FLOW CYTOMETRY Flow cytometry working principle:

Index Flow cytometry is based on the principle of passing individual cells or particles in a
fluid suspension through a focused laser beam. This laser illumination causes the
1. Introduction to Flow Cytometry: cells to scatter and emit fluorescent light, and the instrument's detectors capture
 Brief overview of flow cytometry and its applications in biology and this light. By measuring the intensity and wavelengths of the scattered and
medicine. emitted light, flow cytometry provides valuable information about the properties
2. Flow Cytometry Working Principle: of each cell or particle.
 Detailed explanation of the fundamental principles of flow cytometry,
including laser illumination, light scattering, and fluorescence Flow cytometry instrumentation:
emission.
Flow cytometry instrumentation consists of several key components that work
3. Flow Cytometry Instrumentation:
together to analyse and sort cells or particles in a fluid stream. The primary
 Overview of the key components and instruments used in flow
components include:
cytometry, such as fluidics systems, lasers, detectors, optics, and data
acquisition systems. 1. Fluidics System: The fluidics system controls the flow of the sample
4. Flow Cytometry Procedure: through the instrument. It ensures that cells or particles pass through the
 Step-by-step explanation of the flow cytometry procedure, from laser beam in single-file fashion, preventing clogs or coincident events. It
sample preparation to data analysis and cell sorting. also manages the sheath fluid, which helps focus the sample stream.
5. Flow Cytometry Applications:
 A list of various applications of flow cytometry in different fields, such 2. Laser(s): Flow cytometers use lasers as the light source for excitation.
as immunology, haematology, cancer research, microbiology, stem cell Different lasers with specific wavelengths are used to excite the
research, and more. fluorophores attached to the cells or particles. The choice of laser
Introduction depends on the specific fluorophores used.

Flow cytometry is a fast and precise way to study and sort cells or particles in a 3. Optics: Optics, including lenses and mirrors, direct and filter the emitted
fluid. It uses lasers and detectors to measure their different properties, making it light from the sample to the detectors. They also focus the light onto the
useful for understanding complex mixtures in biology and medical research. It detectors for accurate measurements.
measures various characteristics, including size, fluorescence, and granularity,
4. Detectors: Detectors capture the emitted light, and there are typically
facilitating detailed examinations of heterogeneous biological samples and aiding
several detectors with specific bandpass filters to detect different
in diagnostics, research, and cell sorting.
fluorophores. Detectors are used to measure forward scatter (FSC), side
Flow cytometry is a powerful technique used in biology and medicine to analyse scatter (SSC), and various fluorescent channels.
and sort cells and particles based on various physical and chemical characteristics.
It is widely used in fields such as immunology, haematology, microbiology, and
cancer research.
 5. Flow Cell: The flow cell is a small, narrow channel through which the  Ensure the sample is free of clumps or aggregates to prevent clogs in the
sample flows in a single-file manner. It is designed to ensure that cells or instrument.
particles are properly aligned as they pass through the laser beam.  If necessary, label the cells with fluorescent markers, such as antibodies
or dyes, to target specific molecules or characteristics of interest.
6. Data Acquisition System: This system collects and digitizes the signals  These labels allow for the detection and quantification of various
from the detectors in real-time. It records the intensity and wavelengths properties, such as cell surface proteins, DNA content, and cell viability.
of the emitted light, as well as information about cell size and granularity.
2. Instrument Setup:
The data is stored for subsequent analysis.  Prepare the flow cytometer by turning it on and allowing it to warm up.
 Set the appropriate laser(s) and detector(s) configurations depending on
7. Electronics and Software: The flow cytometer is controlled by electronic
the fluorophores used for labelling. Each fluorescent marker has a specific
systems and specialized software. The software allows the user to set excitation and emission wavelength.
instrument parameters, control data acquisition, and perform real-time  Calibrate the instrument using calibration beads or particles with known
data analysis. properties.
3. Sample Injection:
8. Display and User Interface: Most flow cytometers have a graphical user  Load the prepared sample into the sample injection port or well.
interface (GUI) that allows users to monitor the experiment, set  Ensure that the sample is well mixed, as flow cytometry requires a
instrument parameters, and visualize data in real-time. homogeneous suspension.
 Set the sample flow rate, typically measured in events per second (EPS),
9. Sorting System: Some flow cytometers are equipped with a sorting to ensure that cells or particles pass through the laser one at a time.
mechanism that allows for the physical separation and collection of 4. Laser Illumination:
specific cells or particles based on their characteristics. This is particularly  As the sample flows through the flow cell, it encounters the laser
beam(s).
useful for cell sorting experiments.
 The laser excites the fluorescent labels on the cells, causing them to emit
10. Waste and Sample Containers: These containers collect waste materials fluorescent light.
and sorted samples, respectively. Proper disposal and sample collection
5. Data Collection:
are essential for maintaining laboratory safety and data integrity.  Detectors capture the emitted light, including forward scatter (FSC), side
scatter (SSC), and fluorescence.
Flow cytometry procedure:  Forward scatter provides information about cell size, while side scatter
relates to granularity.
The procedure for flow cytometry is a detailed and precise process that involves
 Fluorescence detectors record the intensity and wavelengths of the
several steps:
emitted light.
1. Sample Preparation: 6. Cell Sorting:
 Collect and prepare your biological sample, which can be a suspension of  If cell sorting is required, a flow cytometer with sorting capabilities can be
cells, tissues, or particles. used.
 Cells or particles of interest are typically suspended in a fluid (often called  Based on the data collected, the instrument can sort and collect cells with
the sheath fluid) specific characteristics into separate containers.
  Use specialized flow cytometry analysis software to interpret the data.
 Analyse the data to identify and quantify different cell populations, assess
the presence or absence of specific markers, and determine various
characteristics of the sample.
8. Data Presentation:
 Visualize and present the flow cytometry results using graphs, scatter
plots, histograms, or other graphical representations.

Flow Cytometry applications:

1. Immunology:
 Immunophenotyping
 Cytokine analysis
 Cell activation studies
2. Haematology:
 Blood cell analysis
 Detection of blood disorders
3. Cancer Research:
 Tumour characterization
 Minimal residual disease detection
 Apoptosis and cell cycle analysis
4. Microbiology:
 Microbial cell counting
 Microbial viability assessment
5. Stem Cell Research:
 Stem cell identification and sorting
 Differentiation studies
6. Cell Viability and Apoptosis:
 Cell viability assays
 Apoptosis studies
7. Cell Cycle Analysis:
 Studying cell cycle phases and DNA content
8. Virology:
 Virus quantification
7. Data Analysis:  Detecting viral antigens
 After data collection, the instrument generates a dataset for each cell or 9. Neuroscience:
particle analysed.  Neuronal and glial cell analysis
 Neuronal activation studies
 10. Environmental Science:
 Environmental microbiology
 Water quality assessment
11. Clinical Diagnostics:
 Disease diagnosis
 Transplantation compatibility testing
12. Cell Sorting:
 Isolation of specific cell populations

These applications cover a wide range of fields, from medical diagnostics and
research to environmental monitoring and cell biology studies.