Aap and Profiler Brochure

7/29/2019 Aap and Profiler Brochure

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Union Biometrica, Inc. y 84 October Hill Rd, Holliston, MA 01746 USA y Tel: +1-508-893-3115 y Fax: +1-508-893-8044

European Office Cipalstraat 3 B-2440 , Geel, Belgium y Tel: +32 (0) 14-570628 y Fax: +32 (0) 14-570621

Email: [email protected] y Internet: http://www.unionbio.com

Union Biometrica now offers two optional packages to significantly expand dataacquisition and analysis capabilities with your COPAS Large Particle FlowCytometer.

Advanced Acquisition Package

The Advanced Acquisition Package (AAP) includes advanced electronics and enhanced softwareanalytical tools that permit significantly greater data acquisition rates, increased data resolution, andobject flow rates.

Higher signal resolution is achieved with 16-bit data resolution (1:65536) to offer 32 times moredetail in intensity and size than the standard COPAS software. Increased resolution makes itpossible to distinguish size differences as small as 5 m.

Increased sensitivity -- AAP also offers the ability to detect smaller objects and sense lower

intensity fluorescent signals.

Higher throughput -- The increasedobject acquisition rate permits samplesto be run four times faster thanstandard rates.

Additional software features: improveddata displays including linear andlogarithmic scaling; extended sortingcapabilities; fluorescence compensa-tion for increased fluorescencediscrimination; user-defined mathe-

matical functions for advanced real-time data manipulation; and expandeddata storage capabilities fullycompatible with FCS 3.0 standard.

Advanced Acquisition andProfiler IIOptions --

Expand the Capability of Your COPAS

System by Increasing Speed, Sensitivity andResolving Positional Fluorescence Information

27um

42um

16um

10umAdvanced

AcquisitionPackage

Standard COPASsoftware

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AcquisitionPackage

Standard COPASsoftware

Figure above illustrates improved resolution

between 10 and 16 particles by using AAP.


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Profiler II

The Profiler II option package, which includes and builds upon AAP, can take your sorting and analysisdecisions to an entirely new level.

What if you could . . .

Record up to 8,000 optical slices per modelorganism (or other object) thereby elucidatingcell or organ level positional information?

Detect weak fluorescence signals even in thepresence of much stronger signals?

. . . or in the presence of background auto-fluorescence?

Both the standard COPAS software and the moreadvanced AAP provide a single integrated signalmeasurement for each parameter of an object, andthese signals are used for analysis and for sortingdecisions.

The Profiler II option package (which includes AAP)takes this to the next level by simultaneouslydetecting and recording up to 8,000 data points perobject for each of the four optical parameters:extinction and three fluorescence channels.

Profiler II offers:

4 channels of simultaneous profiles, one for each of the 4 optical parameters (Extinction +3

channels of fluorescence) Independent channel scaling

Enhanced sorting based on user-definable sort criteria for:o Peak heighto Peak widtho Relative peak locationo Number of peaks for each parameter

Banded Drosophila Embryo (head on left)

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Nematode with green fluorescent marker expressing

in both head and male tail.

Profiler II digitizes the object into a succession ofpeaks and valleys that directly trace thefluorescence intensity of the object as it passesthrough the flow cell. Profiler II also includesadvanced imaging to graphically and numerically

display subtle variations in fluorescence andextinction intensity along the length of an object.

The result is an optical profile of each objectgraphically showing the location and intensity of allfour optical parameters. Profiler II also enablesusers to optimize COPAS systems by visualizingdata, resulting in better detection of strong versusweak signals.

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Detection of Weak Localized Fluorescencevs. Auto-fluorescence (Background)

In the figures shown at right, when using the basicCOPAS for this type of experiment, notice that the twopopulation types are not differentiated on the scatterplot graphing FLU1 (green fluorescence) and FLU2(red fluorescence or auto fluorescence). The Profiler IImodule however, provides enhanced sensitivity and

enables detection of a weak localized fluorescencesignal even against an auto-fluorescence background,showing the two population types on the line graphs onthe right.

This is an example where sorting can be done on thebasis ofPeak Height above a Threshold.

Examples of how some researchers are using Profiler

Strong Red signal

Weak Yellow

Strong Red signal

Weak Yellow

Mapping the Fluorescence Profileof a Zebrafish

Axial profile (red-fluorescence and blue-extinction, shown at right, of a stained 4-day old wild type zebrafish larva overlaidon the corresponding image.

This is an example where sorting could bebased on Number of Peaks for eachParameter.

Detection of weak fluorescent signalseven in the presence of much strongersignals

The figure shown at left demonstrates that withoutthe positional data, the presence or absence of theweaker signal would just be lost or buried in theaverage presence of the stronger signal.

This is an example of sorting based on Relative

Peak Location.

C. elegans N2 Profile

PY1089 Profile

FLU2

FLU1

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Email: [email protected]

y Internet: http://www.unionbio.com

Profiling Expression ThroughoutDevelopment

For a single gene, the change in expressionthrough development can be shown in terms ofthe amount of expression, when expressionbegins, or dim expression. Figure at left showsexamples of these expression terms: amount of

pharyngeal expression displayed increases,expression of the vulvae begins to appear on thegraph at a certain maturity, and weakfluorescence of the anal muscle is alsodetectable using this analysis method. A moreextensive analysis is described in: DuPuy, et al.,Nature Biotechnology, 25, 663-338, 7 May 2007.gy, 25, 663-338, 7 May 2007.

Three-Color Profiling

The KS329 strain (shown at left) was constructed andkindly provided by Yanping Zhang and Mike Hermanof Kansas State U. The GFP expression in the headneurons and the yellow protein expression in the

vulva can be used as landmarks for assessing theposition of the cells expressing red fluorescingprotein from the mab-5 promoter.

This is also another example of sorting based onRelative Peak Location.

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Hydrogel beads as a

matrix for cell growth

Profilerdata

L1 larvae

Adults

Size of pharyngeal

signal increases

with age

Vulvae appear

with maturity

Weak signal,

anal muscle

age

Worm profiles stacked

up head (left) to tail in

order of increasing size

(age).

Rev: May-2008

Cell clusters growing inside Hydrogel beads

Beads formed around cells can be inspected by Profiler II. COPAS can then be used to sort beads containingonly one cell. As these cells grow & divide they form monoclonal clusters. (Courtesy of S.Panke and M.Walser,ETH, Zurich.) Here sorting can be accomplished based onPeak Width as a sign of cell growth.

edges of bead

Cell cluster

Cells growing inside bead sorted by COPAS

Hydrogel beads as a

matrix for cell growth

Profilerdata

edges of bead

Cell cluster

Profilerdata

edges of bead

Cell cluster

Cells growing inside bead sorted by COPASCells growing inside bead sorted by COPAS

Aap and Profiler Brochure

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