Introduction to Flow Cytometry

An electronic book introducing flow cytometry

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Are you interested in quickly learning the basics of an important and rapidly developing area of instrumental biology, flow cytometry?

Have you always wondered how to efficiently, accurately and precisely gather data from populations of biological cells?

Would you like to learn the basics of flow Cytometry quickly and in the privacy of your own home?

If your answer to any of these questions is "yes," ATP hopes you will enjoy Introduction to Flow Cytometry!

Introduction to Flow Cytometry is an electronic book and educational .pdf file specifically designed to assist students understand the biology, chemistry, physics, theory, instrumentation and practice of flow cytometry. Since modern pdf readers are capable of text-to-speech functions, Introduction to Flow Cytometry can be listened to as well as read. </p>

Thank you for your interest in Introduction to Flow Cytometry. Please peruse the detailed information about the book below.

Chapter 1, Page 12, sample introduction.

1.1 WHAT IS FLOW CYTOMETRY?

The process of flow cytometry is a synthesis of many technologies. Chemical tissue disruption and fluorescent labels provide the ability to separate and label cells and their characteristics for study. Fluid dynamics provides rapid delivery of individual cells into a small region for sensing. Laser light sources and optics illuminate the cells and excite their photochemical labels. Photosensors and electronics create electronic signals that are proportional to the characteristics of each cell. Computer data collection and software data analysis perform statistical analysis and display the population's properties revealed by the characteristics of each of its members. Finally, computer and electronic control systems use the physical principles of piezoelectricity, fluid dynamics, and electricity and magnetism to sort subpopulations. A block diagram of a flow cytometer is shown in Figure 1.1.

Flow cytometry is a process that senses the characteristics of each cell in the population, stores data about each cell, and displays data about the population as a histogram. This process allows the rapid creation of an accurate graph of population data in an afternoon that might take months with conventional microscopic or biochemical methods. This is especially important in the analysis of tissues such as tumors and blood that contain large numbers of cells. In addition, the study of a wide variety of physical and chemical characteristics of the cells is possible.

Chapter 1, Page 19-20, sample overview.

1.4 THE FOUR SYSTEMS OF FLOW CYTOMETRY

Construction of flow cytometers varies with each manufacturer, but all flow cytometers have a group of common functions. This section discusses these common functions in the context of four interdependent systems described above: the delivery system, the illumination and detection system, the data collection and analysis system, and the sorting system. Block diagrams of each of these systems are found at the beginning of each part of this book.

1.4.1 THE COMPLETE CYTOMETER

Modern flow cytometers are complex instruments consisting of a synthesis of many individual components. A block diagram of a laser based flow cytometer is shown in Figure 1.1. Successful operation of flow cytometers involves a variety of complex tasks including the correct design of cytometric studies, actual operation of the instrument, the collection of data, and the interpretation of data. To accomplish these tasks, the operator must understand how physical processes, biochemical techniques, and cellular morphology interact with the hardware components of the cytometer to yield data.

1.4.2 THE DELIVERY SYSTEM

To prepare a population for analysis, each cell must be separated from other cells in the population through the creation of a single cell suspension. A single cell suspension is a liquid containing only individual cells. In a fluid tissue such as blood, the natural state of the sample is a single cell suspension. In this context, the operator usually separates the unwanted cells (such as red cells) from desired cells (such as white cells) prior to analysis. In contrast, the separation of cells from a solid tissue such as a tumor requires filtration, enzymatic digestion, or mechanical disruption. When tissues are solid, the addition of a liquid aids in the analysis of the population by providing a medium for transportation.

Usually, the liquids that suspend and transport cells in flow cytometry are isotonic. The isotonic liquid provides a chemically neutral media for the transportation of cells. In addition, the presence of dissociated ions such as sodium and chloride allow the sorting system to impart an electrical charge to droplets of the liquid.

If the cells are to undergo an intrinsic analysis, the creation of a single cell suspension is the only requirement before analysis. If the cells are to undergo an extrinsic analysis, labeling of the cells occurs before placement in the flow cytometer. Often, the correct concentration of the fluorescent label is simply added to the single cell suspension.

The flow cytometer transports cells to the flow chamber in a liquid flow called the sample flow. The sample flow is injected into the center of the sheath flow in the flow chamber. The concentric liquids allow rapid transportation of the cells and provide a method for accurate and precise localization of the cells during measurement at the interrogation point.

1.4.3 THE ILLUMINATION AND DETECTION SYSTEM

The illumination and detection system illuminates the cells with a very bright light and detects each cell's interaction with that light. Sensors detect light scattered from the surfaces and inner structure of the cell, and fluorescence signals emitted from the labeled characteristics of the cell.

These tasks are accomplished with lasers, photodiodes, and photomultiplier tubes. At the interrogation point, a tightly focused beam of very bright light illuminates the cells. Usually, cytometric illumination sources are lasers. The light scatters from the surface of each cell and from structures inside the cell. The light also excites any fluorescent stains in the cell and causes them to fluoresce.

A photodiode detects the light scattered from the cell surfaces and a series of photomultiplier tubes detects the various fluorescence wavelengths emitted from labeled cellular components. In some types of analyses, photomultiplier tubes also detect scattered light from cellular components.

A series of optical filters and dichroic mirrors guide illumination source light, scattered light, and the fluorescence emission from labeled components into the photomultiplier tubes. The filters and mirrors separate and direct different wavelengths of light into separate photomultiplier tubes. This separation of individual wavelengths into dedicated photosensors allows the cytometer to detect the amplitude of each wavelength, and thus, the relative quantity of the labeled characteristic in each cell.

The light emitted from the interrogation point may be light scattered from the edge of the cell, light scattered from cellular components within the cell, or fluorescence emitted from labeled cellular components. Both photodiodes and photomultiplier tubes emit analog electrical signals that are proportional to the light received from the interrogation point.

1.4.4 THE DATA COLLECTION AND ANALYSIS SYSTEM

In the data collection and analysis system, the electrical signals from the photosensors undergo amplification, digitization, and storage on floppy or hard disk. A dedicated computer then presents a frequency distribution of the data for interpretation.

Transampedance amplification converts the electrical current output of the sensors into electrical voltage in preparation for digitization. Analog to digital converters convert the analog voltage signals to digital representations of the signals. The signals are sent to a computer, where tape, floppy disk, or hard disk media store these digital representations of the analog signals for future reference and analysis.

Interpretation of the data employs statistical analysis or a graphical display called a histogram. The computer retrieves the data stored in the cytometer's memory and performs mathematical or non-mathematical analyses for redisplay or printing. This allows the operator to reproduce analyses or perform new analyses on old data.

1.4.5 THE SORTING SYSTEM

The sorting system uses the population data collected by the data collection and analysis system to isolate subpopulations of cells. Selection of a subpopulation for sorting occurs after cytometric analysis of the complete population.

A single channel pulse height analyzer is set to match the waveform of the cells desired for sorting. The operator replaces the population in the delivery system which returns the cells to the interrogation point. As each cell passes through the interrogation point, the waveform it generates is electronically compared with the waveform in the single channel pulse height analyzer. If the waveforms match, the cell is selected for sorting.

As the cells pass out of the interrogation point, a piezoelectric material vibrates the flow, breaking it into droplets. A charging collar charges the droplets that contain cells selected for sorting with an electrostatic charge as they break off of the flow. Finally, pair of electrostatically charged plates deflects the charged droplets that contain selected cells into separate collection vessels.

Chapter 3, page 57-58, sample chapter summary.

3.7 SUMMARY

Proper sample preparation is essential to the correct collection and analysis of data in flow cytometry. The creation of single cell suspensions, elimination of sources of error, and proper labeling of cell characteristics laid the foundation for all cytometric analyses.

THE SINGLE CELL SUSPENSION AND LABELING

The first step of sample preparation is the creation of a single cell suspension. The single cell suspension permits the transportation of cells in concentric liquid flows that provide positional certainty. The cells may also undergo chemical treatment to label cell characteristics of interest, and to remove any molecules that might cause a spurious result. The fluorescent molecules bind stoichiometrically to cellular characteristics so their fluorescence emission is proportional to the quantity of the labeled characteristic. If the fluorescent molecule does not form a stable stoichiometric complex with the characteristic, the proportionality of light emission from the characteristic will be lost.

LIGHT AND LUMINESCENCE

Light has higher energy when the wavelength is short and the frequency is high. Coherent light is in phase and monochromatic light of a single wavelength. Luminescence describes all light emission except incandescence (light emitted from a heated object), and chemiluminescent molecules emit fluorescence or phosphorescence when they absorb light of the proper wavelength.

FLUORESCENCE AND PHOSPHORESCENCE

Certain molecules with free electrons in double bonds, amino groups, hydroxyl groups, aromatic rings, or heterocyclic rings absorb light, causing their electrons to move into higher energy levels. Fluorescence is rapid, high energy emission of light as electrons move from the S1 energy level to the S0 ground state. Phosphorescence is slow, low energy emission of light as electrons move from the T1 triplet energy level to the S0 ground state. Phosphorescence may continue for as long as ten minutes after the exciting radiation has ceased, while fluorescence ceases within 10E-7 seconds.

FLUORESCENCE QUANTUM YIELD

The amount of fluorescence a molecule emits in response to radiation absorption is its fluorescence quantum yield. If a molecule fluoresces without electrons proceeding through radiationless transitions or intersystem crossing, the quantum efficiency is 1. If energy is lost to quenching mechanisms, the quantum efficiency is less than 1. Flexible fluorescent molecules such as propidium iodide, exhibit an increase in quantum efficiency when bound to a cell characteristic.

FLUORESCENCE QUENCHING

Quenching occurs when molecules lose their energy to other molecules through one of four mechanisms: self quenching, energy transfer, charge transfer, or intersystem crossing.

BACKGROUND FLUORESCENCE AND AUTOFLUORESCENCE

Spurious data collection may be due to background fluorescence or cell characteristics that are autofluorescent. Background fluorescence may occur as a result of fluorescent molecules binding debris or from fluorescent fixatives. Fixatives with very low fluorescence such as paraformaldehyde can decrease background fluorescence.

REFERENCES

Becker, R.S., Theory and Interpretation of Fluorescence and Phosphorescence, Wiley Interscience, New York, 1969.

Geacintov, N.E., Swenberg, C.E., Magnetic Field Effects in Organic Molecular Spectroscopy, in

Lumb M.D., Luminescence Spectroscopy, Academic Press, New York, 1978.

Parker, C.A., Photoluminescence of Solutions, Elsevier Publishing Company, New York, 1968.

Rhys-Williams, A.T., An Introduction to Fluorescence Spectroscopy, Perkin-Elmer.

Shapiro, H.M., Practical Flow Cytometry, Alan R. Liss, Inc., New York, 1988.

-- End Sample Excerpts --

Introduction to Flow Cytometry is a detailed introductory textbook for beginning student of flow cytometry. It is written, designed and notated for rapid communication of the founding flow cytometric principles, and should be useful to students, rabid insatiable learners and anyone else who wishes to learn the fundamentals of flow cytometry rapidly

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