FLOW CYTOMETRY  Definition: Measuring properties of cell as they flow in a fluid suspension across an illuminated light path.

Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence

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 analog fluorescent signals to digital signals

A flow cytometer is made up of three main systems: 1.Fluidics : The fluidics system transports particles in a stream to the laser beam for interrogation 2. Optics: The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. 3. Electronics: The electronics system converts the detected light signals into electronic signals that can be processed by the computer.

The scattered and fluorescent light is collected by appropriately positioned lenses. A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them. FSC

Fluidics The purpose of the fluidics system is to transport particles in a fluid stream to the laser beam for interrogation. the stream transporting the particles should be positioned in the center of the laser beam. In addition, only one cell or particle should move through the laser beam at a given moment. To accomplish this, the sample is injected into a stream of sheath fluid within the flow chamber. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis

The Flow System  When a sample is injected into a flow cytometer, it is ordered into a stream of single particles.  Central channel/ core - through which the sample is injected. Outer sheath - contains faster flowing Sheath fluid (0.9% Saline / PBS), enclosing the central core.

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 Sheath Tank Waste Tank Line Pressure Vacuum Sample Pressure (Variable) Sheath Pressure (Constant) Sample Tube Hydrodynamic Focusing cont…..

The sample pressure and the sheath fluid pressure are different from each other. The sample pressure is always greater than the sheath fluid pressure. The sample pressure regulator controls the sample flow rate by changing the sample pressure relative to the sheath pressure. Hydrodynamic Focusing cont…..

A lower flow rate decreases the width of the sample core and restricts the position of the cells to a smaller area. The majority of cells passes through the center of the laser beam; thus the light illuminating the cells and emitted from the cells is more uniform. A lower rate is generally used in applications where greater resolution is critical, such as DNA analysis. Hydrodynamic Focusing cont…..

A higher flow rate is generally used for qualitative measurements such as immunophenotyping. The data are less resolved, since the cells are less in line in the wider core stream, but are acquired more quickly Proper operation of fluidic components is critical for particles to properly intercept the laser beam. Therefore, the operator must always ensure that the fluidics system is free of air bubbles and debris and is properly pressurized at all times. Hydrodynamic Focusing cont…..

Optical System The optical system consists of 1.excitation optics 2.collection optics

1. excitation optics: The excitation optics consist of i.Laser: the most commonly used wavelengths of lasers used in flow cytometers are i.ARGON Lasers 488nm (blue), ii nm (red), iii nm (violet), iv. 514nm (green). ii. lenses that are used to shape and focus the laser beam.

2. collection optics: The collections optics consist of i.collection lens to collect light emitted from the particle–laser beam interaction ii. optical mirrors: a flow cytometer uses a combination of long pass, short pass, band pass, and dichroic mirrors to direct specific wavelengths of light to a particular detector

ii. optical mirrors cont…..  Different wavelengths of light are 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 they absorb or reflect some wavelengths of light, while transmitting others. Types of filters. 1. Long Pass 2. Short Pass 3. Band Pass 4. Dichroic 1

Optics- Long Pass Filters  Transmit all wavelengths greater than specified wavelength  Example: 500LP will transmit all wavelengths greater than 500nm 400nm 500nm 600nm 700nm Transmittance Types of filters

Optics- Short Pass Filter  Transmits all wavelengths less than specified wavelength  Example: 600SP will transmit all wavelengths less than 600nm. 400nm 500nm 600nm 700nm Transmittance Types of filters cont…..

Optics- Band Pass Filter  Transmits a specific band of wavelengths  Example: 550BP Filter will transmit wavelengths of light between 540nm and 560nm 400nm 500nm 600nm 700nm Transmittance Types of filters cont…

Optics- Dichroic Filters  Placed at a 45º angle of incidence  Part of the light is reflected at 90º, and part of the light is transmitted and continues. Dichroic Filter Detector 1 Detector 2 Types of filters cont…..

When a light intersects a laser beam at the so called 'interogation point' two events occur: a) light scattering b) emission of light (fluorescence ) OPTICS

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. Photodiode

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, membrane permeability.

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.) Side scatter detector Measuring cell granularity

FSC Detector Collection Lens SSC Detector Laser Beam

FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells Study of FSC and SSC allows us to know the differentiation of different types of cells. Why FSC & SSC?

 The light scattered in the forward direction is proportional to the square of the radius of a sphere, and so to the size of the cell or particle. FSC cont….

Optics B) EMISSION OF FLUORESCENT LIGHT (FLUORESCENCE)  The cells are labelled with  fluorochrome-linked antibodies or  stained with fluorescent membrane, cytoplasmic or nuclear dye.  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 wave length.

Fluorescence A fluorescent compound absorbs light energy over a range of wavelengths that is characteristic for that compound. The excited electron quickly decays to its ground state, emitting the excess energy as a photon of light. This transition of energy is called fluorescence.

The argon ion laser is commonly used in flow cytometry because the 488-nm light that it emits excites more than one fluorochrome. One of these fluorochromes is fluorescein isothiocyanate (FITC). The combination of FITC and phycoerythrin (PE) is used Fluorochromes  The amount of fluorescent signal detected is proportional to the number of fluorochrome molecules on the particle.

When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic surface markers of the cell (Figure 3-5). In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations.  The staining pattern of each subpopulation, combined with FSC and SSC data, can be used to identify which cells are present in a sample and to count their relative percentages. The cells can also be sorted if desired.

Commonly used Fluorochromes FLUOROCHROMESEMISSION MAXIMUM Fluorescein Isothiocynate (FITC)530nm Phycoerythrin (PE)576nm Peridin-chlorophyll alpha complex (PerCP)680nm Allophycocyanin (APC)660nm Texas red620nm ECD( PE - Texas Red Tandem)615nm PC5 (PE - cyanin 5 dye tandem)667nm

OPTICS - DETECTORS  The photodetectors convert the photons to electrical impulses.  Two common types of detectors used in flow cytometry:  Photodiode used for strong signals, when saturation is a 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.

1.Optical Filters Once a cell or particle passes through the laser light, 1.emitted SSC and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and 2. a photodiode collects the FSC signals. All of the signals are routed to their detectors via a system of mirrors and optical filters.  PMTs detect fluorescence signals, which are often weak. The specificity of a detector for a particular fluorescent dye is optimized by placing a filter in front of the PMT, which allows only a narrow range of wavelengths to reach the detector. Such filters are called bandpass (BP) filters.

3. ELECTRONICS  The electronic subsystem converts photons to photoelectrons.  Measures  amplitude,  area and  width of photoelectron pulse.  It amplifies pulse then digitalizing the amplified pulse.

List mode data are collected on each particle or event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer. This data can be analyzed to provide information about subpopulations within the sample

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