Flow Cytometry: History, Principles, and Applications

• Introduction to flow cytometry as of 2025

Summary of Topics

• Overview of historic and defining milestones in flow cytometry
• Flow Cytometry is built on some basic, but important principles to allow simultaneous data collection
• The Applications
• Other, special cases

Historic and Definitions

• In 1934, Moldovan created the first device to count cells by capillary/photoelectric detector
• Wallace Coulter patented a device in 1949 for counting and measuring cell size
• Kamentsky added cellular constituents analysis in 1965
• In 1969, Herzenberg wrote one of the first articles to describe mammalian cell sorting, followed by its application in 1973 and Marvin Van Dilla started to use lasers as a light source
• Becton-Dickinson commercialized the FACSII in 1974
• 3 fluorescence parameters per cell could be analyzed by 1980 for the first time
• By 1990, 7 fluorescence parameters could be analyzed
• 11 fluorescence parameters could be analyzed by 2001
• 2004 saw Robinson describe spectral approaches
• 18 fluorescence parameters could be analyzed by 2006
• Sony Biotech launched the first commercial spectral flow cytometer in 2012
• 20 fluorescence parameters could be analyzed by 2016
• 2020 saw the use of artificial intelligence to help analysis
• Precise study of isolated cells through a liquid flow defines flow cytometry
• Flow cytometry characterizes individual particles suspended in a liquid, both quantitatively and qualitatively, as well as assessing their function
• Flow cytometry analyzes optical or physical signals emitted by a particle
• This cutting happens via the light beam from either a laser or arc lamp

Basic Principles

• Simultaneous measurement of different characteristics of a cell or particle
• Measurements are carried out as cells pass one by one through an analysis chamber at varying speeds
• The data obtained for each cell includes:
  • Size, via forward scatter (FSC)
  • Granularity or internal complexity, via side scatter (SSC)
  • Fluorescence intensity depending on fluorochromes
• Each event will diffract light as it passes in front of the laser beam
• Cell size (FSC) is proportional to diffraction
• Cell structure (SSC) is proportional to cell granularity or complexity

Forward Scatter

• Light is diffracted at small angles
• Diffracted light is collected at a small angle, between 1 and 10 degrees
• The signal is proportional to cell size
• A detector is aligned with the laser, in the cone of diffracted light

Side Scatter

• Light is diffracted at large angles
• Diffracted light is collected at 90° to the laser axis
• The signal is proportional to cell granularity and complexity
• The detectors are perpendicular to the laser beam
• They are located in the zone of widely diffracted light
• Representational Scatter plots of lysed normal whole blood
• FSC vs SSC alone can distinguish cell types
• Cell size measures particles from 100 nM to 100 μM

Fluorescence

• Fluorescence is measured at 90° when an event passes in front of a laser
• Photoluminescence is radiative phenomenon from the light excitation (visible or UV photons)
• Chemiluminescence is a radiative phenomenon resulting from a chemical reaction
• Many things can produce natural fluorescence including several minerals
  • Adamite fluoresces green
  • Hemimorphite fluoresces blue
  • Calcite fluoresces red or pink
• There are also examples of imported fluorescence such as in the GFP-Rabbit, GFP-mice, and GFP-fly fruit due to animals carrying the GFP ubiquitary promoter

Equipment: Function

• Fluidics introduce and position cells
• Optics produce and collect light signals
• Electronics convert optical signals into electronic signals and digitize them for computer analysis

Equipment: Fluidics

• Cytometers analyze one cell at a time by placing the cells in suspension and driving them with a pump, one by one, in front of one or more lasers
• Centering of cells in laminar flow comes from hydrodynamic focusing
• Cell separation achieved via progressive acceleration
• Cells pass through a nozzle for channeling and centering
• Hydrodynamic focusing centers and aligns cells in laminar flow
• Sheath liquid stretches the sheath liquid to achieve cell separation via progressive acceleration
• Cells then move through a nozzle to channel and center it

Controlling the Speed of Flow

• Low Pressure uses 10μL/mn
• Medium Pressure uses 30 µL/mn
• High Pressure uses 60 µL/mn
• Sheath liquid pressure remains constant, unlike the sample
• Increase in pressure leads to an increase in flow
• The higher the pressure, the greater the freedom of movement of the cells

Equipment: Optics

• Necessary to focus the light source (laser) on the cells
• Illumination must excite dyes at a wavelength close to their excitation maximum
• Lasers are commonly used as powers source and offer stability
• Vapor lamps such as mercury and xenon, are expensive but precise

3-Emission Optics

• A thin laser beam allows focus on one cell at a time
• Background noise is reduced
• Energy concentrates on a small surface
• Monochromatic light excitation becomes very powerful
• Light coming from a laser is monochromatic and unidirectional
• Common lasers and their output include:
  • UV Laser 325 nm: UV
  • Laser 405 nm: violet
  • Laser 488 nm: blue
  • Laser 532 nm: green
  • Laser 561 nm: yellow
  • Laser 592 nm: orange
  • Laser 635 nm: red

4-Reception Optics

• Optical signals emitted by the cells must be focused and separated
• The aforementioned signals need to be routed to detection systems: via photomultipliers or photodiodes
• Selections should be performed by different optical circuits composed of mirrors and filters
• A dichroid mirror directs each signal to the chosen detector
• The signal to pass in one direction is transmission, while being sent in another is reflection

4-Reception Optics: Optical Filters

• Optical filters are composed of materials that absorb certain wavelengths and transmit others
• The three modalities are:
  • LP 500 (Longpass)
  • SP 500 (Shortpass)
  • BP520/20 (Bandpass)

4-Reception Optics: Light Conversion

• Light is collected and converted to an electrical signal after it passes through the series of mirrors and filters
• This can occur via:
  • a Photomultiplier (PMT), which are very sensitive and used for weak signals and high gain, but can assess fluorescence/structure
  • a Photodiode (PD), which has lower sensitivity, for strong, very intense signals and if detector saturation is a potential issue. These are a means to asses size

5-Electronics

• The objective is to transform optical light signals (photons) into electrical signals to be digitized
• These are PMTs (photomultipliers) or photodiodes that convert a photon into an electric current
• A pulse can be created to help collect the data with a laser
• The pulse is characterized by:
  • Height, signifying pulse height which measures the intensity of the scattered light from the particle
  • Area, showing pulse area that indicates the overall fluorescence of the particle
  • Pulse duration to show pulse width
• Each numerical value corresponds to the channel whose counter is incremented by the number of events
• A single-parameter histogram graphically describes the number of events per channel

Electronics: Data

• The simplest method of data collection is a histogram to collect relative fluorescence in number of events on one parameter
• A Dot plot is a characteristic to see relative characteristics in multiple other parameters
• This allows one to use a good way to detect where the number of cells are, and where populations are well separated
• Density Plot is the measure of relative density
• Pseudo colors plots can be used to simulate 3D representation where the third parameter is the number of events while also highlighting the discrete populations
• Contour plots show 2D representation of populations with similar events, and will show high percentages, but some low percentages may disappear
• Three-parameter histograms generated from a two-parameter plot help provide an idea of the proportion of different cell categories.

Applications : Fluorochromes

• Fluorochromes are molecules capable of emitting fluorescent light after excitation
• The emission wavelength is longer than the excitation wavelength

Fluorochromes over Time

• Late 1800s saw the basis for development of future fluorescent probes
  • Chemicals used included: Methyl violet, malachite green, safranin O, methylene blue, fluorescein, rhodamine, or acridine orange
• Early 1930s used fluorescence microscopy: first vital stains for bacteria and protozoa
• Early 1940s saw the development of techniquesto label secondary antibodies coupled with a large variety of fluorochromes
• 1992: Cloning of the GFP-coding gene enabled development of techniques for producing fusion proteins where numerous spectral variants of GFP could be used
• Far-red and near-infrared fluorophores are used for deep tissue imaging, most notably Cy7 and Alexa Fluor dyes
• Advanced super-resolution microscopy using fluorochromes -Spectral cytometry is used, including instruments with 5 lasers and 40 colors
• Advanced synthetic fluorophores are now available, with enhanced brightness and photostability for long-term imaging
• Fluorogenic probes that are only activated with a specific protein improve signal-to-noise ratios
• Machine learning optimizes flourescence signal by having improved sensitivity and resolution
• The discovery of new flourescent proteins are optimized for highly specific wavelengths.
• Fluorescent molecules can combine to form multiple aromatic rings composed of one or more π bonds
• Excitation is usually activated by a certain wavelength but emit other specific ones

Applications: Fluorescence Intensity & Tandem

• Tje amount of Fluorescence intensity emitted is proportional to number of binding sites
• Tandem describes two different molecules covalently attached to each other allowing signal amplification
• Limits to tandem molecules involve the possible degradation of one of the dyes due to environment or reagents used
  • metabolic activity can also degrade them

Applications: How to best use Tandem dyes

• Keep antibody vials and labeled samples at 4°C
• protect from light
• Fix and read within a maximum of 30 minute windows of reading
• Compensate for spectral spillover using single stain controls read with with the same machine settings
• Keep in mind, using tandem configurations may force the need to compansate to achieve clean data to offset "spillover"
• Match color choice in your assay to the specific equipment used and the biological question that you require answering
• Make sure to also pay attention to brightness as well as spectral overlap and other detectors

Applications: Fluorochrome Characteristics

• The number of photons emitted over photons absorbed characterizes how well photons can be delivered
• Each fluorochrome is characterized by an excitation spectrum and an emission spectrum of fluorescence

Applications: Fluorochrome Compensation

• If there is a partial overlap of fluorochrome, it can cause fluorescence issues
• This in turn gives off artifact fluorescence picked up erroneously at the the wrong detection gates
• For this reason, a certain percentage of flourescence should be removed, and this is termed COMPENSATION

Applications: Compensations - A How To

• It is important to have proper controls during compensation setup and execution
• These controls should include Running of the tubes containing many cells such as:
  • Unstained cells for threshold gates
  • Cells stained with cells of the same type used in assay to eliminate background
• Use PMT settings for SSC/FSC gate determination
• Be aware of cells single stained, as they should be accounted for
• When performing FITC, it alligns with FL2, and this is referred to a fluorescence
• Remember*, correct compansation can only be done if the median of the positive to the negative population are relatively similar

Applications: Mulitcolor Considerations

• Match brightness to antigen density in a multi staining experiment
• Use HIGH STAIN for low antigen density, LOW stain for HIGH antigen.
• Minimize overstaining
• Pay attention tandem dye use when doing a multi color experiment

Application: Multi panel

• Understand the limitations of the instruments used
• Understand if antibodies used are able to activate your specific assay target (proliferation or inhibition) It is highly recommended to use pre-made assay/instrument builder websties such as the BD spectrum viewer and select compenstaition These speciffically highlight where spillover will occur
• Pro Tips*
• Isotype: Use with same type if assay is looking at single cell activity
• PMT settings: are needed to adjust data properly
• Gating: Know how your instrument works and plan accordingly

Positivity Threshholds

Is an assay positive that you were trying to stain for?

• Control autogflourescence to ensure what is true signal an what is backgroun
• Can use a control
• can use non reactive antibodies to the test sample, directly with use of antibody
• IMPORTANT*- Can use any cells that are missing fluorescence for new combinations when trying to determin threshold

Applications: Examples

• Clinical labratory Use:*
• HIV identifcation
• Absoute Counts
• Cell Enumeration
• Lemphoima Assays
• Research lab Use:*
• Hematopioteic sorting
• Cell Sorting

Examples: Viability & What things can be studied in flow

• Viability at mortatliy
• PH change
• Apotosotos

Reagents for certain viability

• DNA
• Live and dead