Assay and Drug Development Technologies - 8

GIULIANO ET AL.
preparation methods, whether manual or automated, must
be validated prior to use and monitored for systematic errors,
including plate edge effects, and plate-to-plate and temporal
variability. Once the automated protocols are developed
and validated, the consistency of automated sample preparation
contributes significantly to the consistency of assay
results.
Additional challenges are faced when preparing cells for
live cell and kinetic analysis. Many fluorescent indicators for
live cells are organic dyes. Dye concentrations must be carefully
titrated to ensure cell viability throughout the exposure
period, and to minimize potential phototoxic responses.20,50
Dyes must be additionally evaluated when used in combination
(e.g., multiplexed) within the model system.8 Therefore,
proper sample preparation is perhaps the most important
consideration for successful implementation of HCS. Proper
use of automated sample preparation systems allows the
production of the most reproducible plates of cells.
INSTRUMENTATION
Instrumentation for conducting ''small-scale'' humaninteractive
imaging fluorescence microscopy has been available
for years. The initial focus of HCS implementation has
primarily been the development of a wide range of assays for
secondary screening based on fixed-endpoint and kinetic live
cell measurements. To address these needs, Cellomics produced
the first bench-top HCS instruments (ArrayScan and
KineticScan HCS Readers) that could be used in a standalone
manner or incorporated into automation systems. Recently,
additional manufacturers have entered the market with
HCS instrumentation optimized for speed of image acquisition.
It should be noted that collecting images is only the first
step in the multistep process of HCS data analysis. To efficiently
complete the analysis, the instrumentation must be
offered as a fully integrated package with analysis and informatics
software.
Fixed-end-point HCS assays require instrumentation that at
a minimum provides a white light source with full field illumination,
multi-spectral filters, and a CCD camera. Their
configuration should permit any available fluorescent probe
to be used in a combination of up to six distinct probes. Options
for plate stackers permitting up to 80 microplates to be
automatically analyzed, including different assays on each
plate in the stack, should also be enabled.
Performing HCS assays in live cells requires instrumentation
with additional unique features. It has been demonstrated
that photobleaching and the subsequent phototoxic responses
of cells are minimized with the use of full field illumination
compared to with laser scanning confocal microscopy.51
8 ASSAY and Drug Development Technologies
Therefore, more time points can be measured with full field
illumination than with laser scanning confocal systems
without causing serious damage to the cells under study. This
kinetic measurement capability is critically important for
developing temporal assays and for physiologically relevant
time studies. Onboard fluid addition from one to eight channels
at once allows the measurement of the response of cells
before, during, and after the addition of treatments. An integral
environmental chamber allows cellular studies under
physiological conditions. Furthermore, the incorporation of a
specular-reflectance-based autofocus system significantly
speeds up the scanning of microplates.
The early success ofHCS in secondary screening has created
an interest in moving some cell-based assays upstream to
primary screening, target identification, and target validation,
as well as downstream to absorption, distribution, metabolism,
and excretion/toxicology. Higher-throughput instruments
are desired for primary screening, and some
manufacturers have now produced faster scanning instruments
based on laser scanning confocal microscopy designs.
There is a trade-off among speed of image acquisition, cost,
flexibility, photobleaching, and the resultant phototoxicity.51
However, if only a single time point is required for a primary
screen measurement, then the photobleaching and phototoxicity
are not serious problems.
The next generation of HCS instrumentation is currently in
development. These developments should permit selection ofthe
optimal speed, type of illumination, higher multi-spectral selection,
and optical sectioning as required for particular assays.
BIOAPPLICATION SOFTWARE
Development of HCS assays is compririsesd of integrating
an optimized biological system, including the cells and reagents,
plus image processing and analysis software modules
that will automatically measure defined cellular properties
based on morphometry and fluorescence.
With the introduction of HCS, it was apparent that a new
generation of software modules was required to simplify and
automate the process of developing and optimizing HCS assays.
The first of these software modules, or BioApplications,
was optimized for specific target biologies that measured
defined cellular parameters. These ''specific'' BioApplications
were designed for screening implementation where features
such as turnkey operation and rapid startup time through
validated protocols and specific assay classification features
were essential. This class of BioApplications provides added
value by integrating the logic required to interpret results in
the biological context into the software. This is in contrast to
''scripted'' research imaging software modules that require
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