the

answer is
with
IN
Cell Analyzer 3000
high-throughput sub-cellular imaging system

18-1175-31 AA
 If your CD is missing, please request your copy

www
(quoting reference code 18-1177-12) on:

.amershambiosciences.com
/incellanalyzer
or contact your local sales office.
Minimum system requirements: WindowsTM
You will require a minimum of Windows 95 or later,
16 bit color 800 × 600 pixels
and double speed CD Rom
IN Cell Analyzer

3000
is a purpose built sub-cellular screening system. Combining
sophisticated scanning and optical technologies with
high-throughput capacity, the system provides advanced
multiplexing capabilities and real time, cell-by-cell image
analysis routines for live- and fixed-cell assays.

Designed to run continuously: a 950 000-compound library can be screened and

analyzed in 6 weeks
Precision optics and lasers: generate high-content screening images from assays
with sub-micron resolution
Dedicated image analysis modules: perform rapid imaging and quantitative
analysis of sub-cellular components in a range of applications
High-speed throughput and analysis: scans 30 000 standard images in 16 hours
Straightforward set up: scans standard 96- and 384-well microplates

1
applications
The IN Cell Analyzer 3000 enables high-content z Lead compound discovery
cellular screening of live- and fixed-cell assays in the
z Target validation
following areas:
z Functional genomics and proteomics
z Metabolism and toxicology

technology
IN Cell Analyzer 3000 achieves high-resolution imaging
and analysis through a combination of optimized
confocal optical pathways, dynamic autofocus,
and cooled digital cameras. The environment of the
sample chamber can be set to maintain temperature,
humidity, and CO2 levels at optimal cell culture
conditions for live-cell assays. The graphical user
interface combines flexible and intuitive software and
analysis modules capable of rapid scan rates and
quantitative data analysis.

Fig 1. IN Cell
Analyzer 3000
enables high-content
sub-cellular imaging
and analysis in a
high-throughput
screening
environment.

2
Confocal
line-scanning
beam
and large field
of view
The configuration of the scanning beam (0.6 x 750 µm) (Fig 2)
and the very short sample exposure time allows live cells to be
scanned at high resolution in less than 3 s without adversely
effecting their physiology or photo-bleaching fluorescent
molecules. This means that cells can be scanned repeatedly during
kinetic studies or re-scanned if necessary in static studies.
Confocal optics collect images from a defined focal plane, which
reduces sample background, increases sensitivity, and can
eliminate liquid handling steps in assay processing, facilitating the
performance of homogeneous assays.
A large field of view (750 x 750 µm) ensures that a high cell
count can be achieved in every scan (up to 500 cells per field at
40× magnification). This means statistically significant data are
rapidly acquired from cell populations.

Fig 2. Confocal line scanning with parallel pixel illumination and detection
enables rapid imaging without probe or sample degradation. The arrow
describes the passage of the scanning beam over the cells and the yellow
line defines scanning laser length and width.

3
Dynamic
autofocus
IN Cell Analyzer 3000 incorporates an infrared laser z Continuous, infrared laser autofocus ensures rapid
line that automatically detects the interface between progression from well to well because the sample is
the liquid in the well and the bottom of that well always in focus.
(Fig 3). The system then focuses the objective lens on z The system automatically adjusts the focal plane to
cells that are in a user-defined focal plane above this a user-defined offset level that generates optimal
interface. As a scan proceeds, tracking autofocus images for quantitative analysis.
continuously adjusts the focus of the objective lens on
this defined focal plane, ensuring that all scanned cells
are in focus. This process reproducibly generates images
that are optimal for high-content cellular analyses.

Fig 3. Dynamic, continual autofocus enables very rapid plate scans and cellular screens by ensuring imaged samples are constantly in focus.

4
Multiplexed, simultaneous
3 -color
imaging
Multiplexing facilitates the simultaneous investigation
of more than one event during an assay (Fig 4). IN Cell
Analyzer 3000 is the only high-throughput screening
platform to incorporate three high-speed, cooled CCD
cameras for the simultaneous imaging of red, blue, and
green emissions from samples that have been triple
laser line-scanned.
z Analyzes complex multi-parametric cellular assays
by excitation at three different wavelengths.
z Generates three-color images enabling sub-population
analyses on data generated from individual cells, on
a cell-by-cell basis.
To determine the effect of cell cycle position on
ligand binding a GFP-based cell cycle phase marker
Fig 4. Multiplexed analysis of ligand binding and cell cycle status using a GFP-based cell cycle phase marker.
(CCPM) was multiplexed with fluorescently labelled
cholera toxin (CTX). Transition of the CCPM through
the G2 and M phases of the cell cycle was then
monitored (Fig 4).
A U-2 OS cell line expressing the EGFP G2/M CCPM
was incubated for 15 min at 0 °C with 0.5 ug/ml Alexa
647 CTX subunit B conjugate; both in the absence
(Fig 4a) and in the presence of 50 ug/ml unlabelled
CTX subunit B (Fig 4b). Cells were then washed to
remove unbound CTX and their nuclei stained with
Hoechst. Fluorescently labelled cells were then imaged
on an IN Cell Analyzer 3000. Figure 4 shows binding of
fluorescent CTX is restricted to cells with low CCPM
expression (i.e. cells in G1).
5
Sample chamber
with
environmental
control
IN Cell Analyzer 3000 incorporates a sample chamber
in which the internal environment can be controlled at
37 °C, 70% humidity, and 5% CO2 enabling live-cell
assays to be performed in addition to fixed-cell assays.
Live-cell assays yield more accurate information about
a compound's true effect within the living cell as
opposed to information from assays that rely on
enzymatic or nucleic acid binding data derived from in
vitro assay systems. Please visit
z Full environmental control maintains optimal cell our Web site at:

www.
culture conditions for the performance of time
dependent kinetic assays and extended analytical
assays on living cells.
z Two pumps in the sample chamber can be
programmed to rapidly administer agonist or
antagonist compounds to cells through
amershambiosciences.com/
'non-contact' dispenser tips in volumes of between
10 and 50 µl, in 10 µl increments. incellanalyzer
or contact your local sales office
for the latest information.

6
 Choice of application is achieved through versatile

Image analysis modules designed for the study of biologically

relevant applications in lead selection, target validation,
and cellular toxicology studies, such as:
z Nuclear translocation

analysis
z Receptor internalization
z Mitochondrial translocation
z Protein expression
z Apoptosis
z Ligand binding

modules z Reporter gene expression

Image analysis modules facilitate high-speed cell-by-
cell image analysis, online, during each scan. Run set up
and execution are performed through an intuitive
graphical user interface that has fully adjustable, user-
defined parameters that ensure optimal, reproducible,
assay analyses.
z Cell cycle analysis
z Neurite extension
z Cell morphology
Flexible software performs quantitative image analysis
routines at the individual cell level and in real time,
during image acquisition cycles. Well images can be
selectively saved via user-defined scripting options.
The current range of Image analysis modules is shown
in Table 1.

Table 1. Image Analysis modules for IN Cell Analyzer 3000.

Module
Object Intensity
Nuclear Trafficking
Plasma Membrane Trafficking
Plasma Membrane Spot
Granularity
Cell Cycle Trafficking
(Static)
Morphology
Granularity Update
(CypHer)
Apoptosis-Nuclear
Trafficking and Granularity
Neurite Extension
Measures
Intensity changes, rapid kinetics
Translocation: cytoplasm, nucleus
Translocation: cytoplasm, cell surface
Subtle changes at cell surface
Discrete granules: transport vesicles,
micronuclei, mitochondria
Distinguishes cells in prophase,
mitosis, G0/G1/S, and G2
Flexible cell shape recognition
Variable grain size recognition,
automated analysis parameter
optimization, improved
background sampling method
Simultaneous quantitation of
nuclear trafficking and/or nuclear
orphology and granularity
Recognizes and quantitates
neurite extensions and cell bodies
Examples of use
G1ST, viability, toxicity, viral binding, calcium flux
NFκB, STATs, SMADs, p53, GR, Erk, MAPKAP-k2, NFAT
PLCδ, Cofilin
Rac, AKT
Receptor internalization, nuclear condensation,
cytochrome C trafficking
G2/M cell cycle phase markers assay employing a
reporter protein comprised of EGFP fused to a
fragment of cyclinB1
Identification of apoptotic and necrotic
cells in screening assays
CypHer based receptor internalization
Various apoptotic marker proteins
Retinoic acid-induced changes in PC12
Neuro-2a cells

7
Examples
of
analysis
module
function
Granularity
Nuclear Trafficking
Quality metrics
for HCS data

8
Granularity
Analysis
Module
A variety of biological stimuli result in the association
or dissociation of proteins or other molecules with
grain-like objects within cells. When such proteins or
molecules are tagged with an appropriate fluorescent
or fluorogenic molecule, these granular objects can be
imaged on the IN Cell Analyzer 3000.
Changes in the granular appearance of a cell can be
associated with key processes, such as endocytosis,
receptor activation, receptor down-regulation,
secretion, and activation or deactivation of intracellular
enzymes. The Granularity Analysis Module provides a
method to identify, count, and analyze grain-like
structures within cells (Fig 5).
A B
Fig 5. Analysis of TRHR-1 internalization using the Granularity Analysis
Module. CHOK1 cells expressing VSV-G epitope-tagged thyrotropin releasing
hormone receptor-1 (TRHR-1). Cells were pre-incubated with 5-mg/ml
CypHer5-labelled anti-VSV-G antibody and then incubated for 30 min at
37 °C with (A) no TRH or (B) 10-mM TRH. Bitmaps overlaid on the images
demonstrate how the Granularity Analysis Module analyzes CypHer5
internalization. The analysis routine identifies a measurement region for each
cell (large boxes), and then searches within each measurement region for
fluorescent grains of a user-specified size. No CypHer5 is detected in unstim-
ulated cells (A). After agonist-induced receptor internalization (B),
CypHer5 is detected in large perinuclear recycling endosomes (smaller boxes).
The system software generates cell-by-cell and
population-averaged results for each well to provide
you with complete assay information. The flexibility
of the Granularity Analysis Module enables
quantitative analysis of a range of assays that involve
changes in granular fluorescence, dissociation of
reporter molecules from cytoplasmic endosomes
(Fig 6), and internalization of plasma membrane
receptors (Fig 7).
A B
Fig 6. Image of living cells expressing a fluorescently tagged endosomal
reporter protein. (A) Endosomes within U2OS (human osteosarcoma) cells
are decorated with enhanced green fluorescent protein (EGFP) fused to
tandem FYVE domains. Location of the reporter is dependent on activity of
a phosphatidlyinositol 3-kinase, and the cell line can be used to screen for
inhibitors of this class of enzyme. (B) After 30 min treatment of cells with a
PI3-kinase inhibitor (100 nM Wortmannin), the EGFP-2xFYVE dissociates
from the endosomes and distributes diffusely throughout the cell
cytoplasm. Green = endosomes (EGFP-2xFYVE), Blue = nuclei (Hoechst
nuclear dye).
9
 A B

Fig 7. Graphical user interface of Granularity Analysis Module showing (clockwise from top left hand corner (A) Plate map. Yellow wells indicate cells that
contain granules and green wells indicate absence of granules. (B) Image with bitmap defining cells present in well 'A1' and the vesicles contained within them.
The degree of granularity is quantitated and recorded individually for each cell in the image. (C) Population scatter plot in which the points indicate
population-averaged granularity measurement for each well (in this case, one image per well). Yellow, prior to addition of agonist and green after addition of
agonist. (D) Population Summary table containing detailed, population-averaged granularity measurements for each well. Individual cell data are also recorded,
but not shown here.

10
Nuclear
Trafficking
Analysis
Module
The Nuclear Trafficking Analysis Module provides a
method to quantitate the movement, in either direction,
of the target molecules between the cytoplasm and
the nucleus. To do this, the quantitative image analysis
routine measures the fluorescence intensity of a target
molecule in discrete nuclear and cytoplasmic regions,
and then calculates the ratio of the sampled intensities
as an index of translocation (Fig 8).
Fig 8. 96-well live-cell EGFP-SMAD2 assay. CHO-hiR stable cell line expressing
EGFP-SMAD2 fusion protein was stimulated for 60 min in (A) the absence
or (B) presence of 3 ng/ml transforming growth factor-β1 (TGF-β1).
(B) Following activation with TGF-β1, the EGFP-tagged transcription factor
translocated from the cytoplasm to the nucleus. Hoechst 33342 nuclear
stain also shown. Images shown are 0.25 of the actual image size acquired
by the IN Cell Analyzer 3000. Error = +/-SD, n = 48 replicates per data point.
Quality
metrics
for
HCS data
IN Cell Analyzer 3000 hardware and software combine
to generate accurate, quantitative, robust, and
reproducible assay results, which can be used to derive
quality metrics that define how an assay is performing
as it proceeds (Fig 9).
Fig 9. Two quality control inhibition curves obtained on consecutive days
of a screen using the GFP-MAPKAP-k2 cell line and assay. Cells were
treated with increasing concentrations of a p38 MAPK inhibitor (SB203580)
in the presence of agonist (300-nM anisomycin). Comparable IC50 values,
hill slopes, and signal windows were obtained on the two days. Each data
point represents the percent maximal response measured from eight
replicate wells.
11
Table 2. Technical specifications.
Computer specifications 150 GB hard drive - Microsoft Windows 2000
Software control Graphical user interface for IN Cell Analyzer, Windows 2000 MFC application
Light sources Krypton laser: red (647 nm)
(two lasers generate three Argon Laser: UV(364 nm) and green (488 nm)
excitation laser-line wavelengths) Red, green, and blue LEDs (transmission mode)
Sample detection Selected wavelengths between 420 and 720 nm
High speed autofocus Dynamic, continuous infrared laser autofocus
Optics Automated optical pathway control with user controllable attenuation of excitation,
band pass filters for each camera and scanning mode
Imaging Three high-speed, TE-cooled, 12-bit CCD cameras.
Separate grayscale diagnostic camera
Field of view 0.75 × 0.75 mm, with 40× objective
Spatial resolution 1.2 µm
Scan time ≤ 2.5 min per 96-well microplate, at 2.4-µm resolution
Standard image size 1250 × 1250 pixels
Pixel size 0.6, 1.2, and 2.4 µm at high, medium, and low resolution respectively
Multiple field option Up to 30 individual fields can be imaged per well on a 96-well microplate, and up to
9 fields can be imaged per well on a 384-well microplate
Dispenser 10 to 50 µl in 10-µl steps
Run capacity (unattended) 216 microplates, 96- or 384-well format
Sample chamber for Internal environment controlled at: 37 °C, ≥ 70% humidity, 5% CO2,
live-cell kinetics (Ambient conditions are obtained when control is switched off)
Automation Compatible with externally scheduled automation packages
Plate format Designed to scan standard 96- or 384-well microplates

Ordering information
Product Product code
IN Cell Analyzer 3000 instrument 25-8010-11
(includes three analysis modules)
Object Intensity Analysis Module 63-0048-93
Nuclear Trafficking Analysis Module 63-0048-94
Plasma Membrane Trafficking Analysis Module 63-0048-95
Plasma Membrane Spot Analysis Module 63-0048-96
Granularity Analysis Module 63-0048-97
Cell Cycle Trafficking (Static) Analysis Module Available at end of 2003
Morphology Analysis Module Available at end of 2003
Granularity Update CypHer Analysis Module Available at end of 2003
Apoptosis Analysis Module Available at end of 2003
Neurite Extension Analysis Module Available at end of 2003

12
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go to www.amershambiosciences.com/incellanalyzer

or contact your local office:

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CypHer is a trademark of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc.
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