BD LSR II and FACSDiVa Software. Dr. Jens Fleischer, Basel Dr. Norbert Leclere,, Berlin

Transcription

1 BD LSR II and FACSDiVa Software, Basel Dr. Norbert Leclere,, Berlin

2 Overview Electronics Covers Fluidics Connectors Controlpanel Sample Port On/Off

3 The Control Panel

4 Ease of Use The basic procedures to use the BD LSR II are quite similar to those for FACScan and FACSCalibur Controlpanelp with RUN, STDBY and PRIME. Flowrates LOW, MED and HI adjustable Sample injection port with sleeve needle and tube support arm

5 Optics The BD LSR II in your lab has three lasers: 488 nm Coherent Solid-State State (DPSS) 635 nm JDS Uniphase HeNe 407 nm Coherent Vioflame Diodelaser Using all three excitation line allows the user to select the best dyes for the applications

6 Optics The BD Octagon technology directs the collected light via fibres into dedicated detection blocks per laser:

7 Simple schematics

8 What goes where

9 Default Filters and Mirrors Changes in the configuration should be done with care. Use the charts in the lab to check the setup before starting your measurements!

10 Sheath and Waste The LSR II is equipped with an automated FACSFlow Supply System 20 L FACSFlow cubitainer as Sheath 20 L empty cubitainer for waste Level sensors with audible alarm Easier to use than refilling and emptying small tanks all 3 hours

11 Startup Check Sheath cubitainer, replace if necessary Check Waste cubitainer, replace if necessary First, turn on the BD FACSFlow Supply System Second, turn on the BD LSR II Vent sheath tubing at the side of the instrument.. Be careful not to spill PBS over monitor and keyboard! Take off the cleaning tube Press PRIME three times Put a tube with DI onto the SIP and run it for 10 minutes in HI and RUN System ready for measurements

12 Shutdown Run a tube with FACSClean for 10 minutes in HI Put the tube support arm to either side and empty the remains of the FACSClean solution This will disinfect the needle and the outer sleeve Run a tube with FACSRinse 10 minutes in HI Put the tube support arm to either side and empty the remains of the FACSRinse solution until appprox.. 1 ml is left. This will wash out dead cells and debris from the needle and the outer sleeve Put a tube with DI onto the SIP and out the instrument to STDBY Turn the BD LSR II and the FACSFlow Supply System off

13 FACSDiVa Software The new digital acquisition platform introduces a complete new software concept from BD The software runs on a Windows XP computer,, Macintosh systems are no longer available

14 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.

15 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, of selected objects Compensation

16 Floating windows (always( on top) Acquisition Status Acquisition Control Counters for events per second, Starts and stops Acquisition and Aborted events, data recording. Elapsed time

17 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.

18 Log-in Each time the software is started, the log-in menu appears:

19 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

20 How to start? New experiments can be created via the associated browser icon or via the Menu bar. New Experiment Folders can be used to sort data sets. The folders exist only in the database!

21 Global Worksheets Be default Global WorkSheets offer the possibility to re 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 datasets you can create multiple global sheets.

22 A new experiment A new experiment contains a standard 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" Template" for your Experiment (created from your older experiments).

23 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 associated tubes. Pre-definition is not required.

24 The first plots At the top of the Worksheet Window you will find the "Tool Palette" Clicking on the DotPlot DotPlot icon activates the creation of DotPlots.. Just click once on the worksheet to create a dotplot of standard-size. 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.

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

26 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.

27 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

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

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

30 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.

31 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.

32 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.

33 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

34 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.

35 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.

36 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.

37 Working Without Global Worksheets In order to switch to the Standard WorkSheets, 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. Standard Worksheets are good for a long list of tubes with different analysis requirements, or if you want to put plots of different tubes onzto the same print page However, be careful which mode is currently active: : A green tab means "Global Worksheet", a grey tab Normal Worksheet".

38 User Preferences Tube specific Worksheet: Each Tube gets its 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: Always 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

39 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

40 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 can occur that you get black dots on black background, always open the Population Hierarchy to check this!

41 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.

42 Exporting Templates After choosing "Export" the user can select the folder in a drop-down box. If you type in a new name, the folder is generated.

43 New Inspector view for recorded tubes The new inspector view for recorded tubes shows the instrument settings in a different way compared to the live settings shown before It is not possible to change the volate settings or to delete / add parameters AFTER acquisition It is possible to chnage compensation offline

44 New scaling In the digital electronics there are no errors in the determination of the logarithmic value for low intensities. Indeed, it always looks up the mathematically correct value. However, the logarithmic scale can only display numbers from 26 to in the 4-decade display (which is recommended). In the 5-decade display it is 2.6 to channels. The electronics actually calculates intensities from 2600 to Everthing below 26 in the 4-decade scaling therefore is an "on" 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.

45 Example 1 Normal log-scale

46 Example 1 Bi-exponential

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

52 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. Population borders 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.

53 Density Plots Densities and Contour Plots can be generated with the same icon Final formatting is done via the Inspector Window. Note that density plots can also be calculated on bi-exponential scaling, showing the population distribution.

54 Copy and Paste... The FACSDiVa Software now supports Copy and Paste to generate PowerPoint Presentations or Word- Documents (or any other format) with flow cytometry data. Alternatively, Plots can be exported as graphics files. However, only JPG-format is supported, leading to loss of quality if resized after import to PowerPoint.

55 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 need to create four different polygon regions.

56 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.

57 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!

58 Final recommendation: Just Do It!