BD FACSDiVa 4.1

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BD FACSDiVa 4.1

• An introduction

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FACSDiVa Software

• The new digital acquisition platform introduces a
  complete new software concept from BD
• The software runs on a Windows2000 computer,
  Macintosh systems are no longer available

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FACSDiVa Software

• The software uses a database server software to
  manage the flow cytometry data.
• All data, plots, gates, instrument settings, and
  statistics are saved inside the database.
• The user cannot specify any path for saving,
  everything is done automatically in the
  background.

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The main layout
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Floating windows (always on top)
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Log-in

• The softwares requires a user log-in. Users can
  only see and manipulate their own data.
• Only the Administrator can define new users and
  their rights.
• The Administrator can Share data with other
  users.

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Log-in

• Each time the software is started, the log-in
  menu appears

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Log-in

• Depending on the log-in, the user can only see
  the appropriate datasets

A user can only see his own data.
The Administrator can see everything
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How to start?

• All data are stored in an Experiment inside the
  database. Each time you start a new measurement,
  you either have to create a new Experiment or use
  an old one as template for new data.
• A new experiment can be created via the
  associated browser icon or via the Menu bar.

New Experiment Note that you also can create
folders like on a harddisk for better data
management. The folders exist only in the
database!
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Global Worksheets

• Global Templates offer the possibility to
  re-use plots like in CellQuest.
• When pressing the NEXT button, the same plots
  are used for data display.
• Also saved data can be loaded into plots.
• For different views on the same data you can
  create multiple global sheets.

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A new experiment

• A new experiment contains a standard instrument
  setting and a single tube in a so-called
  specimen.
• When selecting "New Experiment" from the menu bar
  you can select a "Template" for your Experiment
  (created from your older experiments).

A selected and activated element is always
highlighted in blue in the browser window.
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Specimens

• The FACSDiVa software uses Specimens to
  associate a series of tubes as a pattern or
  panel.
• Existing Specimens can be used as a template for
  the next, similar tube pattern.

It is possible to pre-define a tube list, but
you can always duplicate a specimen after
measurement of all tubes. Pre-definition is not
required.
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The first plots

• At the top of the Worksheet Window you will find
  the "Tool Palette"
• Clicking on the DotPlot icon activates the
  creation of DotPlots. Just click once on the
  worksheet to create a dotplot of standard-size.
  Dragging and holding the mouse creates a dotplot
  with the size of cour choice.
• In addition do dotplots, the software offers
  contour/density plots and histograms.

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DotPlots
The DotPlot can be activated by clicking onto the
white border area.
The Inspector window shows informations about
the activated plot(s)
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Acquiring Data

• Data acquisition is controlled via the
  Acquisition Control window

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Acquiring data
The acquisition control window shows also the
tube name, an event counter and the elapsed time
since the acquisition has started. The user also
controls which events are counted and
stored Storage Gate Which events are recorded
and saved Stopping Gate Which cells are
counted Events To Record How many cells should
be recorded (in the Stopping Gate) Events to
Display How many events are shown on-line in
the acquisition plots
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The instrument control

• The Instrument window controls all parameters
  of the instrument. It consists of different tabs,
  which all relate to specific instrument
  functions.

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Instrument - Parameters

• The Parameter tab shows the instrument parameters
  and their settings. The user can change the PMT
  voltage, the linear/logarithmic settings, and can
  select whether Area, Height and Width for the
  parameter should be acquired. The digital
  electronics has no parameter limitation!

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Instrument Parameters
The Add and Delete buttons allow the user to
delete unwanted parameters, thus saving space for
the recorded data sets. Note that the parameters
correspond to fluorochrome names, not channel
numbers. The association between channels, lasers
and fluorochromes is explained later.
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Instrument - Threshold
The digital electronics allows the user to define
multiple thresholds. Thresholds can be set on
any parameter from any laser. Threshold can be
connected with logical OR and logical AND, so
that either one of the threshold conditions is
fulfilled or all conditions must be met by an
event.
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Instrument - Compensation
The digital electronics allows the compensation
of all parameters against each other. Thus, the
compensation starts to get increasingly complex
in multicolor applications. The software allows
Autocompensation if single color stains are
available, reducing the workload for instrument
setup during multicolor experiments.
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Instrument - Ratio
The instrument can automatically calculate the
ratio between any two parameters, even from
different lasers. Calcium fluxRatio
Fluoro3/FuraRed Ratio Indo(bound)/Indo(Free) Absor
ption ratio for dyes excitable by different
wavelengths
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Instrument - Lasers
Users should in most cases not change these
settings. All values are setup during
installation of your machine. Please use this tab
only if a BD representative asks you to do so.
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Instrument - Status
The Status tab shows any errors that the
instrument encounters. If an error occurs, the
color of the tab changes to red, signalling that
something is wrong. The Clear button at the lower
right corner deletes the warning messages.
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Acquisition Status
The Acquisition Status window included a counter
for events over the threshold (Events/Second) and
a sum counter (all events since Acquire has been
pressed). The Abort Rate and Abort Count show how
many cells are lost from the analysis because
they were too close together to be separated as
single events. Processed Events shows how many
cells have been analysed and is always close to
the Threshold Count. Elapsed Time shows how long
you have been acquiring the data.
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Working Without Global Worksheets

• In order to switch to the "old" DiVa way of
  working, click on the small green triangle in the
  upper left corner of the tool palette.
• Now you can create different plots for each tube.
  In addition, each tube now has its own set of
  population.
• For a long list of tubes with different analysis
  requirements the old way may be the better way
  for data analysis.
• However, be careful which mode is currently
  active A green tab means "Global Worksheet", a
  grey tab "Classic Worksheet".

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User Preferences

• Tube specific Worksheet Each Tube gets ist own
  Worksheet
• Start Acquisition on pointer change Saves one
  mouseclick on Acquisition
• Show GUID Unique ID for each tube (useful?)
• Remove Tube-Specific Instrument Settings on
  Duplicate Use the same Instrument Setting in an
  Experiment, even when one tube is changed.
• Save Analysis after Recording Copies all Plots
  from the Template onto the normal Worksheet after
  Recording
• Automatically Update Instrument Setting Use
  always the latest setup even in "old" experiments

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User Preferences

• Interval Gate Default Color Histogram Gates per
  Default have no color to reduce clutter
• Quadrant Gate color Quadrants are also not
  colored to reduce data cluttering

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User Preferences

• Default Background Color since version 4.1 is
  White (equal to printout)
• Can be set to any color by the user
• In some instances, especially when updating
  existing databases, it may occur that you get
  black dots on black background, always open the
  Population Hierarchy to check this!

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User Preferences

• Each user can define his own default templates
  for new experiments.
• Default Global Worksheet BD recommends to use
  the global worksheet as standard method to reduce
  screen clutter.

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Templates
In the "BDEXPORT\Templates\Experiment" folder
users can add additional folders as necessary.
They are NOT created by the software. However,
the software uses the folders and displays them
correctly when exporting or importing templates.
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Exporting Templates
After choosing "Export" the user can select the
folder in a drop-down box.
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New Inspector view for recorded dtubes

• The new inspector view for recorded tubes shows
  the instrument settings in a different way.
• It is no longer possible to change the voltages
  or thresholds on saved data.
• In the previous versions the corresponding
  buttons were just "greyed out", still leading to
  confusion.
• As before, changing linear and logarithmic
  display and changing the compensation is still
  possible.

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New scaling

• In the digital electronics there are no errors in
  the determination of the logarithmic value for
  small intensity values. Indeed, it always looks
  up the mathematically correct value.
• However, the logarithmic scale can only display
  numbers from 26 to 260.000 in the 4-decade
  display (which is recommended).
• In the 5-decade display it is 2.6 to 260.000
  channels.
• The electronics actually calculates intensities
  from 2600 to 260.000. Everthing below 26 in the
  4-decade scaling therefore is an "on-axis" event,
  as there is no logarithmic value to plot.
• The "way out" Use a different axis scaling. The
  "bi-exponential" sclaing is linear at the
  beginning and converts to logarithmic at a
  certain point.

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Example 1 Normal log-scale
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Example 1 Bi-exponential
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Example 2 Normal log-scale
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Example 2 Bi-exponential
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Example 3 Normal log-scale
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Example 3 Bi-exponential
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Bi-Exponential

• Bi-exponential scaling shows also nicely the
  effect of compensation.
• The upper panel is over-compensated, the effect
  is clearly visible in the bi-expoential display.
• Correct Compensation can be nicely seen in the
  lower panel.

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Bi-Exponential Where to activate?
The bi-exponential scaling is activated for each
plot in the Inspector. Thus, it is possible to
have bi-exponential and normal log scale on the
same page. Populationborders drawn in
bi-exponential displays cannot be displayed in
"normal" plots, but are always calculated on
bi-exponential scaling. The same holds true for
the borders of population from "normal" log
scales. They are not drawn in bi-exponential
plots, but calculated based on log scale.
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"Real" Density Plots
In previous versions the "density plots" were
just colored contour plots. Now "real" densities
can be calculated and plotted. Note that density
plots can also be calculated on bi-exponential
scaling, showing the population distribution.
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Copy and Paste...

• Finally, the FACSDiVa Software now supports Copy
  and Paste to generate PowerPoint Presentations or
  Word-Documents (or any other format) with flow
  cytometry data.
• It is still possible to export the graphics as
  files, but still only as JPG files in the screen
  resolution.

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Hinged Quadrants
In many cases rectangular quadrants are not the
correct solution. The FACSDiVa Software now
offers "hinged" quadrants, which can be moved
individually to gate the cells of interest
without the neeed to create four different
polygon regions.
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Hinged Quadrants
In many cases rectangular quadrants are not the
correct solution. The FACSDiVa Software now
offers "hinged" quadrants, which can be moved
individually to gate the cells of interest
without the neeed to create four different
polygon regions.
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Offset Quadrants

• Sometimes it may be useful to offset one side of
  the quadrants to adjust clearly overlapping
  populations.
• You can either offeset quadrants or use the
  hinged version. It is not possible to use both
  versions simultaneously!

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Enough of the Talking
Just Do It!