Download BD FACSDiVa Software

BD FACSDiVa 4.1
An introduction
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
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.
The main layout
The Browser
Management of your
experimental data and settings
Worksheet
The area for plots, histograms,
Statistics and everything else
Inspector
Instrument
Always shows the properties
Voltage settings, Threshold,
Compensation of selected objects
„Floating windows“ (always on top)
Sort control
Defines all settings for
Frequency, amplitude, and
Stream deflection
Acquisition Status
Acquisition Control
Counters for events per second,
Starts and stops Acquisition and
Aborted events,
data recording.
Elapsed time
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.
Log-in
• Each time the software is started,
the log-in menu appears:
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
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!
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.
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.
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. Predefinition is not required.
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.
DotPlots
The DotPlot can be activated by
clicking onto the white border area.
The Inspector window shows
informations about the activated
plot(s):
Acquiring Data
Data acquisition is controlled via the „Acquisition Control“ window
Data acquisition is started and stopped with the „Acquire“ Button
During acquisition, data saving is initiated by pressing the
„Record“ button.
The „Restart“ button is available during acquisition to restart
the acquisition and/or data recording.
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
The instrument control
The „Instrument“ window controls all parameters of
the instrument. It consists of different tabs, which all
relate to specific instrument functions.
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!
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.
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.
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.
Instrument - Ratio
The instrument can
automatically calculate the
ratio between any two
parameters, even from
different lasers.
Calcium flux:
Ratio Fluoro3/FuraRed
Ratio Indo(bound)/Indo(Free)
Absorption ratio for dyes
excitable by different
wavelengths
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.
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.
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.
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".
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
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
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!
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.
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.
Exporting Templates
After choosing "Export" the user can select the folder in a
drop-down box.
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.
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.
Example 1 – Normal log-scale
Example 1 – Bi-exponential
Example 2 – Normal log-scale
Example 2 – Bi-exponential
Example 3 – Normal log-scale
Example 3 – Bi-exponential
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 biexpoential display.
Correct Compensation
can be nicely seen
in the lower panel.
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 biexponential 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.
"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.
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.
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.
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.
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!
Enough of the Talking
Just Do It!