1. These particles have something in common
Blood cells
Chromosomes
Algae
Protozoa
Certain parameters of these particles can be measured with a flow cytometer 1

2. Which parameters can be measured?
the relative size (Forward Scatter - FSC)
the granularity or complexity (Side Scatter - SSC)
the fluorescence intensity (FL1, FL2, up to FL X) 2

3. Characteristics of FSC and SSC
Coherent lightsource (488 nm)
Forward scatter Cell size (488 nm)
Side scatter Granularity (488 nm)
Forward scatter (FSC)
measured along the axis of the incoming light
proportional the the cell size / cell surface (only true for perfect round cells)
Side scatter (SSC)
measured in 90° direction to the excitation light
proportional to cell „complexity“ or granularity 3

4. An example of light scatter:
Granulocytes
Side scatter
Monocytes
Lymphocytes
Debris
Forward scatter 4

5. Fluorescence
l=488 nm
l=530 nm
Excitation light
Emission light
The fluorochrome molecule absorbes the energy of the incoming light
It releases the absorbed energy by:
vibration and dissipated heat
emission of a photon with a higher wavelength ( = less energetic) 5

6. Fluorescence intensity Relative fluorescence intensity
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
FITC
FITC
101
102
103
104
Relative fluorescence intensity 6

7. Parts of a flow cytometer
Fluidics
Provide a constant stream of sheath
Transport the sample to the interrogation point
Arrange and focus the cells to the laser intercept
Optics
Focus the excitation light
Collect the emitted light
Electronics
Convert the optical signals into electronic signals
Send the signals to the analysis computer
Computer
Display data graphically
Control instrument settings

8. What a flowcytometer is
Very basically, a flow cytometer is an automated fluorescence microscope (in fact, that is how the first prototype instruments looked like).
Like a microscope, some adjustments have to be made to optimally illuminate and collect the light.

9. The basic microscope
In a standard microscope, the operator uses the XY-stage to screen the sample and detect cells of interest.

10. The automated Microscope
Detector
& Counter
This primitive diagram shows the principle: Cells are passing the microscope objective, and an electronic circuit decides whether the cells is fluorescent or not. This is how a flow cytometer works!
Waste
Sample

11. Basic fluidics of the FACSAria
Fluidics Cart
Plenum
Cuvette
Pressure
Sheath
Sample
tube
Waste

12. Hydrodynamic focussing in the cuvette
Sample
Sample
Sheath
Sheath
Sample pressure low, small core stream. Good for DNA analysis
High sample pressure, broader core stream.
Bad for DNA analysis 1 1

13. Summary
Pressure (= Sheath Pressure) drives the sheath buffer through the cuvette, and the higher pressure in the sample tube (= Sample Differential) delivers the sample to the cuvette.
In the cuvette the principle of hydrodynamic focussing arranges the cells like pearls on a string before they arrive at the laser interception point for analysis
Hydrodynamic focussing cannot separate cell aggregates! Flow cytrometry is a technique that requires single cell suspensions 2

14. Basic optics
Somehow the light from the laser(s) must be directed to the cuvette to illuminate the cells.
At the same time, the emitted light must be collected to analyse the signals from the cells.
The alignment of the system is performed during installation. 3

15. Basic optics
A system of prisms and lenses directs the laser light to the interrogation point in the cuvette

16. Basic Optics
The emitted light induced from each laser is focussed onto separate glass fibers.

17. Optical filters Longpass Shortpass Bandpass LP 500 SP 500 BP500/80
LP 500
SP 500
BP500/80 5

18. Octagon Detection System
PerCP-Cy5.5
FITC
695/40
655 LP
SSC PE
PE-Cy7

19. Summary
Excitation light is steered with prisms and lenses to the interception point
Emitted light is collected using lenses and is split up with dichroic mirrors and filters 7

20. Tasks for the electronical system
Convert the optical signals into electonic signals (voltage pulses)
Digitise the data
Analyse Height (H), Width (W) and Area (A) of the pulse
Send the data to the analysis computer 8

21. How a voltage pulse from the PMT is generated
Laser t
Voltage 1. 2. 3.
Voltage 9

22. Height, Area, and Width Pulse area(A) Voltage Time (µs)
Pulse Height (H)
Voltage 40
Pulse Width (W)
Time (µs) 12

23. Threshold
The threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower intensity are not displayed and not recorded for later analysis. 13

24. Summary
During passing the laser voltage pulses are generated at the PMT
Amplifiers enhance the signals
The electronics digitizes the pulse using 10MHz sampling
Only signals passing the desired threshold(s) are analysed and recorded
The data are finally passed to the analysis computer connected to the cytometer 15

25. Instrument settings
the exact values for PMT voltages and thresholds are depending on the applications (type of cells, staining methods) and the specific instrument.
Displaying the data in a linear fashion or using the logarithmic form is also depending on the application. 1

26. Workstation
The connected workstation is designed for instrument control, data acquisition, -storage and -analysis.
OS is Windows2000 Professional running on a IBM-compatible computer platform.
Software
DiVa application: Instrument connectivity, Data-acquisition and analysis system
DiVa Data Manager: Backup and Restore the database. 3

27. Data saving
All data are saved directly into a special database. Every plot is connected with its corresponding datafile. All tubes carry a copy of the instrument setting that was active during acquisition.
Due to this, there are no special save commands in the software. Every action is recorded in the database. When you quit and re-start the software, it will open the last experiment exactly at the position you left it. 5

28. Visualization of data
1) Histograms - single parameter, intensity plotted as frequency distribution 6

29. Visualization of data Listmode file
2) Dotplot - two parameter are plotted on X and Y
FSC
SSC
FL1
FL2
Event 1 30 60
638
840
Event 2
100
160
245 85
Event 3
300
650
160
720
200
400
600
800
1000
840
FL2-H 85
200
400
600
800
1000
FL1-H
245
638 7

30. Let`s have a look at an example from real life
Enough theory of flow!
Let`s have a look at an example from real life 8

31. Example: Determine the percentage of CD3, CD4, and CD8 populations from whole blood
Material
Mouse splenocytes
Method
Three-colour immunofluorescence
Preparation
Staining of freshly isolated splenocytes
Stainings
Isotype controls
Single-colour stainings for CD3-FITC, CD3-PE, CD3-PerCP und CD3-APC to determine suitable instrument settings 9

32. Prepare the instrument10

33. Proper adjustment of FSC and SSC voltage
FSC und SSC are optimally adjusted when the population of interest (i.e. Lymphocytes) can be resolved from all other populations
The threshold on FSC is adjusted so that most of the debris is excluded from the data acquisition. 11

34. Parameters (I)
FSC and SSC n are depending on cell type and cell state (activated, resting) n depend on the preparation method (Ficoll, LW, LNW, fixation method etc.) n are normally used to define the population of interest for further analysis 12

35. Parameters
Fluorescence channels (FL1, FL2, FL3, FLX) n depending on the specific staining (conjugate) antibodies, propidium iodide for DNA-labelling, etc.) n most of the time fluorescence serves as marker for the statistical analysis 13

36. Defining the population of interest (often just named „gating“)14

37. About „Gating“ selectively analyse defined cell populations
Gates can be set manually or automatically by software
multidimensional gating with hierarchical gates
too narrow gates may lead to the loss of cell populations
too wide gates enhance the number of unwanted cells
during analysis of the desired cell population the cells in the gate are considered to be the 100% 15

38. Adjusting the fluorescence settings
A) Adjusting PMT voltages
Sample: Isotype control
The observed fluorescence is considere to be unspecific background fluorescence,
Setup is done „gated“ on the lymphocyte population
Try to put the background into the first decade (only a rule of thumb!)
B) Defining quadrants
Traditionally, a „Quadrant“ is set to define the possible four populations in two-colour experiment. Later we will see that quadrants are not the appropriate way for multicolour analyses. 1

39. Theory of quadrant analysis
FITC + PE
negative Q2 Q4 Q3 Q1
FL2-H
FL1-H 2

40. Real life: FITC-fluorescence overspill
650nm
700nm
500nm
550nm
600nm
Relative Intensität
Wellenlänge ( n m )
FL1
530/30
FL2
585/42 3

41. FITC Compensation Detektor - … % Signal FL1 FL2 530/30 585/42
Relative Intensität
500nm
550nm
600nm
650nm
700nm
Wellenlänge (nm) 4

42. FITC Compensation Lowering the FITC-population is achieved by...
... Subtracting a percentage of FITC- intensity from the affected PE-channel ...
FL1
530/30
FL2 5 8 / 4 2
… because 25% of the FITC-signal are actually detected in the PE channel ...
Relative Intensity
500nm
550nm
600nm
650nm
700nm
Wavelength (nm) 5

43. PE-fluorescence overspill
650nm
700nm
500nm
600nm
Wavelength (nm)
550nm
Relative Intensity
FL3
größer 650
FL1
530/30
FL2
585/42 6

44. Automatic Multicolour Compensation
Multicolour compensation with more than three colours can become very time-consuming because each channel has to be compensated against each other.
Automatic compensation offers the possibility to run single-color controls and let the software calculate all overspills.
Mathematical calculation results in the correct spillover values for all channels. However, to the user the visual data may look undercompensated. This will be discussed in detail during the training course. 7

45. Summary What we have seen:
the emission spectra of common fluorochromes (FITC, PE)
the spectral overlap of fluorochromes into neighbouring channels depending on the emission spectra and filtersets
how spectral overlap can lead to misinterpretation of multicolour stainings
How compensation can correct the spectral overlap of fluorochromes 10