3. INTRODUCTION
● Flow : the motion characteristics of fluids.
● Cyto : combining form of cells or cells.
● Metry : measurement / count.
● Hence , measurement of physical and /or chemical
characteristics of cells,or in general,biological articles is known
as CYTOMETRY.
● When such measurements are performed while the cells or the
biological material pass in a fluid stream across an illuminated
light path,the process is known as FLOW CYTOMETRY.
4. FLOW CYTOMETRY
● This method allows the quantitative and qualitative analysis of several
properties of cell populations from virtually any type of fresh unfixed
tissue or body fluid.
● The properties measured include a particles relative size , relative
granularity or internal complexity , and relative fluorescence intensity.
Most commonly analyzed materials are :
● Blood
● Bone marrow aspirate and
● Lymph node suspensions
5. PRINCIPLE
● The basic principle of flow cytometry is the passage of cells
in single file in front of a laser so they can be detected,
counted and sorted. Cell components are fluorescently
labelled and then excited by the laser to emit light at varying
wavelengths.The fluorescence can then be measured to
determine the amount and type of cells present in a sample.
6. ● Prepare single cell or particle suspension is necessary for flow cytometric
analysis.
● The suspension of cells or particles is aspirated into a channel surrounded by a
narrow fluid system.
● They pass one at a time through a
Focused laser beam.
● The light is either scattered or absorbed
When it strikes a cell.
● Light scattering is dependent on the
Internal structure of the cell and its
size and shape.
7. ● Absorbed light of the appropriate wavelength may be re-
emitted as fluorescence.
● Light and fluorescence scatter signals are detected by a series
of photodiodes and amplified.
● Optical filters are essential to block the unwanted light and
permit light of the desired wavelength to reach the
photodetector.
● Fluorescein isothiocynate (FITS),Texas red and phycoerythrin
(PE) are the most common fluorescent dyes used in the
biomedical sciences.
● Large number of cells are analysed in a short period of time
(>1000/sec).
10. WORKING OF FLOW CYTOMETER
A flow cytometer is composed of three main systems :
1. FLUIDICS : Transport cells in a stream to the laser beam for
interrogation.
2. OPTICS : Consist of laser to illuminate the cells in the sample
stream and optical filters to direct the resulting light signals to the
appropriate detectors.
3. ELECTRONICS : Converts the detected light signals into electrical
signals that can be processed by computer.
11. FLUIDICS SYSTEM
❖ When a sample is injected into a flow
cytometer,it is ordered into a stream
of of single particles.
❖ The fluidic system consist of a FLOW CELL
❏ Central core : through which the sample is
Injected.
❏ Outer sheath : contains faster flowing fluid
k/a sheath fluid (0.9% saline /PBS),
Enclosing the central core.
12. Hydrodynamic focusing
Once the sample is injected into a stream of sheath fluid within the flow
chamber , they are forced into the center of the stream forming a single file
by the PRINCIPLE OF HYDRODYNAMIC FOCUSING.
‘Only one cell or particle can pass through the laser beam at a given
moment’.
● The sample pressure is always higher than the sheath fluid pressure,
ensuring a high flow rate allowing more cells to enter the stream at a
given moment.
● High flow rate -Immunophenotyping analysis of cells.
● Low flow rate - DNA analysis.
13. OPTICS
★ After the cell delivery system, the need is to excite the cells
using a light source.
★ The light source used in a flow cytometer :
➔ Laser (more commonly)
➔ Arc lamp
★ Why Lasers are more common ?
➔ They are highly coherent and uniform. They can be easily
focused on a very small area (like a sample stream).
➔ They are monochromatic ,emitting single wavelengths of light.
14. ★ ARGON Lasers -488nm wavelength (blue to blue green).
★ When a light intersects a laser beam at the so called ‘interogation
point’ two events occur :
➔ Light scattering
➔ Emission of light (fluorescence).
Fluorescence is light emitted during decay of excited electron
to its basal state.
15. a) LIGHT SCATTER
● When light from a laser interrogates a cell, that cell scatters light in all
directions.
● The scattered light can travel from the interrogation point down a path to a
detector.
16. OPTICS - FORWARD SCATTER (FSC)
● Light that is scattered in the forward direction (along the same axis the laser
is traveling)is detected in the forward scatter channel.
● The intensity of this signal has been attributed to Cell size , refractive index .
17. OPTICS - SIDE SCATTER (SSC)
❏ Laser light that is scattered at 90 degrees to the axis of the laser path is
detected in the side scatter channel.
❏ The Intensity of this signal is proportional to the amount of cytosolic structure
in the cell (eg:granules,cell inclusions etc.)
18.
19. Why FSC and SSC?
Study of FSC and SSC allow us to know the differentiation of different type
of cells.
20. Commonly used Fluorochromes
FLUOROCHROMES EMISSION MAXIMUM
Fluorescein Isothiocynate (FITC) 530nm
Phycoerythrin (PE) 576nm
Peridin-chlorophyll alpha complex
(PerCP)
680nm
Allophycocyanin (APC) 660nm
Texas red 620nm
ECD (PE - Texas Red Tandem) 615nm
PC5 (PE-cyanin 5 dye Tandem) 667nm
21. b) Emission of light (fluorescence)
● As the fluorescent molecule present in or on the particle is
interrogated by the laser light , it will absorb energy from
the laser light and release the absorbed energy at longer
wavelengths.
● Emitted photons pass through the collection lens and are
split and steered down specific channels with the use of
filters.
22. Optics : Filters
● Different wavelengths of light scattered simultaneously from a cell.
● Need to split the light into its specific wavelengths in order to
measure and quantify them independently.This is done with filters.
● The system of filters ensures that each photodetector receives
light bands of various wavelengths.
● Optical filters are designed such that absorb or reflect some
wavelengths of light,while transmitting others.
● Types of filters :
1. Long pass
2. Band pass
3. Short pass
4. Dichroic
23. Long pass filters
❖ Transmit all wavelengths greater than specified wavelengths.
❖ Example :500LP will transmit all wavelengths greater than 500nm.
24. Short pass filters
❏ Transmits all wavelengths less than specified wavelengths.
❏ Example :600SP will transmit all wavelengths less than 600nm.
25. Band pass filters
● Transmits a specific band of wavelengths.
● Example : 500/20BP filters will transmit wavelengths of light between 540nm
and 560nm .
26. Dichroic filters
● Long pass or short pass filters.
● Placed at a 45degree angle of incidence.
● Part of light is reflected at 90degree , and part of the light is transmitted and
continues.
27. OPTICS :DETECTORS
● The photodetectors convert the photons to electrical
impulses.
● Two common types of detectors used in flow cytometry:
❏ Photodiodes:used for strong signals,when saturation is
potential problem(eg:forward scatter detector).
❏ Photomultiplier tube (PMT) : more sensitive than photodiode
but can be destroyed by exposure to too much light.
❏ Used for side scatter and fluorescent signals.
28. ELECTRONICS
● The electronic subsystem converts photons to photoelectrons.
● Measure amplitude,area and width of photoelectron pulse.
● It amplifies pulse either linearly or logoarithmically and then
digitalizing the amplified pulse.
29. Data Analysis -PLOT TYPES
There are several plot choices :
● Single color Histogram : Fluorescence intensity (FI) versus the
number of cells counted.
● Two color dot plot : FI of parameter 1 versus FI of parameter 2.
● Two color contour plot : concentric rings form around populations.
The more dense the population , the closer the rings are to each
other.
● Two color density plot : Areas of higher density will have a different
color than other areas.
