Transcript These particles have something in common

These particles have something in common
Blood cells
Algae
Chromosomes
Protozoa
Certain parameters of these particles can be measured with a flow
cytometer
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)
Characteristics of FSC and SSC
Forward scatter
Cell size (488 nm)
Coherent lightsource
(488 nm)
Side scatter
Granularity (488 nm)
Forward scatter (FSC)
Side scatter (SSC)
 measured along the axis of the
 measured in 90° direction to the
incoming light
excitation light
 proportional the the cell size / cell
surface (only true for perfect round
cells)
 proportional to cell „complexity“ or
granularity
An example of light scatter:
Side scatter
Granulocytes
Monocytes
Lymphocytes
Debris
Forward scatter
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)
Fluorescence intensity
FITC
FITC
Number of Events
FITC
FITC
FITC
FITC
101
102
103
Relative fluorescence intensity
104
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
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.
The basic microscope
In a standard
microscope, the
operator uses the XYstage to screen the
sample and detect
cells of interest.
The automated Microscope
Detector
& Counter
Waste
Sample
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!
Basic fluidics of the FACSAria
Fluidics Cart
Pressure
Plenum
Cuvette
Sheath
Sample
tube
Waste
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
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
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.
Basic optics
A system of prisms and
lenses directs the laser light
to the interrogation point in
the cuvette
Basic Optics
The emitted light induced from each laser is focussed onto
separate glass fibers.
Optical filters
Longpass
460
Shortpass
500
LP 500
540
460
500
540
SP 500
Bandpass
460
500
540
BP500/80
Octagon Detection System
PerCP-Cy5.5
695/40
655 LP
SSC
PE
PE-Cy7
FITC
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
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
How a voltage pulse from the PMT is generated
t
1.
Laser
Voltage
t
2.
Laser
Voltage
t
3.
Laser
Voltage
Pulse Height (H)
Voltage
Height, Area, and Width
Pulse area(A)
0
40
Pulse Width (W)
Time (µs)
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.
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
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.
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.
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.
Visualization of data
1) Histograms - single parameter, intensity plotted as frequency distribution
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
1000
FL2-H
840
800
600
400
200
85
0
0
200 400 600 800 1000
FL1-H
245
638
Enough theory of flow!
Let`s have a look at an example from real life
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 CD3APC to determine suitable instrument settings
Prepare the instrument
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.
Parameters (I)
• FSC and SSC
 are depending on cell type and cell state (activated,
resting)
 depend on the preparation method (Ficoll, LW,
LNW, fixation method etc.)
 are normally used to define the population of
interest for further analysis
Parameters
• Fluorescence channels (FL1, FL2, FL3, FLX)
 depending on the specific staining (conjugate)
antibodies, propidium iodide for DNA-labelling, etc.)
 most of the time fluorescence serves as marker for
statistical analysis
the
Defining the population of interest
(often just named „gating“)
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%
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.
Theory of quadrant analysis
Q2
FL2-H
Q1
FITC
+
PE
PE
Q3
negative
FITC
FL1-H
Q4
Real life:
FITC-fluorescence overspill
FL2
530/30
585/42
Relative Intensität
FL1
500nm
550nm
600nm
Wellenlänge (nm)
650nm
700nm
FITC Compensation
Detektor - … % Signal
FL1
FL2
Relative Intensität
530/30 585/42
500nm
550nm
600nm
650nm
Wellenlänge (nm)
700nm
FITC Compensation
Lowering the FITCpopulation is achieved
by...
... Subtracting a
percentage of FITCintensity from the affected
PE-channel ...
FL1
530/30
FL2
585/42
Relative Intensity
… because 25% of the
FITC-signal are actually
detected in the PE
channel ...
500nm
550nm
600nm
Wavelength (nm)
650nm
700nm
PE-fluorescence overspill
FL2
530/30
585/42
FL3
größer 650
Relative Intensity
FL1
500nm
550nm
600nm
650nm
Wavelength (nm)
700nm
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 singlecolor 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.
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