Assay and Drug Development Technologies - 6

GIULIANO ET AL.
users the flexibility to design their own analysis, but can become
limiting when more complicated processing techniques
are required to optimize data collection. Implementation of
challenging assays often requires an expert script programmer
and can be prohibitive for some users.
One characteristic common to research imaging methods
has been the requirement for extensive interaction between
among the researcher, instrumentation, and software tools.
This approach has allowed researchers to perform a small
number of experiments on a small population of cells or
samples per day. Automation of image acquisition and some
simple measurements were the only aspects of the process
that avoided human interaction.16 Furthermore, the workflow
of these experiments included acquiring images of cells
in small chambers, allowing only one treatment per experimental
sample. The protracted task of post-processing images,
usually done offline, was based on interactive image
analysis, data reduction, and visualization. Finally, the
process included manually archiving the data to magnetic or
optical media. Thus, the process of imaging cells and extracting
data was comparable to the time-consuming and
labor-intensive steps involved in traditional DNA sequencing
on the bench-top.
When combined with the various modes of fluorescence
microscopy, a variety of novel fluorescence-based reagents
permitted a high degree of specificity and sensitivity in
measuring the temporal and spatial activities of cellular
constituents in fixed and living cells.29,30 Functional labeling
of specific cell constituents including proteins, called fluorescent
analog cytochemistry,31,32
was made a generally
available tool with the development of GFP as a ''genetic''
marker for proteins.33,34 Physiological indicator dyes for
membrane potential,35 free calcium ions,36 and other cellular
parameters37-39 accelerated the use of imaging technologies
to study the functions of living cells. The development of
''fluorescent protein biosensors,'' which are cellular reagents
formed by labeling proteins with either organic dyes or GFPs,
that ''report'' biochemical or molecular changes helped to
make fluorescence imaging microscopy a powerful tool in
molecular cell biology.40-46
THE NEED FOR SYSTEM INTEGRATION AROSE
EARLY IN THE DEVELOPMENT OF HCS
The development of HCS can be compared with the development
of automated DNA sequencing. Research scientists
were sequencing pieces of DNA long before Applied Biosystems,
Inc. (Foster City, CA) developed the automated DNA
sequencer. Traditional DNA sequencing involved sequential
steps including the manual loading of gels, running the gels,
reading the ladder patterns on the gels, and then manually
entering the sequence into a database. The data in this early
process ofDNA sequencing were the ladder patterns in the gel.
The information was the DNA sequence that was observed
after entering the ladder information into a database and
viewing the sequence. New knowledge was created by visually
searching the sequence data to ''find'' new potential genes. The
Human Genome Project required an integrated solution that
automated the whole process of generating data, producing
information, and creating knowledge. A combination of the
automated instrumentation, reagents, and genomic software
was integrated to rapidly identify new genes. This integrated
solution accelerated the discovery of new genes47,48 and
formed the basis for ''large-scale biology.''49
The development ofHCSmade the revolutionary step from
human-interactive imaging fluorescence microscopy to
creating a fully integrated and automated platform to rapidly
generate data, produce information, and create new knowledge
about cell functions. In comparison, traditional research
imaging instruments, with the interactive software
tools as described above, require extensive human interaction
to setup, acquire, process, analyze, visualize, and archive
images. The data from traditional methods are images
or pictures that the instrument acquires. The information
produced is the measurement of some specific cell parameter
such as cell number or shape that required the use ofhumaninteractive
software tools. New knowledge, such as the effects
of specific treatments have on cell division, is created
by performing a large number of experiments on small
numbers of cells and then correlating the information in
some type of manually managed dataset. While these traditional
imaging tools continue to generate great value in
small-scale biological research, the recent combination of
automated HCS instrumentation, reagents, and informatics/
bioinformatics software now permits the rapid characterization
of the functions of specific target proteins and other
cellular constituents, as well as wholecell functions, in largescale
biological research.
The HCS workflow schematic in Fig. 1 illustrates the importance
of integration to systematic knowledge building
from information extracted from living cells. First, the components
of the HCS platform are used to define and build a
screening assay based on a combination of informatics tools
and a repertoire of cell-based reagents and automated sample
preparation tools. Second, the HCS platform provides instrumentation
to generate cellular response profile data in either
fixed-end-point or kinetic modes of acquisition. It is important
to note here that the data from an HCS assay are not
collections of images, but rather a set of cellular response
6 ASSAY and Drug Development Technologies
ª 2022 MARY ANN LIEBERT, INC.
Assay and Drug Development Technologies

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