an introduction to flow cytometry

2. By:
Saberzadeh

3. INTRODUCTION
Technique for counting and examining
microscopic particles.
Particles are suspended in fluid.
Very powerful tool for analyzing multiple
parameters of cells in a heterogeneous
population

5. FACSCalibur
1-2 Lasers, 3-4 Colors
FACSCanto II
2-3 Lasers, 6-8 Colors Up to 8 Colors
LSR II
Up to 7 Lasers, 18 Colors
LSRFortessa
Up to 5 Lasers, 18 Colors

6. Generaly:
Each cell is subjected to a laser beam.
The light reflected from each cell is captured.
Information is then interpreted statistically by a
software

7. HISTORY
Mark Fulwyler was the inventor of the forerunner of
the modern flow cytometer.
This was developed in 1965
First fluorescence based flow cytometer was
developed in 1986by Wolfgang Gohde.

8. HISTORY
The flow cytometer was originally called
pulse cytophotometry.
In 1988, the name was officially changed to
“flow cytometry” at the Conference of the
American Engineering Foundation in
Pensacola, Florida.

9. THE INSTRUMENT
Modern flow cytometers can analyze several
thousand particles every second.
It can also actively separate and isolate particles
having specified property.

10. COMPONENTS
Liquid stream system: carries and aligns cells so that they
pass through a single file.
Measuring system: measure the impedence.
optical system: lamps, high power and low power lasers
Detector and analogue system
computer

11. PRINCIPLE
Beam of light of single wavelength is directed onto
hydrodynamically focused stream of liquid
Detectors are placed where the stream passed through the
light beam.
The light scattered by the cells are detected.
The amount of light scattered is measured.
This is analysed and results are interpreted.

12. HYDRODYNAMIC FOCUSING
Cells to be analyzed are suspended in liquid and
forced through a small aperture.
A tube is used through which the sheath fluid is
pumped.
Cells along with the fluid are forced through this
narrow aperture.
Cells move in a single file or line.
Laser hits each cell and data from each cell can be
read.

13. 1. Cells to be analyzed are suspended in
liquid and forced through a small
aperture.
2. A tube is used through which the
sheath fluid is pumped.
3. Cells along with the fluid are forced
through this narrow aperture.
4. Cells move in a single file or line.
5. Laser hits each cell and data from
each cell can be read.
HYDRODYNAMIC FOCUSING

14. SCATTERING
Cells pass through the laser.
Light gets refracted or scattered in
all angles.
2 types of scatter are analyzed:
Forward scatter: scatter in the forward
direction
Side scatter: light scattered in very
large angles.

15. Principles of Flow Cytometer
Immunobiology Janeway et. al.

16. FORWARD SCATTER
Forward scatter is the light that is scattered by the cell in
the forward direction.
Magnitude is proportional to the size of the cell.
The detector converts intensity of light into voltage or an
electric pulse.

19. FLOURESCENCE IN FLOW
CYTOMETRY
Most common method of studying cellular characteristics.
Antibodies are tagged with a flourophore.
The antibody binds to cells.
When laser hits the flourophore, the molecule gets
excited.
Signal is emitted which can be detected.

20. Energy State of Fluorescence during Excitation and
Emission
http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

21. Commonly Used Fluorochromes forFACS Analysis
Fluorochromes
Emission
Max (nm)
Fluorescein
isothiocyanate (FITC)
519
(green)
488
R-phycoerythrin (R-PE) 578 (orange-yellow) 488
Propidium iodide (PI) 620 (red) 488
PerCP 678 (red) 488
APC 660 (red) 633
Laser (nm)
APC-Cy7 785 (infra red) 633
PerCP-Cy5.5 695 (far red) 488

23. DETECTION OF FLOURESCENCE
Fluorescent t light is travels the same path as side
scatter.
The light is directed through a series of filters and
mirrors.
Particular wavelength of light is detected by the
appropriate detector.

25. TWO COLOUR EXPERIMENT
Cells are labeled with two different fluorophores.
The fluorophores must have compatible spectra.
Eg: AlexaFluor 488 ans Phycoerithrin (PE) have
compatible spectra.
Both have an excitation peak at 480- 520 nm.
The excitation peaks are far away so that discrete
emission can be collected

26. FLUORESCENCE ACTIVATED CELL
SORTER
(FACS)
It is a specialized form of cell sorting.
Provides a method for sorting heterogeneous mixture
of biological material like cells based on light
scattering and fluorescent characteristics.

29. MEASURABLE PARAMETERS
Volume and morphological complexity of cells
Cell pigments such as chlorophyll
Cell cycle analysis, cell kinetics
Chromosomal analysis
Protein modification
Antigens
Enzymatic activity
Membrane fluidity

30. Terminology of Flow Cytometer
Operation
• Laser excitation
• PMT
• Optical filters
Data analysis
• Electronic compensation
Curr. Protocol. Immunol. 2002 Chap. 5

31. LaserExcitation
•488nm Blue Laser
–Basic laser which is equipped in almost all Flow Cytometers
–Generates FSC/SSC
–Fluorochromes excited:
•FITC, Alexa488, GFP, CFSE
•PE and PE tandems
•PI
•PerCP and PerCP tandems, 7-AAD

32. Optical Filters
Light Transmittance through Longpass, Shortpass (Cutoff) and
Bandpass (Centerpoint) filters
www.bdbiosciences.com

33. Emissions from fluorescent dyes bound to individual cells are
detected by photomultiplier tubes (PMTs) which convert and
multiply light signals (analog) as much as 100 million times into
electronic signals (digital(
What is a PhotomultiplierTube (PMT)?
http://micro.magnet.fsu.edu/primer/digitalimaging/con
cepts/photomultipliers.html

34. FSCLaser
FACS Instruments Generate Three Types of Data
Forward scatter (FSC) Approximate cell size
Side scatter (SSC) Cell complexity or
granularity
Fluorescence To investigate cell
structure and function
SSC
SSC

35. Excitation and Emission
 Use the maximum excitation wavelengths to determine
lasers that can be used to excite the fluorochrome
 Use the maximum emission wavelengths to determine
filters and PMTs that can be used to measure the signal

36. In order to properly analyze multicolor
flow cytometry experiments it is
necessary to employ a mechanism
called color compensation.
Specialized circuitry in the flow
cytometer is used to subtract a portion
of one detector's signal from another,
leaving only the desired signal. In the
above example, region A represents
unwanted FITC fluorescence
appearing in the FL2 detector.

37. THANK YOU