
future reanalysis can prove advantageous. Visual evaluation of images of the primary
screening hits allows for manual rejection of assay artifacts not identified automati-
cally by the image quantification algorithm. In some cases, changes deviating from the
