The world leader in serving science
GSK, Shanghai New User Training
Cellomics ArrayScan VTI HCS
Reader
August 2008
version x.6.1.4
Helen Ho
Field Application Scientist, West
Cell: 415-902-8734
Email: helen.ho@thermofisher.com
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
1
HCS Data “Quality”
 “GIGO”: “garbage in, garbage out”
 HCS assay data’s “legitimacy” depends on the
suitability & quality of an assembly of steps.
 “A chain is as strong as its weakest link.”
• Quality and suitability of…
specimen
image
analytical input
(parameters)
output
(features)
 After good sample preparation is achieved, the 1st step in
HCS is getting a suitable image. But this is
circular/bidirectional between adjacent links…
specimen  image  image analysis input  output
 HCS principles don’t differ from scientific assay
development generally:
• maximize/optimize signal:noise ratios
• maximize/optimize max:min response windows
• maximize/optimize speed vs. resolution of S:N & max:min
 …these are determined by sample quality,
imaging, and analytical input parameters
 images’ foreground:background  output
max:min responses
• images’ foreground:background depends on both…
• specimen quality, including labeling reagents and support
matrix (e.g., plastic vs. glass well bottoms)
-and• imaging exposure times (eventually, background will rise
with foreground, approaching point of “diminishing returns”)
• output min/max response windows depend on both …
• image foreground:background (sample quality, exposure
times, etc.)
-and• analytical input parameters
Typical assay development principles apply:
The outputs are not meaningful unless the inputs are.
Certain input/outputs can be progressively optimized. For
example, Reference Level-based, derived, non-intrinsic features
(e.g., %HIGH_, %Responders, %<2N…) can be attended to
after “raw”, intrinsic features are solid based on robust input
settings.
©1999-2008 Thermo Fisher Scientific
imaging exposure
time
max:min output
window
image
fore- : background
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2
A Brief Cellomics Glossary
 Object- the item(s) in an image that you
wish to analyze. This can be a cell, a
nucleus, a colony, or an organism
 Object Identification- the process of
determining which pixels in an image are
part of an Object or part of the
Background. This is done by creating or
modifying a Threshold.
 Threshold- the intensity value that
delineates object pixels from background
pixels. Maybe specified directly or
indirectly.
 Field- from microscopy’s “field-of-view’the area of a Well visible by the camera
 Channel- a collection of image
acquisition settings for a single image;
including exposure time, Dye selection
(wavelength filter configuration)
 Image Set- one or more images acquired
from a single field, comprising one image
per channel
 Primary Object- generally the objects in
the first Channel
 Sub-objects- an informal term for objects
that belong to a primary object- such as
neurites or spots assigned to a single
cell.
 Object Segmentation- the process of
separating objects that are very close to
one another, for the purposes of
analyzing them individually
 Parameter- generally, an input that you
make to the software
 Feature- an output data point based on
the image and data analysis process
• Cell Features- numeric data points
describing the measured properties of a
single Object
• Well Features- numeric data points
describing the measured properties of a
Well of cells/objects- most often, statistical
calculations (Mean, SD, % responders, etc.)
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
3
Getting Started
1.
Power on instrument.
•
•
2.
4.
ArrayScan VTI: Single green switch on right side
or front of instrument.
For VTI Live cell chamber module, see
Appendix.
Power on computer.
•
•
•
3.
Order of 1 & 2 does not matter. But…
Do NOT power off instrument (or components)
while ArrayScan software is open.
If do, close software & re-boot computer.
•
•
•
May not be able to use same login as at your
desk PC.
May need to be part of a Cellomics domain
group.
Only YOUR network administrator/IT support
staff can help you with this. That person is:
____________________________________
THIS LOGIN IS VERY IMPORTANT for full
functionality.
Never let IT staff change name of computer
without consulting Cellomics. Some functions
(e.g., entire Twister) could be lost & require full
re-installation.
Instrument: Shortcut like “Cellomics ArrayScan
Instrument”
To start software in Disk Mode, have instrument
powered off.
b) vHCS:View
Either (a) or (b) will prompt you for a Cellomics login.
•
NOT same as Windows login.
•
Default is “cell/cell”. Yours?:
______________________
•
Your system admin may have/should set up each
user with their own Cellomics login (see ArrayScan
Administrator tab in the ArrayScan Operator’s Guide)
to personalize your files, results, & activities on the
Cellomics system.
•
NOT as secure as Windows (or your network’s) login,
so keep simple, and know others may see it.
Suspect problem? Use HCS Administrator software.
a)

Log on to Windows on the computer:
•
Launch Cellomics software.
5.
•
•
•
©1999-2008 Thermo Fisher Scientific
If some Cellomics software behavior is suspect, launch your
HCS Administrator> Diagnostics> Run Diagnostics.
All Results? You’re OK. Otherwise, may need your IT staff’s
support. Email Report, or drag down row buttons to select all,
then Ctrl+C to copy (then paste and email, etc.). Or PrtSc
keyboard button (then paste, etc.)
Info also useful for learning AppSvr name, acct name, network
bandwidth, server path & free space, database name &
location…
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4
ArrayScan VTI Light Source and Camera
 LIGHT SOURCE: EXFO
 http://www.exfo-lifesciences.com/products/X-Cite120.html
• Turns on via main ArrayScan power switch. Stable in
90s. For max life, keep on ≥ 5 minutes.
• Some users turn lamp off (inside ArrayScan right front
door), but leave instrument on, to save bulb life –
though potentially confusing.
• If turned off, and try to re-light before fully cooled,
flashing bulb with “no” symbol may appear on lamp
display (inside ArrayScan right front door); auto-restrikes when cooled.
• bulb rated for 1500h, but note drop-off begins ~1000h
in plot.
• bulb is 100% user-changeable (requires no alignment)
• instructions come with replacement bulb ordered from
Cellomics
 Recommendation: always have a spare on hand
•






(order one now? P/N = IC001001)
CAMERA:
Hamamatsu ORCA-ER (for specs see http://www.leedsmicro.com/hamamatsuorcaer1.asp)
12-bit: this means 4096 grey levels
Usually images are acquired with the camera Binned 2x2, resulting in an image of 512x512pixels
Power is automatically controlled, no user intervention required
See Appendix for resolution information
©1999-2008 Thermo Fisher Scientific
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5
Overview of Create Protocol View
ArrayScan / vHCS:Scan software
anatomy, top to bottom
Title bar: Software name, version & build
(important), current view.
Here showing
vHCSTM:Scan Version 6.1.0 (Build 6018) – [Create
Protocol]
Menu bar: Little redundancy with other
controls.
File Options View Tools Window Help
Toolbar: The 1st three icon buttons are the
main “views” in the software (shown here is
Create Protocol view).
Main GUI body (all pale blue background
canvas). Notice the Main GUI in this [Create
Protocol] view has multiple panes:
•
•
•
•
Image Acquisition
Scan Limits
Assay
Channel Specific Parameters
• Object Identification
• Image Display Options
Status bar (gray bar along bottom)
Here showing: unlocked protocol icon,
Protocol Modified; Operator: cell.
©1999-2008 Thermo Fisher Scientific
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66
Scan software’s 3 views
Lesson 1: Assay Protocols
How do the 3 main views of
ArrayScan/ vHCS:Scan
software present Assay
Protocols?
Create Protocol view
Access all settings of an Assay
Protocol
Protocol Interactive view
 Interact with sample plate to
adjust various imaging input
parameters of an Assay Protocol
and temporarily acquire image
sets.
 With temporarily acquired image
sets, adjust various analysis
parameters and visualize
example results.
Scan Plate view
Execute an Assay Protocol (run
an assay).
What is in an Assay Protocol?







Objective (magnification)
Imaging resolution
What color fluorophores to image
Exposure (CCD integration) times
Autofocusing settings
How many images to take per well
BioApplication image processing
algorithm settings
 Kinetic and Motility Feature Setup
What’s NOT in an Assay Protocol?
 Plate Type Form Factor
 Which wells of a plate to analyze
 Which field of a well to start in
 Whether to save images
 Maximal image displays during scan
 Reference Well selections
©1999-2008 Thermo Fisher Scientific
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7
Create Protocol View
Settings accessible in Create Protocol view are the only settings saved with an Assay Protocol.
(Some settings are redundantly accessible in Protocol Interactive view; if changed in that view, they’re also saved when an Assay Protocol is saved
because they’re the same settings, just a different view).
©1999-2008 Thermo Fisher Scientific
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8
Create Protocol View: Image Acquisition pane
(except for Configure Focus, these settings accessible only in Create Protocol view)
a) Camera Configuration- on an Instrument you will have only one choice; on Toolbox clients choose the camera that you have or plan to
acquire images with
b) Objective
• All objectives installed on system appear in the drop-down list.
• Assay Parameters: PixelSize auto-computed. Understand benefit of UseMicrometers=1 and impact of Objective setting on area- & length-based
Object Selection Parameters. See BioApplication Guides.
c) Acquisition Camera Mode (Generally, “Standard”)
d) Autofocus Camera Mode: “AutoFocus” is likely specified in Assay Protocols provided by Cellomics. “Standard” may yield faster scan
times if the Acquisition Camera Mode is also Standard, but it depends on the samples imaged in the Focus
Channel; determine empirically or consult Cellomics Support.
e) Intra-well Autofocus Interval
•
•
•
•
Illustrated in ArrayScan HCS Reader User’s Guide (in ArrayScan HCS Reader Operator’s Guide).
Determines the points within a well where a focus adjustment is performed.
Conservative generalizations to start: 4 fields@5x; 3 fields@10x; 2 fields@20x; 1field@40x.
Affects scan times & quality, depending on #fields scanned per well (dependent on Scan Limits).
a. Therefore, best optimized during development of an assay by careful review of all images acquired during a scan
(at least review images from wells with highest # of fields acquired).
b. Variables to consider include
1.
2.
3.
4.
5.
6.
sample’s cell density (too sparse? “empty” fields take extended time)
sample’s quality (occasional debris or stacked cells? maybe more frequent Interval)
objective magnification and NA (inversely proportional to Interval)
standard- or thin-bottomed plates (thin wavier? maybe more frequent Interval)
plate’s well density (384, 96, etc.; esp. if thin-bottomed, lower density wavier)
users’ and algorithms’ analytical tolerances…
f) Configure Focus… Generally, use defaults.
• Autofocus may consider a Focus Channel image out-of-focus (/sparse) & prevent BioApplication analysis (though save the field’s
images). Relax… avoids this prevention (the case for all Disk Scans, since Autofocus isn’t used).
• Relax the Pass/Fail Criteria may be useful when:
1.
2.
3.
4.
High-NA objective on standard thick plates (see User Guide Appendix "Plate Types & Objective Selection");
Object intensities in Focus Channel vary widely;
Sparse fields are potentially common;
Multi-layer cells are potentially common;
• Relax the Pass/Fail Criteria also has additional effects (e.g., focus accuracy may be less), so it’s not recommended as default.
©1999-2008 Thermo Fisher Scientific
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9
Create Protocol view – Scan Limits pane
Scan Limits pane
Some logic is built in among checkbox selections.
 Scan Limits pane, Intra-Well Stopping Criteria section:
•
When will the ArrayScan stop scanning a well, and proceed to the next well?
• When at least one check-able Intra-Well Stopping Criteria is met or exceeded.
•
A “Sparse Field” has fewer objects than the Min Objects For Field (always keep >0).
•
Max Sparse Fields For Well: Sparse Fields must be contiguous to hit this limit.
•
All Scan Limits can have dramatic impacts on scan time. Think through logic very carefully.
•
How many objects is enough?
• Must be answered by assay developer’s statistical requirements, which can be arbitrary and vary between
organizations..
• Cellomics has some recommendations, but these are in the context of specific assay statistical requirements,
arbitrarily selected, though common.
• Here’s a Good Practice:
Early in assay development, scan all possible fields in model wells to create a comprehensive, stored image set.
Create a series of batch rescans using the Disk Scan Wizard (or ArrayScan software in Disk Mode) to rescan the
images, where the only variable between scans is decrementing # of fields per well. Such a batch is programmed
using Plate Protocols, where, in this case, the Plate Protocols differ only in one regard—their Assay Protocols
differ only in Max Fields For Well, with all other Scan Limits boxes unchecked. Examine the results of such
batches to determine a minimum sample size at which StdDev is stable for principal output well features.
• NOTE: An “object” in the context of Scan Limits is BioApplication-dependent.
 Scan Limits pane, Intra-Plate Stopping Criteria section:
What use is Max Sparse Wells For Plate?
•
In mass sample prep operations (e.g., automation), if most cells are lost by unsuitable liquid handling or a treatment
undergone by all wells, or a necessary labeling regent was not suitably applied…
•
When scanning a stack of plates, more efficient not to scan “bad” plates.
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
10
Create Protocol view – Assay pane
Assay pane
All subsections present in every BioApp, but options within subsections may
vary.
 Assay Algorithm: NOTE: Generally, do not change the Assay Algorithm
selection unless you are familiar with its impact. You will not be prompted that
changes not saved before altering this selection are irrecoverable.
 No. of Channels: BioApplication- dependent (see BioApplication Guides).
Best practice: begin with a default
assay protocol already written for
the BioApplication you want to use
• For some, the standard # of channels can be scaled back.
• For some, additional “gating” channels can be added.
 Focus Channel
• Specifies in which channel Autofocusing will be performed during a scan
• Generally, choose a channel imaging a dye whose quantitative
fluorescence is not critical and/or is relatively photostable (e.g., a nuclear
Hoechst stain or demarcating the nucleus).
 Features to Store: default, and a good place to start, is to store all features.
Select features to Store when screening or doing intensive kinetic assays
 Kinetics Checkbox: used to set up time course experiments. Configure
button becomes active when checkbox is ticked.
 Assay Parameters: List is BioApp-specific (see Guides) & varies with No. of
Channels.
 Hide Advanced Parameters: BioApp-dependent (see respective User’s
Guides). Generally, parameters that refer to Data Analysis (Levels, _CC) are
Advanced Parameters
 Default Well Feature: see Scan Plate view window (and plate diagram coloring
in vHCS™:View).
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
11
Channel Specific Parameters- “Dye” and Filter Selection
1. The Label is an image caption; for
your record keeping only (Displayed
as Caption in vHCS:View)
2. Dye refers to the filter configuration
to be used in that channel
Dye/ Fluorophore Color Classes
Hoechst
FITC
TRITC
DAPI
fluorescein
rhodamine
DRAQ5
GFP, EGFP
Whole Cell Stain
Orange
Whole Cell Stain
Red
Whole Cell
Stain Blue
Coumarins
®
Texas Red
Cy5
Alexa Fluor 488
Whole Cell Stain
Green
Alexa Fluor 546
Alexa Fluor 594
Alexa Fluor 555
~Alexa Fluor 568
YOYO®-1
DyLight™ 549
YO-PRO®-1
~BOBO™-3
~YO-PRO™-3
SYTOX® Green
Cy3™
~SYTO®17
Fluo-4
Dil (DiIC18(3))
Calcein
BODIPY-TMR
Alexa Fluor 647
®
~TOTO -3
DiD (DilC18(5))
BODIPY®-FL
XF100
XF93
XF53
Hoechst
good
good
good
FITC
good
good
good
TRITC
good
Texas Red
Cy5
XF32
XF110
sensitive
good
good
©1999-2008 Thermo Fisher Scientific
3. Consult Appendix B of your
ArrayScan User’s Guide tab within
your ArrayScan Operator’s Guide
binder, which explains why scans
are faster if all fluors in a sample
can be imaged by the same XF
set.
4. All else being equal, Cy5 channels
are usually the weakest (because of
both excitation and detection optics).
Therefore, use a Cy5-like dye only
if a 4th color is needed, and use
to label/ report the most abundant
target, etc.
5. Other dyes/fluorophores are
certainly possible; consult Cellomics
Support or
http://probes.invitrogen.com/resourc
es/spectraviewer/ or
https://www.omegafilters.com/front/
curvomatic/spectra.php.
sensitive
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12
Imaging: AutoExpose and Fixed Exposure
Types of Exposure Methods:
 Fixed: Specify a time in seconds for exposure in each channel- that exposure time will be used throughout
 AutoExpose: still must specify an Initial Exposure Time. That exposure time will be used as a starting point to
identify a time that meets your criteria (see below)
 Key image analysis steps are tightly dependent on image characteristics that are, in turn, largely determined by
exposure times.
• Matching exposure times to existing input analysis parameters is often more productive that resetting analysis
parameters.
• Instrument components change over time, so autoexpose options that can compensate will prove useful, even when
using Fixed exposure during plate scanning.
• Even when stored images are re-loaded, their % of dynamic range will be reported by the Autoexpose routine.
How bright should my images be?
Here are some guidelines for what % of dynamic range may be suitable for a few classes of fluorescence labels.
25%: Good for mainly “structural” labels (e.g., counter-stained nuclei). Still affords some range for differentiation between, e.g.,
interphase, mitotic/apototic nuclei. ~20% can be tried for minimizing autofocus time & maximizing speed. Typically also sufficient
for “binary” assays where the interest is whether the label is there or not, yes/no—e.g., mitotic index, negative viability (“dead
dyes”), micronuclei, and others.
35-40%: For “intensity” quantifications. Localized intensity differentials may reflect >40%. Also reasonable starting range for
“structural+intensity” labeling—e.g., nuclei in assays needing more discrimination (e.g., DNA content/cell cycle).
55-65%: Good for multi-modal structural labels where accurate identification of more than one intensity level is of interest—e.g., some
neuronal cell bodies are considerably brighter than neurites that also must be accurately identified. High dynamic range may also
be required for some indicators of cellular metabolism, physiology, health, etc.
Best Practice: When trying higher %, be on guard for saturation. Optimal exposure ranges are worth careful investigation—esp. in
“intensity” assay, large improvements often emerge. If your BioApplication has multiple channels measuring the same thing, fill
them all with variable exposure times during assay development pilots.
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
13
AutoExpose Options and Procedures
AutoExpose Options
1.Whereas Background Target(Mode) Method refers to
image histograms, “Peak” does not refer to a
histogram peak; after Skipping specified brightest pixels,
Peak is X-axis intensity of remaining brightest pixel. “Max
Intensity”
2.Peak methods good for targeting brightest signals as %
of camera’s 4095 grey-level dynamic range.
3.Background… method: When signal is expected to be
absent, still can get useful exposure time (typically low %
so when fore-ground appears in unknowns, it extends
into useful range).
If a scan is executed with an Assay Protocol specifying AutoExpose as
Exposure Type, before the whole scan begins, the system…
 Goes to 1st well in List of wells to Autoexpose.
 Goes to 1st field in List of fields to Autoexpose.
 Autofocuses (using Configure Focus…> Autofocus Options> Camera
Exposure Time> Focus Exposure Time For Autoexpose—note,
AutoFOCUS setting!).
If Autofocus fails, so does that field! Proceeds to next.
 Execute Autoexposure Method.
a. Failed any channel? Try in next specified field
(i.e., in example, field 5 is used only if field 4 fails).
b. Succeeded for all channels? Temporarily hold determined exposure time(s) in
memory.
 Go to next well, as applicable, repeat, hold time.
 Compute mean of all “held” times (I.e., where succeeded) & use it for
respective channel(s) for whole plate scan.
Benefits:
 If reagents, optics, etc. vary over time or from plate to plate, this
Exposure Type may be compensatory.
Caveats:
 No automatic way to know if specified wells/fields are in good
condition in every plate. And final exposure time(s) is only from
successful wells.
 Screeners may want not to have assay variables masked by system
compensations.
 Users need to remember what Assay Protocols have set as
wells/fields to autoexpose and whether the same for every plate used
with that Protocol.
An Intermediate – Best Practice:
Use Autoexpose in Protocol Interactive on fields deemed representative by
you, but specify Fixed ultimately in the saved Assay Protocol.
©1999-2008 Thermo Fisher Scientific
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14
Choose your BioApp by choosing
an Assay Protocol
Summary of Create Protocol functions
Advanced training topics. Retain settings
specified…
• in Cellomics-provided Assay Protocol
(likely Standard & Autofocus respectively)
• …or… by Cellomics support
• Saved with the Assay Protocol (User Guide error says
“stored with results for the plate”).
• Admin. changes to read/write status auto-written here.
• BioApplication Event Wizard entries auto-written here.
• Max 4000 characters, but keep <255 if possible.
BioApp.-specific.
Advanced training topic
• Do not change (default=25)
unless advised by Cellomics
• Depends on plate type, objective (NA
& mag), etc.
• Best determined empirically.
• Starting pointers (conservative):
5x, 4 fields
10x, 3 fields
20x, 2 fields
40x, 1 fields
Must choose both a Method and a Value here;
best optimized in Protocol interactive
BioApp.-specific
If both checked,
whichever comes
first. “Objects” is
BioApplication-specific.
contiguous
“Objects” is
BioApp.-specific.
See Scan view window
(& plate diagram coloring in vHCS:View).
©1999-2008 Thermo Fisher Scientific
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15
Scan software’s Anatomy Lesson 2: Protocol Interactive view
How do the 3 main views of ArrayScan/
vHCS:Scan software present Assay
Protocols?
Create Protocol view
Access all settings of an Assay Protocol
Protocol Interactive view
 Interact with sample plate to adjust various
imaging input parameters of an Assay Protocol
and temporarily acquire image sets.
 With temporarily acquired image sets, adjust
various analysis parameters and visualize
example results.
Scan Plate view
Execute an Assay Protocol (run an assay).
What’s Protocol Interactive view for?
 Select a Plate Type Form Factor
 Navigate to a well and field (within a well)
 Autofocus (& adjust if desired, + DZ’s)
 Alter Dyes (filter sets) if desired
 Image Fields (1 at a time)
 Alter Exposure times (& Auto settings if desired)
On an Acquired Image Set…
 Alter overlays & composite & colors
 Edit Assay parameters
 Run Algorithm to see results of current settings
 Identify Objects and adjust settings, plus modify Object
Selection Parameters
 Double-click image to get “pop-ups” and zoom in, see
numerical results, toggle overlays.
What parts of an Assay Protocol are NOT accessible in
Protocol Interactive view?
 Objective (magnification)
 Camera settings: Acquisition Modes (Binning) & Gain
 Autofocus Interval
 Scan Limits
 Assay Algorithm selected
 No. of Channels, Image/Channel Labels
 Default Well Feature & Extents
 Kinetics Setup
 Select Features to Store
©1999-2008 Thermo Fisher Scientific
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16
Protocol Interactive View Screenshot
Object Identification Settings
Object Selection/ Validation Settings
Image Display Settings
Most bioapplication-specific image and
data analysis parameters are accessed
by this button
©1999-2008 Thermo Fisher Scientific
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17
17
I-V-S : Identify, Validate, Select
Intra
Venous
Saline
I’m
Very
Smart
It’s
Very
Simple
Object Identification
IN “PRIMARY” CHANNEL(S)**
Object Identification
 Separate foreground from background.
 intensity-based (Some Assay Params can
alter raw intensities [e.g.,
BackgroundCorrection] or shapes/sizes of
Identified Objects [e.g., Smoothing].)
 Controlled by Object Identification Value
• Results depend on Method.
Object Validation
Object Validation
Object Selection
• “Rejected” overlay*
• Must be  in current Ch
to see
• Algorithm ignores these
objects, so no data is
reported.
“Selected_” overlays
 “Is this identified object valid?” Criteria are
BioApplication-specific.
 Objects in primary channel(s) must also be
Selected to be Valid.
Object Selection
 As noted above, in primary channel(s), Object
Selection Parameters also Validate objects*
Most V3 BioApps have an Assay Parameter for
RejectEdgeObjects
** Some BioApps have >1 primary channel (e.g., Cell
data results
Spreading, Neuronal Profiling, Micronucleus).
* BioApp-specific (a few cases
have no “Rejected_” overlay).
©1999-2008 Thermo Fisher Scientific
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18
Object Identification Methods
IsoData Method
Peak Triangulation Method
Threshold
Threshold
Object
Number of Pixels
Background
Background
Peak
Object
Peak
Background
Peak
0
Object
Peak
4095
0
4095
Object Identification Value shifts auto-computed threshold by Value’s %age
• How do you think FixedThreshold Method works?
• What do you think the impact of BackgroundCorrection is on these methods?
• What do you think happens if there is an extraordinarily bright artifact (lint, debris, etc.) in an image?
©1999-2008 Thermo Fisher Scientific
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19
Common V3 Assay Params: BackgroundCorrection
BackgroundCorrection Best
Practice: Use…Whenever you can!
Background image constructed
essentially by blurring Raw
Image.
Value inversely proportional to
how much blurring.
 Always use it, whenever you can! And, when you can,…
Best Practice:
Use Fixed Threshold for Object Identification Method (if you can).
 When can I not use it?
• When any image may have near-confluent fluorescence (maybe):
1.Imagine the Raw Image is full of objects; what would the
blurred Background image look like?
2.…And the Background Corrected Image?
3.In sum, has to be some background in every image, in every
channel… Why every image/channel?…
 BackgroundCorrection is a “global” Assay Parameter—i.e., when
activated (non-zero Value), it is performed on every image in that
channel
 Adjustable individually in each channel in V3 Bioapplications
 High impact on all aspects of processing & analyses
1.Performed BEFORE Object Identification
2.All output feature values, etc. vary with Value
 Choosing a Value
1.“Aggressive” Values (low)
• ~ diameter largest typical object.
• for reliably monodisperse objects/signal.
• affords “localized” correction
2.“Conservative” (large Values) for heterogeneous objects/signals.
• Greater than 2x largest diameter?
• Heterogeneity too unpredictable? Consider Max Value (255)
©1999-2008 Thermo Fisher Scientific
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20
Scan Plate View
Select which
Wells to Scan
Select starting
Field
Type any text desired, for descriptive purposes. When scan
completed, this text will be searchable, too. Generally, keep this
field <255 characters (though not necessary) and use simple
alpha-numeric characters.
In the plate
Avoid non-simple characters in Plate
ID (Barcode) or Plate Name. The
Scan software will accept them;
however, subsequent searches in
vHCS:View using characters like “#”
will fail.
You can change the feature to display
here but it is NOT saved with the Assay
Protocol.
©1999-2008 Thermo Fisher Scientific
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21
Reviewing results in vHCS:View
Launch via Start> Program Files> Cellomics>
vHCS:View.
 Highly developed software with user-friendly GUI
(graphical user interface):
• Almost complete redundancy between menus, toolbar
icons, and right-click context-sensitive pop-up menus.
• Uses Windows conventional Ctrl+click & Shift+click
functions for selections on text lists, spreadsheets, etc.
• Uses Windows conventional Ctrl+C (copy) & Ctrl+V
(paste) from spreadsheets.
• Full on-line Help menu.
 Best Practices – Tips for fast, easy use &
learning
• Frequently right-click on GUI elements (graphs, images,
diagrams, spreadsheets) for command options on
single GUI elements.
• Use the Windows menu on the menu bar for managing
arrangements of multiple windows/views and navigating
between them.
• Check menu bar menus for additional command
options, and when multiple GUI element types are
displayed (e.g., a view with multiple graphs).
• Notice the interconnections of interactive GUI elements
(e.g., click a point on a graph, and note spreadsheet
highlight changes with it).
• Be fearlessly exploratory—except for Delete Plate(s).
Aside from Delete, you cannot change any data using
vHCS:View: It’s only a tool for viewing data in different
ways.
Find a Plate result of interest:
 Well Details
•
•
•
•
•
•
Row/Col mode (so lay out plates accordingly)
Use down arrow key to “flip” through Feature list
double-click anywhere on graph (except points) to Customize
Copy/export spreadsheets or graphs
Methods (bar, points, points+lines)
File> Report
 Cell Details
• toggling overlays
• Maximize images (and de-max to restore; don’t click  or Cell Details
closes)
• making New plots
• Scatter plots vs histograms
• maximize, copy, export spreadsheets/graphs
• select spreadsheet column heading, right-click, Sort…, useful to find where
to use subpop cutoffs or other assay params, as are histograms/scatter
plots.
• show can call Cell Details from multiple highlighted Well Details wells
(contiguous)
Other common uses besides Well & Cell Details:
 Multi-Plate View
• Graph menu> Plot on Single Graph
• Spreadsheet menu> Export to…
 Multi-Feature View
• Graph menu> Plot on Single Graph (only good for similar scales, and
rough with multiple rows)
• Spreadsheet menu> Export to…
©1999-2008 Thermo Fisher Scientific
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22
Quiz 1: Assay Protocol
Starting with a provided Assay Protocol for the Target Activation BioApplication, 10X objective (NA
0.3), build an Assay Protocol to accomplish the following:
1. Magnification & resolution (see Appendix pages of this training packet)
1. ArrayScan 3.5/4.5, ArrayScan VTI (1.0x coupler), 0.645 micron/pixel, 512x512 pixel images
2. ArrayScan VTI (5.5) with 0.63x coupler, 1.024 micron/pixel, 512x512 pixel images
2. Analyze 93 cells in each well, but don’t take more than 3 fields per well to do it, and don’t try for
a 2nd or 3rd field if the 1st field has <50 cells.
3. 2 channels, focusing on nuclei
1. Ch1: Hoechst; Exposure time that will bring foreground intensity to ~1/4 dynamic range (±0.4%, ignoring
0.25% of the brightest pixels)
2. Ch2: Alexa Fluor 594; Exposure time that will bring background intensity to ~200 intensity units (±0.4%,
using smoothing Kernel Size 25)
4. In RGB composites, make Hoechst appear in blue and AF594 in red.
5. In Ch1, overlay analyzed objects in blue and Rejected cells in yellow
6. In Ch2, show Rejected object overlays, plus Modify the Ch1 mask to make it 2 pixels wider in
Ch2 and show it in Ch2 as red.
7. Don’t use Illumination Correction in any channel.
8. In image captions, call Ch1 “Nuclei” & Ch2 “Target”
©1999-2008 Thermo Fisher Scientific
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23
Redundant Access in Protocol Interactive View
Redundant access in Protocol Interactive view
These 4 boxed regions in this Create Protocol view window are accessible
from within Protocol Interactive view too.
Changes made in either view can be/are saved with the Assay Protocol.
• Configure Autofocus
• Number of Channels
• Assay Params. (including Hide…)
• Except Kinetics configuration, Select Features to Store
• Channel Specific Parameters,
• including all Object Identification parameters
• including all Image Display Options parameters
• except Label, Apply Illumination Correction, Gain Options
©1999-2008 Thermo Fisher Scientific
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24
Pop-up windows in
Protocol Interactive
Best Practices rely heavily on productive uses of popups.
Use Window menu inside ArrayScan software to
manage pop-ups productively. Involves using
Windows TaskBar to switch between main software
window and popups. Both are shown in this
illustration.
 Single images at a time:
Double-click anywhere on
embedded image to release a
copy of the image in separate
pop-up window
• Draw box to zoom in for details of overlay accuracy, etc.
• Use overlay toggle button to see underlying structures.
• Brighten images to avoid missing accuracy detail.
 Series of same image, varying input parameters.
• Don’t move the window once popped up.
• Change a parameter, Run Algorithm, & pop up result; stacks
on top of prior popup. Now use Windows’ TaskBar to flip back
& forth between them to detect subtle changes in overlays.
• Repeat with multiple popups, investigating a range of values
for the same parameter. Type each value into popup’s text
field to track which value is best.
[But read note typed into text field in example shown here.
Images can be saved, but not text.]
 Pop up images from multiple fields for side-by-side
comparisons.
•
•
•
•
Tile Popups Vertically (Ctrl+T).
Select equivalent Bits To Display.
Click Well Features button to compare numerical results.
Same approach for fields from multiple wells—replicates or
opposites (positive/max & negative/min).
[Quiz 1 answers (2 pages) in Appendix!]
©1999-2008 Thermo Fisher Scientific
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25
Contrast Stretching – Common Illusions
 There are only 2 images on this page – top & bottom row. Across each
row are different display adjustments of the same image. The images
in the top row are from a field right next to the field in the bottom row!
 In 1st column – Full or Max – do you think the bottom field is dimmer?
Do the 12-bit histograms suggest that?
 The 1st column illustrates the common illusion of Contrast-Stretching—
an auto-adjustment of brightness/contrast done on an image-by-image
basis. Contrast-stretched images canNOT be legitimately compared for
visual intensity differences!
or
 Beware: Full Contrast Stretch is the default display adjustment!
 For image-to-image intensity comparisons, Contrast-Stretching
should be deactivated, and Bits-To-Display selected.
 Histograms under images are 8 bits—256 gray levels—the maximum
# of grays display-able on a monitor. Bitshift 3 (i.e., bits 3-10, or grays
8-1024 from the 12-bit source image) illustrates the best spread of the
gray levels across the 8 display bits. For the top field, Bitshift 4
resembles the Contrast-Stretched image, whereas for the bottom
field, it’s Bitshift 5.
Display Options
12-bit histogram
y stretch zoomed
to see low
frequency pixels
12-bit histogram
y auto-scaled
8-bit histograms
 In the bottom field, the bright spot shows up on
the 12-bit y-stretch histogram. When auto-remapped to 8 bits, those high values prevent
expansion of the display histogram.
12-bit histogram
y stretch zoomed
12-bit histogram
y auto-scaled
©1999-2008 Thermo Fisher Scientific
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26
FAQ’s- Protocol Interactive View
Q. How can I see my numeric data (cell and well details) interactively?
A. Use icons below image (shown at right) after Run Algorithm.
• All cell and well details are shown, regardless of what you’ve selected to
Store
• Field details are only available if the BioApplication reports them. Consult
the User’s guide.
Well details
Field details
Cell details
Q. How can I tell what the intensity in a region of the image is? Where can
I tell at what Z position I am?
A. Status Bar/Tray: Lower left corner of GUI. Information is only displayed for
a few seconds.
Windows TaskBar hiding
Status Bar/Tray
Status Bar/Tray info when
pointing at image
Status Bar/Tray info when
moving stage (incl. Focus)
•Top fig.: Window's TaskBar hiding Status Bar/Tray. Right-click empty space on Windows
TaskBar> Properties> Lock…, Auto-hide,  Keep…on top… Thenceforth, move pointer
all the way to edge to get TaskBar.
•Status Tray when pointing at image:
Intensity (x,y)= raw value (uncorrected) intensity of pixel under mouse pointer.
(x.y) are that pixel’s coordinates.
•Status Tray when moving Stage/Focus (z) motor:
•Position = [x,y,z]. Since only displayed transiently, to see again without moving, use 0 (zero)
for Step Size (microns): and click [–] or [+] button.
•Manual Focus buttons are opposite what some think: [–] moves up, toward/into sample; [+]
down, away from sample.
•System’s current z-position is 3rd Position coordinate shown (in example here, it's 9023.500).
©1999-2008 Thermo Fisher Scientific
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27
Exercise: Interacting with Protocol Interactive
Your trainer shall have helped design an Assay Protocol suitable for an
available training sample and your BioApplication interest.
 When you move(d) from the Create Protocol view to the Protocol
Interactive view, 3 new elements of the toolbar become active: Name
them.
Are these 3 settings saved with an Assay Protocol?
 There are 2 top-level panes: Name them, & the 2 sub-panes of each.
 There are two “orphan” tables with bold headings: Name them.
 What do you see in the Status Bar/Tray?
 Load the training plate: How?
 Go to well ___ (B02?), watching the Status Tray. What do you see
there?
• What Field are you in?
• Move two fields left; now what Field?
• Go back to the original Field without using Select Field buttons.
 Acquire an Image in Ch1, foreground achieving 25% dynamic range,
±2%, skipping brightest 0.05%.
What steps were necessary?
 Is the top portion of the image fully visible? If not, make it so.
How?
 Keep this exposure time Fixed for all subsequent images acquired.
How?
 Acquire an Image in Ch2, again keeping the Exposure method Fixed,
but getting 25% of dynamic range.
How?
 Go to another field and get images in Ch’s 1 & 2 in a single click.
How?
 Run the Algorithm to see the results using the current settings in Ch1.
What’s your Object Identification Method and Value?

Can/should you use BackgroundCorrection on this specimen (see
page elsewhere)?
• If so, use your pointer and the Status Bar in the lower left of the
window to determine a “conservative” value.
• Then use the same to determine a suitable FixedThreshold
(Object Identification Method) Value. (Hint: What will
BackgroundCorrection do to the background pixel values in this
raw image).

Adjust the Contrast-Stretching (brightness binning).

Display the Well Features.

Go back to the Raw Image. Then back to the overlay, then raw, then
overlay. Double-click the image, then, on this “image pop-up
window,”…
• Zoom in on a specific region of interest (hint: drag mouse pointer
to draw box around the region).
• In a single click, restore the zoomed image to its 1:1 resolution.
• Keep this pop-up open and click back on the main window.

Change the color of the Rejected_ overlay, and turn on the Rejected_
overlay in Ch2.
Are the changes immediate?

De-Focus the Ch1 image one Manual Step Size, Acquire an Image
Set, then set a Focus Offset (DZ) in Ch2 to restore focus in Ch2, but
not Ch1.

Go to a different well and re-examine the results of your settings after
Running the Algorithm on an Acquired Image Set.

Compare these results to the one you just did. How?
©1999-2008 Thermo Fisher Scientific
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28
Object Identification Methods & Values
 Object Identification is, fundamentally, identifying foreground objects as patches of pixels that
are brighter than background pixels.
• An image comprises pixels; each pixel has an intensity value.
• A frequency distribution histogram of these pixel intensity values is called the “image
histogram” and is one form of information in an image.
• Does an image histogram represent spatial information from the image?
• Analyze image histogram info  intensity threshold  identify objects as foreground vs.
background. Advantage:
• 100’s or 1000’s of images from one assay have variations in foregrounds and/or
backgrounds, so histograms vary from image to image.
• Flexible, systematic method of analyzing each can still  comparable object
identification results amidst the variations.
 Method: IsodataThreshold
• Assumes bimodal histogram, determines T by iteratively computing threshold between
means on left (m L) and right (mR) sides of histogram until T does not change any more.
• Good for bimodal histograms (expect every image to have similar densities of foreground
objects?)
• What happens in fields that are sparse, or fluorescence very low?
• What happens in fields with bright fluorescent outliers, debris, or artifacts?
 Method: TriangulationThreshold
• On image histogram, hypotenuse “drawn” from mode (tallest peak) to max intensity.
• T is value on x-axis where longest normal (perpendicular line) between hypotenuse &
histogram’s curve occurs.
• Good for potentially sparse fields.
• What are possible outcomes when fields are potentially dense?
• What are possible outcomes when fields have bright fluorescent outliers (consider size
of such objects, and “tightness” [kurtosis] of their peaks)?
 Method: 3sigmaThreshold- calculated threshold is 3 standard deviations above the calculated
Mean of the distribution.
 Value: Shifts auto-computed threshold (Tcalc) according to equation below.
mL
mR
Tfinal = Tcalc x (1+Value)
©1999-2008 Thermo Fisher Scientific
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29
Common V3 Assay Params: ObjectSegmentation
Image illustrates some caveats
of ObjectSegmentation. Some of
such debris segments can be
Rejected if Object Selection
Parameters are optimized, but
not always. Also, other caveats
less obvious include oversegmentation of a large or
elongated single object.
ObjectSegmentation
Just when you must!
ObjectSegmentation
 Use it only when you must.
– It’s an approximation, so subject to imperfections.
 When must I use it?
– When a prohibitive majority of cells are simply too crowded to identify individually,
and individual identification is required.
– e.g. let your biological assay needs guide you.
 How else can I deal with clustered cells?
• Seed wells to achieve lower final densities, and/or more appropriately dispersed
cells (some tips are provided in Cellomics’ FAQs).
• Use a different cell type, if possible (a handful of cell types are known for their
tendencies to cluster; e.g., HEK293, HepG2, MCF7).
• Reject them using Object Selection Parameters. In such a case, weigh…
•
[time to acquire more images] versus [risk of inaccurate Object Segmentation];
•
whether Rejection of clusters introduces bias (e.g., target phenotype expressed only in
clusters?). Compare results using [Object Selection Parameters that Reject all but single
cells] versus [Rejection of only/mostly single cells].
 What Value should I use?
• Radius (1/2 the diameter) of a typical Object.
• Clusters of >2 or 3 cells may best be Segmented using a negative Value.
©1999-2008 Thermo Fisher Scientific
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30
Caveats & benefits of different Object Identification Methods and
Assay Parameters
Images show caveats of Isodata (or PeakTriang or other
histogram-based, dynamic) Object Identification Methods,
as well as a caveat of the ObjectSegmentation Assay
Parameter, and a benefit of the BackgroundCorrection
Assay Parameter.
©1999-2008 Thermo Fisher Scientific
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31
I-V-S in non-1° (2°) channel(s)
1° Object Identification
1° Object Validation fail
includes
1° Object Selection
“1° objects”
with
“1° masks”
“Rejected_” overlay,
usually*.
• If “Rejected_” because
of 1°Ch,
• excluded from
Valid_Count
•  from
Selected_Count
•  from %Selected_
• If “Rejected_” because
of 2°Ch,
• included in
Valid_Count
• excluded from
Selected_Count
• reflected in
%Selected_
( = Selected_ 
Valid_Count)
2° channels
(a.k.a. “downstream/dependent”, “ChN”)
“1° object mask”
“2°/sub-object
masks”
MaskModifierChN
(Assay Parameter)
Object Identification
(often + Assay Params)
fail
Object Selection
(a.k.a. “gating”)
Sub-Object Selection
(same list )
Selected_
overlay
no overlay
data results
* BioApp-specific (a few cases
have no “Rejected_” overlay).
Also, overlay must be .
©1999-2008 Thermo Fisher Scientific
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32
Interactive view after Identify Objects
Interactive identificationProtocol
and
Selection
ofmode:
Objects
This is an interactive
Object Selection
Clicking Identified objects
makes them temporarily Selected, adding their values to the
Object Selection Set Statistics
temporary table that can be transferred to the
Object Selection Parameters
that are applied to all objects each time you
Run Algorithm.
To transfer only a subset, click on the row(s) of the Statistics table to
toggle graying out.
Restore Protocol Values is NOT an undo button!! Restores values from
last saved version of the Protocol only!
Common mistakes
1. Notice scrollbars. Unseen Statistics may be transferred!
a) If zeroes transfer as Max for certain Parameters, all objects will
be rejected!
b) Esp. common in BioApps with parent objects + “child” subobjects: 1st one selected determines mode of accumulating only
object or only sub-object Stats.
2. Statistics table is NOT CUMULATIVE! Many actions buttons clear it
(incl. Run Algorithm, Identify Objects itself, Deselect…, Acquire…,
etc.)
©1999-2008 Thermo Fisher Scientific
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33
More
FAQ’s
Q: “Does my Form
Factor let high
field#s get outside
my wells?”
A: When you pass the
borders specified by the
selected Plate Type
Form Factor, the
displayed Field # goes
blank. To check a corner
of the well, select the
max# in the list, then
learn what corner it is by
moving 1 field in each
direction using the
Select Field arrows until
you find the 2 blanking
directions. You can then
use the arrows to step to
the opposite corner
where a blank appears,
etc.
Q: “I want to look at the
same non-1 field# in
many wells on my
plate. Do I have to
select it every time I
change wells?”
A: No. The remedy is
illustrated on this page!
Movement by Select Field
arrows auto-updates this #.
• And/or, a field # can be
selected from list to move
there.
• Starting Field Offset dialog
changes the default value
here, but must move out of well and
back in to “freshen” changes made in
that dialog.
(This temporary interactive Starting
Field Offset reverts to default 1 when
(a) a new Plate Type Form Factor is
selected, (b) each time the software is
started, and (c) may also when an
Assay Protocol is saved, (d) a scan is
completed, or other actions, so
monitor closely).
©1999-2008 Thermo Fisher Scientific
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34
Plate Protocols
Plate ID / Barcode
Plate Protocol
Remaining scan settings not in Assay Protocol:
• Plate Type (Form Factor)
• Scan Area (which wells on plate)
• Starting Field Offset (within each well)
• Store Images (saves space for quick tests)
• Display More (reduced GUI graphics)
Assay Protocol
©1999-2008 Thermo Fisher Scientific
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35
Anatomy of a Generic Cell
Ch 1
Downstream Channels
Object
Circ
Circ
Spots
Ring
Ring
Spots
Primary Object = Nucleus
Special Design for Morphology Explorer
Ch 1
Ch 2
Ch 3 or 4
Object
Process
Members
Primary Object = Whole Cell
©1999-2008 Thermo Fisher Scientific
SpotFibers
(high LWR)
CONFIDENTIAL
SpotFibers
(low LWR)
36
Masks and Modifications
 Mask: the graphic representation of an object that is applied to separate channel in order to
make a measurement in that channel
 Mask Modification: the enlargement or shrinking of the original mask
• Positive values for the Mask Modifier parameter enlarge the mask (dilation)
• Negative values for the Mask Modifier parameter reduce the size of the mask (erosion)
 Three major types of Mask Modifications:
• Primary object mask (MaskModifier)
• Circ Mask (CircModifier)
• Ring Mask (RingDistance and RingWidth)
 The value of the Modifier is the number of pixels to dilate (positive) or erode (negative) the mask
 Adjustment of Mask and Circ Mask looks like this:
Primary Object (Nucleus)
Mask Modifier
+2
Mask Modifier
+4
Mask Modifier
-2.0
Mask Modifier
-4.0
Channel 1
©1999-2008 Thermo Fisher Scientific
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37
Adjustment of Circ & Ring Regions
 Ring Mask is described by 2 parameters: Ring Distance and Ring Width
 Adjustment is always relative to the Primary Object Mask
CELL
CELL
RING
CIRC
PRIMARY
OBJECT
PRIMARY
OBJECT
Ring
Distance
Circ
Modifier
Ring
Width
©1999-2008 Thermo Fisher Scientific
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38
APPENDIX: ArrayScan resolutions & field sizes
6.45 1
6.45 1
6.45 1
6.45 0.63
6.45 0.63
6.45 0.63
Binning
CouplerMag
Acquisition
Camera
Camera
Configuration
Mode
ORCA-ER
Standard
Autofocus
HiRes
ORCA-ER (0.63x)Standard
Autofocus
HiRes
CamPixelSize
MICROMETERS PER PIXEL
ImagePixelSize = CamPixelSize / CouplerMag * Binning / ObjectiveMag
(in MICRONS)
5x
10x 20x 40x
2
4
1
2
4
1
5
2.580
5.160
1.290
4.095
8.190
2.048
10
1.290
2.580
0.645
2.048
4.095
1.024
20
0.645
1.290
0.323
1.024
2.048
0.512
40
0.323
0.645
0.161
0.512
1.024
0.256
5x
10x
20x
40x
5
1,321
1,321
1,321
2,097
2,097
2,097
10
660
660
660
1,048
1,048
1,048
20
330
330
330
524
524
524
40
165
165
165
262
262
262
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
CouplerMag
Binning
Acquisition
Camera
Mode
Standard
Autofocus
HiRes
ORCA-ER (0.63x) Standard
Autofocus
HiRes
Camera
Configuration
ORCA-ER
CamPixelSize
FIELD WIDTH (in microns)
ImageWidth = ImagePixelSize * 1024/Binning
6.45
6.45
6.45
6.45
6.45
6.45
1
1
1
0.63
0.63
0.63
2
4
1
2
4
1
39
APPENDIX:
Field Scan Patterns
“I’m trying to scan fields at the edges of scan-able areas, but
corners are outside the wells.” See text below, right.
 Pattern always counter-clockwise, but field#2’s position depends on
well’s total scan-able field#: System uses width of field at selected
magnification. to compute how many fields fit in Form Factor’s Well
Height (= Well Width), yielding a square grid of fields. When total
field# is odd, field#1 is center; when even, offset by half field.
 Largest square grid in circular well: Well’s diameter = hypotenuse of
grid. Pythagorean theorem says Well Width = Well Height ~ 70% of
diameter.
28 fields x 28 fields = 784 fields
28

28
27 fields x 27 fields = 729 fields
General pattern when TOTAL number of fields is an ODD number (field #2 is left of field #1).
27  27 = 729
729 728 727 726 725 724 723 722 721 720 719 718 717 716 715 714 713 712 711 710 709 708 707 706 705 704 703
25  25 = 625
626 625 624 623 622 621 620 619 618 617 616 615 614 613 612 611 610 609 608 607 606 605 604 603 602 601 702
23  23 = 529
627 530 529 528 527 526 525 524 523 522 521 520 519 518 517 516 515 514 513 512 511 510 509 508 507 600 701
21  21 = 441
628 531 442 441 440 439 438 437 436 435 434 433 432 431 430 429 428 427 426 425 424 423 422 421 506 599 700
19  19 = 361
629 532 443 362 361 360 359 358 357 356 355 354 353 352 351 350 349 348 347 346 345 344 343 420 505 598 699
17  17 = 289
630 533 444 363 290 289 288 287 286 285 284 283 282 281 280 279 278 277 276 275 274 273 342 419 504 597 698
15  15 = 225
631 534 445 364 291 226 225 224 223 222 221 220 219 218 217 216 215 214 213 212 211 272 341 418 503 596 697
13  13 = 169
632 535 446 365 292 227 170 169 168 167 166 165 164 163 162 161 160 159 158 157 210 271 340 417 502 595 696
11  11 = 121
633 536 447 366 293 228 171 122 121 120 119 118 117 116 115 114 113 112 111 156 209 270 339 416 501 594 695
9

9
= 81
634 537 448 367 294 229 172 123 82 81 80 79 78 77 76 75 74 73 110 155 208 269 338 415 500 593 694
7

7
= 49
635 538 449 368 295 230 173 124 83 50 49 48 47 46 45 44 43 72 109 154 207 268 337 414 499 592 693
5

5
= 25
636 539 450 369 296 231 174 125 84 51 26 25 24 23 22 21 42 71 108 153 206 267 336 413 498 591 692
3

3
=
637 540 451 370 297 232 175 126 85 52 27 10
9
8
7
20 41 70 107 152 205 266 335 412 497 590 691
638 541 452 371 298 233 176 127 86 53 28 11
2
1
6
19 40 69 106 151 204 265 334 411 496 589 690
639 542 453 372 299 234 177 128 87 54 29 12
3
4
5
18 39 68 105 150 203 264 333 410 495 588 689
9
2

2
=
4

4
= 16
640 543 454 373 300 235 178 129 88 55 30 13 14 15 16 17 38 67 104 149 202 263 332 409 494 587 688
6

6
= 36
641 544 455 374 301 236 179 130 89 56 31 32 33 34 35 36 37 66 103 148 201 262 331 408 493 586 687
8

8
4
General pattern when TOTAL number of fields is an EVEN number (field #2 is right of field #1).
= 784
784 783 782 781 780 779 778 777 776 775 774 773 772 771 770 769 768 767 766 765 764 763 762 761 760 759 758 757
= 64
642 545 456 375 302 237 180 131 90 57 58 59 60 61 62 63 64 65 102 147 200 261 330 407 492 585 686
643 546 457 376 303 238 181 132 91 92 93 94 95 96 97 98 99 100 101 146 199 260 329 406 491 584 685
26

26
= 676
10  10 = 100
677 676 675 674 673 672 671 670 669 668 667 666 665 664 663 662 661 660 659 658 657 656 655 654 653 652 651 756
24

24
= 576
678 577 576 575 574 573 572 571 570 569 568 567 566 565 564 563 562 561 560 559 558 557 556 555 554 553 650 755 12
 12 = 144
644 547 458 377 304 239 182 133 134 135 136 137 138 139 140 141 142 143 144 145 198 259 328 405 490 583 684
22

22
= 484
679 578 485 484 483 482 481 480 479 478 477 476 475 474 473 472 471 470 469 468 467 466 465 464 463 552 649 754 14
 14 = 196
645 548 459 378 305 240 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 258 327 404 489 582 683
20

20
= 400
680 579 486 401 400 399 398 397 396 395 394 393 392 391 390 389 388 387 386 385 384 383 382 381 462 551 648 753 16
 16 = 256
646 549 460 379 306 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 326 403 488 581 682
18

18
= 324
681 580 487 402 325 324 323 322 321 320 319 318 317 316 315 314 313 312 311 310 309 308 307 380 461 550 647 752 18
 18 = 324
647 550 461 380 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 402 487 580 681
16

16
= 256
682 581 488 403 326 257 256 255 254 253 252 251 250 249 248 247 246 245 244 243 242 241 306 379 460 549 646 751
20  20 = 400
648 551 462 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 486 579 680
14

14
= 196
683 582 489 404 327 258 197 196 195 194 193 192 191 190 189 188 187 186 185 184 183 240 305 378 459 548 645 750
22  22 = 484
649 552 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 578 679
12

12
= 144
684 583 490 405 328 259 198 145 144 143 142 141 140 139 138 137 136 135 134 133 182 239 304 377 458 547 644 749

24  24 = 576
650 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 678
10
10
= 100
685 584 491 406 329 260 199 146 101 100 99
98
97
96
95
94
93
92
91 132 181 238 303 376 457 546 643 748
8

8
= 64
686 585 492 407 330 261 200 147 102 65
64
63
62
61
60
59
58
57
90 131 180 237 302 375 456 545 642 747
26  26 = 676
651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677
6

6
= 36
687 586 493 408 331 262 201 148 103 66
37
36
35
34
33
32
31
56
89 130 179 236 301 374 455 544 641 746
4

4
= 16
688 587 494 409 332 263 202 149 104 67
38
17
16
15
14
13
30
55
88 129 178 235 300 373 454 543 640 745
2

2
=
689 588 495 410 333 264 203 150 105 68
39
18
5
4
3
12
29
54
87 128 177 234 299 372 453 542 639 744
690 589 496 411 334 265 204 151 106 69
40
19
6
1
2
11
28
53
86 127 176 233 298 371 452 541 638 743
4
3

3
=
9
691 590 497 412 335 266 205 152 107 70
41
20
7
8
9
10
27
52
85 126 175 232 297 370 451 540 637 742
5

5
= 25
692 591 498 413 336 267 206 153 108 71
42
21
22
23
24
25
26
51
84 125 174 231 296 369 450 539 636 741
7

7
= 49
693 592 499 414 337 268 207 154 109 72
43
44
45
46
47
48
49
50
83 124 173 230 295 368 449 538 635 740
9

9
= 81
694 593 500 415 338 269 208 155 110 73
74
75
76
77
78
79
80
81
82 123 172 229 294 367 448 537 634 739
11

11
= 121
695 594 501 416 339 270 209 156 111 112 113 114 115 116 117 118 119 120 121 122 171 228 293 366 447 536 633 738
13

13
= 169
696 595 502 417 340 271 210 157 158 159 160 161 162 163 164 165 166 167 168 169 170 227 292 365 446 535 632 737
15

15
= 225
697 596 503 418 341 272 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 291 364 445 534 631 736
17

17
= 289
698 597 504 419 342 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 363 444 533 630 735
19

19
= 361
699 598 505 420 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 443 532 629 734
21

21
= 441
700 599 506 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 531 628 733
23

23
= 529
701 600 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 627 732
25

25
= 625
702 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 731
27

27
= 729
703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730
 On a micron scale, plates may vary widely from specs,
even in the same lots. Seating on stage and
temperature expansion/contraction also cause
variation. If you have verified…
©1999-2008 Thermo Fisher Scientific
• system is calibrated,
• Form Factor’s First Well X&Y optimally approximates A1’s
center,
• Well Pitch X&Y are optimally approximate across a plate (use
Status Bar/Tray coordinates in Protocol Interactive noting DX’s
and DY’s for A1 vs. distal and corner wells)…
…may be advisable to decrease Well Width & Height
CONFIDENTIAL
40
Appendix: Data Hierarchy and Tiers
Many terms in HCS have historical roots that are blends of
photographic imaging and cell-based screening. Often the
vocabulary works well, but is not a restriction on capabilities.
 Plate level features- E.g., exposure times, z-offsets,
Reference Levels. This “plate” structure is rooted in cellbased assays in multi-well microtitre plates. In HCS, it can
mean a microscope slide, too.
 Well level features- E.g., Z-position, CellCount,
MEAN_ObjectAvgIntenCh2, SD_ObjectTotalInten,
%HIGH_RingSpotCountCh2. A well is merely a subdivision
of a plate. It constitutes a container for materials delivered to
it. It can also be a subdivision of a micro-slide.
 Fields- A term borrowed from microscopy’s “field of view”.
When a Field size is smaller than the image-able surface of
a well, multiple fields may be imaged per well. Some
BioApplications report Field features. All report Cell Features
linked to Wells+Fields.
 Cell level features- E.g., ObjectSizeCh1,
RingSpotCountStatusCh2. Historically, it was cells that were
plated into wells of a plate. In HCS, the fundamental unit of
analysis (primary Object) is most often a Cell, and multiple
features of it are measured. Some of these features may be
sub-objects (e.g., spots, micronuclei, perinuclear rings,
neurites), though not necessarily. So, Cell Features are a
class in this hierarchy, but Cell Features may correspond to
properties of colonies or whole organisms (e.g., C. elegans).
 “Intrinsic/raw” Cell Features- e.g., RingSpotCountCh2 is
a mere count of how many spots have been ascribed to an
object based on sub-objects (spots) identified by Ch2’s label
within a sub-compartment (ring) of an object.
 “Simple Statistical” Well Features based on
intrinsic/raw Cell Features- e.g.,
MEAN_TargetAvgIntenCh2 represents that mean response
of that well’s population of objects. That mean has a
SD_TargetAvgIntenCh2 about it, too. ObjectCount is another
example; it’s the sample size, n, on which the mean, stdDev,
etc., are based.
 Status Cell Features- e.g., RingSpotCountStatusCh2. In
some BioApplications, an object can be assigned to one of
some Cell Feature phenotypes. For example, if an object’s
TargetIntenCh2 exceeds the corresponding Reference
Level, it’s classified by TargetIntenStatusCh2=1; else =0.
 Population Characterization Well Features based on
Status Cell Features- Rather than a metric of a mean
response, these report the fraction of a well’s sampled
population that fall in a phenotypic class. E.g.,
%HIGH_TargetAvgIntenCh2 reports what percentage of the
well’s sampled population exceeded a minimum Response
Level. It’s simply computed by tabulating the corresponding
Status frequencies.
 The meaning & legitimacy of intrinsic/raw features depends
on the quality of input settings that generate them. The
same is true of Population Characterization features. the
%HIGH value is not meaningful if relevant Reference Level
inputs were not used.
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
41
Quiz 1 Solution
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
42
Quiz 1 Solution
©1999-2008 Thermo Fisher Scientific
CONFIDENTIAL
43