Flow cytometry is a technique used in cell biology that allows for the analysis of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It provides rapid multi-parametric analysis of cells based on light scattering, fluorescence, and other optical properties. Flow cytometry gives information about cell size, granularity, and the expression of cell surface markers or intracellular proteins through the use of fluorescent probes. It has many applications in fields like immunology, cancer research, and infectious disease.

1. Flow Cytometry in Cell
Biology
Prepared by:
Amani Alsharidah, 441203711
Aljouhra AlHargan, 442203436
ZOO 642
Advanced Cytology

2. Definition
• Flow cytometry (FCM) is defined as a method for qualitative and quantitative analysis of
biological and physical properties of cells suspended in a liquid, passing through
a laser beam, in a single file.
• It is a complicated technique that works on the principle of light scattering and
fluorescence emission and provides rapid multi-parametric measurements of a single
cell.

3. Flow Cyto - metry
Motion Cell Measurement

4. An Overview
• FCM is a useful tool in cell biology because it combines different cytological examinations.
• It gives information about cell number, size, macromolecular content, in addition to genetic
identity that can be determined by various labels, stains, and probes.
• It is considered as a powerful tool that has applications in immunology, molecular biology ,
pathology, cancer biology and infectious disease.
• It offers several unique advantages as it allows fast multiparametric analysis of cell populations in
addition to enabling physical sorting of the cells to separate the subpopulations.

5. Flow Cytometry
Principle
• Flow cytometers utilize lasers as light sources to
produce both scattered and fluorescent light
signals that are read by detectors such as
photodiodes or photomultiplier tubes.
• These signals are converted into  electronic
signals that are analyzed by a computer and
written to a standardized format data file.
• Cell populations can be analyzed and/or purified
based on their fluorescent or light scattering
characteristics.

6. Traditional Flow Cytometers System
The flow system
Fluidics
The light sensing system
Optical
Signal processing
Electronic

7. Fluidics: The Flow
System
• Cells suspended in fluid (usually buffered
saline) are pressured to pass in a single file
to a laser point where the sample is analyzed.
• Once samples are in the sheath fluid, cells
are forced to pass one by one by
hudrodynamic focusing.
• The result of the hydrodynamic focusing is
that only ONE cell pass through the laser
beam at a given time.

8. Optical: The Light
Sensing System
• It consist of excitation optics (laser), and
collection optics (photomultiplier tubes) that
emit visible and fluorescence light signals to
be used to analyze samples.
• When a cell passes through the laser beam, it
emitts light (fuoresence) and scatters light
(forwad and side scattering).

9. Electronics: Signal
Processing
• The signals are converted into  digital
values that can be read and analyzed by
computer systems.
• It converts photons to  photoelectrons,
then it measure the pulse, then translate it
into a plot.

10. Traditional Flow
Cytometers System

11. FCM samples
Peripheral
Blood
Bone
Marrow
Tissue and
Body fluids

12. Flow cytometry measurement
1. Measurement of forward and side scatter of light.
2. Measurement of scatter light and fluorescence.

13. Measurement of forward and side scatter of
light.
• Cells or particles passing through the beam scatter light, which is detected
as FS and SS.
• Cell populations can often be distinguished based on differences in their
size and granularity.

14. Side scatter (SS)
• The amount of light scattered at right
angle to the indicant light beam depends
on the internal complexity of the particle,
this known Side Scatter (SSC).
• Related to cell granularity and complexity.
• Side scatter detected at 90, to the laser
beam.

15. Forward scatter
(FS)
• Measuring the size of the cell or
particle.
• Related to cell surface area.
• Detection along axis of incident light in
the forward direction.

16. Total blood counting
• Larger and more granular granulocyte cells produce a large population with high SS and FS.
• Monocytes are large cells, but not so granular, so these produce a separate population with
high FS but lower SS.
• Smaller lymphocytes and lymphoblasts produce a separate population with less FS. They
are not granular cells, so also have low SS.
• Therefore, these cells can be separated into different populations based on their FS and SS
alone.

17. Figure: Dot plot of FS versus SS.
Each dot represents a single cell
analyzed by the flow cytometer.
The characteristic position of different
cell populations is determined by
differences in cell size and granularity.

18. Measurement of scattered light and fluorescence
• As well as separating cells based on FS and SS, cells can also be separated by whether
they express a particular protein.
• In this case, a fluorochrome is often used to stain the protein of interest.
• Fluorochromes used for the detection of target proteins emit light when excited by a laser
with the corresponding excitation wavelength.
• These fluorescent stained cells or particles can be detected individually.

19. Flow cytometers
and fluorecent
probes
• Modern flow cytometers use many laser
wavelengths and corresponding fluorescent
probes for simultaneous analysis of 20 or more cell
characteristics.
• Advanced instruments will usually be equipped
with:
1. a cyan laser 488 nm ,
2. a red laser diode ~640 nm
3. a green, green-yellow, or yellow diode-pumped
solid-state (DPSS ) laser ( 532
,
552
,
ro
561 nm
• These lasers all excite a variety of fluorochromes.

21. Flow
cytometry
application
In Clinical Laboratories:
1. Immunophenotyping.
2. CD4 absolute counts.
3. Cell cycle.
4. Residual white blood cell detection.

22. Flow
cytometry
application
In Research Laboratories
1. Immune function studies.
2. Hematopoietic stem cells.
3. Multi-drug resistance studies (cancer).
4. Kinetics studies (cell function) Platelet
analysis (coronary disease).
5. Environmental sample analysis.

24. Result
Flow cytometry method for quantitation of T cell and monocyte
activation status. Representative dot plots from analysis of patient
samples are shown.
CD4+ T cells and CD8+ T cells gated on CD3+ CD4+ (A,C,E) and
CD3+ CD8+ (B,D,F) lymphocytes from (A-B), a healthy control;
(C- D), tuberculosis; (E-F), influenza;
the inserted percentages indicate the fraction of HLA-DR+ cells.
Monocyte plots gated on CD3- CD14+ monocytes from (G), a
healthy control; (H), tuberculosis; (I), influenza;
mfi = mean fluorescence intensity.

25. Thank You..
Any questions?

26. References
• Robinson, R. K. (2014). Encyclopedia of food microbiology. Academic press.
• McKinnon K. M. (2018). Flow Cytometry: An Overview. Current protocols in immunology, 120, 5.1.1–5.1.11. https://doi.org/10.1002/cpim.40
• Chantzoura, E., & Kaji, K. (2017). Flow Cytometry. In Basic Science Methods for Clinical Researchers (pp. 173-189). Academic Press.
• O'Leary TJ. Flow cytometry in diagnostic cytology. Diagn Cytopathol. 1998 Jan;18(1):41-6. doi: 10.1002/(sici)1097-0339(199801)18:1<41::aid-dc7>3.0.co;2-x.
PMID: 9451557.
• https://www.labome.com/method/Flow-Cytometry-A-Survey-and-the-Basics.html
• https://www.laserfocusworld.com/lasers-sources/article/16548156/lasers-for-the-biosciences-novel-ultraviolet-320-nm-laser-source-enhances-flow-cytometry