Flow Cytometry

Daniel GrassoEE 494

02/22/00

Cytometry: An Introduction

• Cytometry is a technique to analyze individual biological cells

• Viewed superficially, it is a process of sorting, counting, and / or sizing of cells or tissue sections

• Cytometry is broadly classified into two categories based on medium through which the sample is analyzed:– Image Cytometry - sample on microscope slide– Flow Cytometry - sample immersed in a stream or flow

• We will be concerned with the latter technique.

Basic System Layout

• A typical flow cytometer is composed of the following:– A biological sample that has been “tagged” or injected with a

fluorescent compound– A flow source and capillary– A light source or illumination device

• Xenon, Mercury, or Tungsten lamp (early systems)• Laser (modern systems)

– A data acquisition system• Electronics, signal processing, data capture• PC, software• Optical components (lenses, mirrors, etc.)

• Cytometry is a highly interdisciplinary science

Historical Review

• (1934) Moldavan describes theoretical device to count red blood cells, but ideas are never brought to fruition

• (1949) Coulter builds first commercially available device for counting blood cells. System is bulky and requires team of experts to maintain.

• (1960s) The laser replaces electric lamps• (1980s) The field of cytometry burgeons, assisted by

related advances in computers, optics and electronics.• Modern systems can fit on a table, and are ubiquitous.

Flow cytometers are used in academic, clinical, and research applications.

Typical 1980s Cytometer (Coulter 753) ($200-300,000)

Lasers

Fluidics

Computers

Detectors

Laser Power Supply

© PUFCL

Modern Cytometer ($90-120,000)FACS = Fluorescence Activated Cell Sorter

© Becton-Dickinson

Details of Operation

• Flow source and sample stream• Lasers and optics of modern flow

cytometers• Electronics and signal processing

The Flow Source

• The sample to be analyzed is injected with a fluorescent compound

• The sample is then forced into a capillary and guided using the sheath-flow principle of hydrodynamic focusing:– An aqueous suspension of the sample is injected into a faster

flowing stream of fluid; this provides a “sheath” which aligns the particles

• Early systems directed the stream through an aperture, and analysis was based on the change in electrical properties at the aperture. Cells are sized and counted in this fashion.

• Modern systems illuminate the flow with light from a laser, and photonics are used to deduce properties

Flow Cell

Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheathfluid

© PUFCL

The Flow Source (contd.)

• When the stream encounters the light source, the samples fluoresce

• The intensity and wavelength can be detected by optoelectronic elements and circuits

• Samples can be sorted with electric plates• The use of proper fluorescent materials needs to be

considered:– Will the chemical interference occur with the sample?– Is the material compatible with the laser wavelength?– Do we have appropriate optical instruments to detect the emission?

488 nm laser

+-

Fluorescence Activated Cell Sorting

Charged Plates

Single cells sortedinto test tubes

Light Sensor

Fluorescence detector

© PUFCL

EthidiumEthidium

PEPE

ciscis--Parinaric acidParinaric acid

Texas RedTexas Red

PEPE--TR Conj.TR Conj.

PIPI

FITCFITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488Common

Laser Lines

© PUFCL

Lasers and Optics of Cytometry

• The laser is the heart of the cytometric system. It is responsible for imaging the sample.

• Other special optical components are required:– Dichroic filters – Photomultiplier (PMT)– Bandpass optical filters (BPF)

PMT

PMT

PMT

PMT

DichroicFilters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

BPF

© PUFCL

Electronics, Signal Processing

• Electronics, data capture and software are the workhorses of the device

• The PMT is the interface between the optical and electronic aspects of the system.

• Need the usual collection of amplifiers, logic, software, etc. to analyze incoming signals.

Design and Maintenance

• Choice of laser is clearly important.– Need to consider power output, wavelength, etc.– Some common lasers used in cytometry:

• typically IR / visible / UV emission• argon ion, HeNe, and solid state lasers are popular

• The system must be kept in a clean and stable environment. Optics and fluids do not like each other!

Applications of Flow Cytometry

• Every major university and hospital has a flow cytometer– used for academic and industrial research, and as a clinical

diagnostic tool

• Examples:– disease detection and immunology

• leukemia, lymphoma, other cancers

– provide qualitative information about DNA and chromosomes• entire subfield known as flow cytogenetics

– characterization of human and non-human cells useful to immunology and preventative medicine

– used in conjunction with other interesting applications • optical trapping, cell manipulation, microrobotics

Conclusions

• Flow Cytometry is a highly interdisciplinary field that involves the automated, quantitative analysis of cells

• The system is a marriage of biological, optical, and electronic components

• The technology admits many interesting medical applications, and will be useful to future scientists and physicians

Sample Cytometric Images

(scale bar = 10? m)

The following were obtained through image cytometry.

Human blood

Chondrocytes (cartilage cells)

Dinoflagellates (single-celled organisms)

Giardia (bacteria)

Bacterial colony

References

• Textbooks:– Givan, Alice. Flow Cytometry: First Principles. New York: Wiley, 1992.– Melamed, Myron. Flow Cytometry and Sorting. New York: Wiley, 1990.– Salzman, Gary. New Technologies in Cytometry. Proceedings of SPIE,

Los Angeles, 1989.– Van Dilla, Marvin. Flow Cytometry: Instrumentation and Data Analysis.

London: Academic Press, 1985.

• WWW sites– Purdue University Cytometry Laboratories

• http://www.cyto.purdue.edu/ (all figures marked PUFCL)

– Becton-Dickinson, Inc.• http://www.bd.com/

– Coherent• http://www.cohr.com/

– Bigelow Laboratory for Ocean Sciences (cytometric images)• http://www.bigelow.org/cytometry/index.html