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.
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.
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.
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.
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
Editor's Notes
Whole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed.
routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method.
The immunophenotyping included the activation markers HLA-DR and CD40.
The immunophenotyping included the activation markers HLA-DR and CD40.
An informative pattern was obtained by combining two of the analysed parameters: (i),
the fractions of HLA- DR-expressing CD4+ T cells and CD8+ T cells, respectively,
and (ii), the level of CD40 on CD14+ CD16- monocytes.
Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect
was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral
agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses.
Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of
CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual
variation.
