Flow cytometry

1. FLOW CYTOMETRY
Subject- Pharmaceutical Biotechnology (P)
DEPARTMENT OF PHARMACEUTICAL SCIENCES
Dr. Hari Singh Gour Vishwavidyalaya
Sagar(M.P)-470003, India
(A Central University)
Submitted By:
DEBASIS SEN
(Y22254008)
Submitted To:
Prof. UMESH K. PATIL
(Professor, DOPS)
Dr. UDITAAGRAWAL
(Guest Faculty, DOPS)
Ms. POOJA DAS BIDLA
(Research Scholar, DOPS)

2. CONTENT
• INTRODUCTION
• PRINCIPLE
• BASIC MECHANISM
• SAMPLE PREPARATION
• INSTRUMENTATION
• DATAANALYSIS
• GATING
• COMPENSATION
• WORKING
• APPLICATION
• LIMITATIONS
• CONCLUSION
• REFERENCES

3. INTRODUCTION
Flow- the motion characteristics of fluids
Cyto- combining form of cells
Metry- measurement
• Hence, flow cytometry is the measurement of cell properties as cell move in a single
hydrodynamic flow and interrupt a beam of light.
• Flow cytometry is a popular cell biology technique that utilizes laser-based technology to
count, sort, and profile cells in a heterogeneous fluid mixture.
• The technique was first described by Wallace Coulter in the 1950s.
• The present flow cytometers are capable of analyzing upto 13 parametrs (forward scatter,
side scatter, 11 colours of immunofluorescence).

4. PRINCIPLE
Flow
Cytometry
Principle of Laminar
Flow
Electrostatics
Optics and Light
Scattering
Coulter Principle

5. BASIC MECHANISM
Biological sample
Label it with a fluorescent marker
Cells move in a linear stream through a focused light source(laser
beam)
Fluorescent molecule gets activated and emits light that is filtered
and detected by sensitive light detectors(usually a photomultiplier
tube)
Conversion of analogue fluorescent signals to digital signal

6. SAMPLE PREPARATION
The exact steps for sample preparation can vary depending on the specific experimental design
and research question. Here is the general steps which are followed for sample preparation in
flow cytometry-
Collect the
cells or
particles of
interest
Prepare the
cells or
particles by
washing them
in a buffer
solution to
remove any
debris or
contaminants
Stain the cells
or particles
with
fluorescent
dyes or
antibodies
Fixation: In
order to stop
any further
degradation
Filtering: to
eliminate any
cellular
aggregates and
debris
Final
resuspension:
involves
resuspending
the cells or
particles in a
buffer solution

7. INSTRUMENTATION
COMPONENTS
Flow Cytometry has three main components-
Fluidics
• Transports cells in a
stream to the laser
beam for
interrogation.
Optics
• It consists of lasers
to illuminate the
particles in the
sample stream and
optical filters to
direct the resulting
light signals to the
appropriate
detectors
Electronics
• The electronics
system converts the
detected light
signals into
electronic signals
that can processed
by the computer

9. FLUIDICS
The fluidic system consists of a Flow cell (Quartz chamber)
• Central core- through which the sample is injected.
• Outer sheath- contains faster flowing fluid i.e; Sheath
fluid, enclosing the central core.
• Once the sample is injected, they are forced into the
centre of the stream forming a single file.

10. OPTICS
Single wavelength, coherent light
Light Source
• Provide energy that excite the fluorochrome of interest, helps to
create stable and reliable signal.
Focus the beam
Lenses
• Keep the beam perpendicular to sample stream flow
• Make beam small enough to illuminate only one cell at a time
Filters
• Ensures that each photodetector receives light bands of various
wavelenghts

11. When a light intersects a laser beam at the so called ‘interrogation point’ two events occur-
A. Light scattering
B. Emission of fluorescent light

12. LIGHT SCATTERING
The light scatters in two ways-
• Forward Scatter (FSC)- The incident light scattered in forward direction (Along same
axis laser is travelling).
• Forward scatter represent the size of the cell.
Smaller cell will scatter less and larger will
scatter more.

13. • Side Scatter (SSC)- The incident light scattered at an angle of 90 degree to the axis of the
laser path.
• The intensity of this signal is proportional to
amount of cytosolic structure in cell (e.g-
granules, cell inclusions etc).
• Side scatter represents the granularity/ internal
complexity of cell.

14. EMISSION OF FLUORESCENT LIGHT
Fluorescence is the property of certain substances by which they absorb light at a certain wavelength
and then emit light at a longer wavelength, this substances are called fluorochromes.
Mainly used fluorochromes in flow cytometry- FITC, PE, ECD, PC5, APC, PERCP
Fluorochrome absorb some light and
become excited
Release energy in form of photons with
specific wavelength (larger than
excitation wavelength)
Photons pass through the collection lens
and splits down specific channel with the
use of filter

15. OPTICAL FILTERS
Many wavelengths of light will be scattered from a cell.
But how to split the light into its specific wavelengths to detect them independently?
Optical filters are designed such that they absorb or reflect some wavelengths of light, while
transmitting others.
Optical
Filters
Short pass
filter
Long pass
filter
Band pass
filter
Dichroic
filter

16. DETECTOR
There are mainly two types of detectors used in flow cytometry-
• Photodiode
• Photomultiplier tube (PMT)
Photodiode
• Used for strong signals
• When saturation is a potential
problem
• e.g- Forward scatter
Photomultiplier tube
(PMT)
• More sensitive and used to
detect small amounts of
fluorescence
• Band pass filter in front to
allow only specific band width
of light reach it

17. ELECTRONICS
• Detectors basically collects photos of light and convert them to current.
• The electronics process that light signal and convert the current to a digitalized value.
• This digitalized value is represented as a graph by the computer.
• Softwares-
1. Kaluza
2. CellQuest
3. FlowJo
4. WinMDI
5. FCS Express

18. DATA DISPLAY
The data are usually presented in the form of single parameter histograms or as plots or
correlated parameters, which are referred to as cytograms.
Cytograms may display data in the form of-
• Dot plot
• Density plot
• Contour plot
• Histogram

19. GATING
Gating is used to isolate a subset of cells on a plot and set a region on a histogram or
cytogram.
It allows the ability to look at parameters only on that subset.

20. COMPENSATION
Why do we need to compensate?
Fluorochromes typically give fluorescence over a large part of the spectrums (100nm or
more).
• Detectors detect fluorescence based on wavelengths
• Thus detects fluorescence from more than one fluorochrome which is called ‘Bleed Over’
• You need to compensate this bleed over so that one detector reports signals from only one
fluorochrome.

21. WORKING OF FLOW CYTOMETRY

22. APPLICATIONS
Erythropoiesis
and red cell
disorder
Platelet
function
analysis
Phagocytosis
analysis
Detection of
apoptosis
Cell
counting
and cell
sorting
Cell cycle
analysis
Staging of
Lymphomas

23. LIMITATIONS
• Flow cytometry is limited by its requirement that analyzed cells be in suspension, making
information on tissue architecture and cell-cell interactions unavailable.
• Cell subpopulations with similar marker expression and difficult to differentiate and
analyses that employ more fluorophores are subject to signal slipover.
• Flow cytometry may generate massive amounts of data, making analyses complicated.

24. CONCLUSION
Flow cytometry is a versatile and powerful technique that has revolutionized our
understanding of cells and their functions. Flow cytometry has a wide range of applications,
including immunology, hematology, oncology, microbiology, and stem cell research. It can
be used to analyze complex mixtures of cells, such as blood or bone marrow samples, to
identify and quantify different cell types. Flow cytometry can also be used to study the cell
cycle, apoptosis, and cell signaling pathways. So it has widespread use in research and
clinical settings has led to numerous discoveries and advancements in medicine and biology.

25. REFERENCES
1. Büscher M. Flow cytometry instrumentation–an overview. Current Protocols in
Cytometry. 2019 Jan;87(1):e52.
2. Nunez R. Flow cytometry: principles and instrumentation. Current Issues in Molecular
Biology. 2001 Apr;3(2):39-45.
3. Hoffman RA. Flow cytometry: instrumentation, applications, future trends and
limitations. Standardization and Quality Assurance in Fluorescence Measurements II:
Bioanalytical and Biomedical Applications. 2008:307-42.
4. Chapman GV. Instrumentation for flow cytometry. Journal of immunological methods.
2000 Sep 21;243(1-2):3-12.

26. THANK YOU