Thermal Pollution

Thermal Pollution: Effects, Causes and Control!

An increase in the optimum water temperature by industrial process (steel fac­tories, electric power houses and atomic power plants) may be called as “Thermal Pollution.” Many industries generate their own power and use wa­ter to cool their generator.

This hot water is released into the system from where it was drawn, causing a warming trend of surface water. If the system is poorly flushed, a permanent increase in the temperature may result. However, if the water is released into the well flushed system, permanent increase in tempera­ture does not occur.


Many organisms are killed instantly by the hot water resulting into a high mor­tality. It may bring other disturbance in the ecosystem. The egg of fish may hatch early or fail to hatch at all. It may change the diurnal and seasonal be­haviour and metabolic responses of organisms. It may lead to unplanned mi­gration of aquatic animals.

Macro-phytic population may also be changed. As temperature is an important limiting factor, serious changes may be brought about even by a slight increase in temperature in a population. For minimising thermal pollution, hot water should be cooled before release from factories and removal of forest canopies and irrigation return flows should be prohibited.

Causes or Sources of Thermal Pollution:

The various causes of thermal pollution are as follows: 

(1) Coal-fired Power Plants:

Some thermal power plants use coal as fuel. Coal-fired power plants constitute the major source of the thermal pollution.

(2) Industrial Effluents:

Industries generating electricity require large amount of Cooling water for heat removal. Other industries like textile, paper, and pulp and sugar industry also re­lease heat in water, but to a lesser extent.

(3) Nuclear Power Plants:

Nuclear power plants emit a large amount of unutilized heat and traces of toxic radio nuclear into nearby water streams. Emissions from nuclear reactors and processing installations are also responsible for increasing the temperature of water bodies.

(4) Hydro Electric Power:

Generation of hydro-electric power also results in negative thermal loading of water bodies.

(5) Domestic Sewage:

Domestic sewage is often discharged into rivers, lakes, canals or streams with­out waste treatment. The municipal water sewage normally has a higher tem­perature than receiving water. With the increase in temperature of the receiv­ing water the dissolved oxygen content (DO) decreases and the demand of oxy­gen increases and anaerobic conditions occur.

Control of Thermal Pollution:

Control of thermal pollution is necessary as its detrimental effects on aquatic ecosystem may be detrimental in the future. Viable solutions to chronic ther­mal discharge into water bodies are as follows: 

(1) Cooling Ponds:

Cooling ponds or reservoirs constitute the simplest method of controlling ther­mal discharges. Heated effluents on the surface of water in cooling ponds maximize dissipation of heat to the atmosphere and minimize the water area and volume. This is the simplest and cheapest method which cools the water to a considerable low tem­perature. However, the technique alone is less desirable and inefficient in terms of air-water contact.

(2) Cooling Towers:

Using water from water sources for cooling purposes, with subsequent return to the water body after passing through the condenser is termed as cooling process. In order to make the cooling process more effective, cooling towers are designed to control the temperature of water. In-fact, cooling towers are used to dissipate the recovered waste heat so as to eliminate the problems of thermal pollution.

(3) Artificial Lake:

Artificial lakes are man-made bodies of water which offer possible alternative to once through cooling. The heated effluents may be discharged into the lake at one end and the water for cooling purposes may be withdrawn from the other end. The heat is eventually dissipated through evaporation.

These lakes have to be rejuvenated continuously. A number of methods have been suggested and developed for converting the thermal effluents from power plants into useful heat resources for maximing the benefits.

Some of the potential physical applications for thermal discharge (rejected heat) of power plants are:

  1. Industrial and space heating.
  2. Biological applications such as soil warming.

iii. Fish culture, livestock shelters and for heating greenhouses.

Most of these potential physical applications are of colder regions or locations.






Microorganisms influence the chemical state – speciation and hence the mobility of the metals by numerous and complex mechanisms ranging from direct processes like metal transformation and intracellular uptake, to more indirect processes via production of substances that render the metals more or less mobile.


Principal ways in which microorganisms can influence metal mobility. M=Metal species, (Ledin and Pedersen, 1996)

The contact of the soluble metal species with the microbial cells results in in-situ transformations of the targeted species. As a consequence, the metals are immobilised by the microbial biomass. Soluble metal and metalloid species may be sequestered from water streams via their interaction with microbial cells by active i.e. metabolic – energy dependent and passive i.e. non metabolic – energy independent processes.

Metabolism mediated immobilisation of metal species by active microbial cells includes different mechanisms such as bioprecipitation and biological reduction / oxidation.

Passive metal uptake by microbial cells is described by the general term biosorption and includes different mechanisms of physico-chemical interaction between the microbial cell biopolymers and the metal species such as complexation, chelation, ion exchange and precipitation.

Both living-metabolizing and non-metabolizing microbial cells, may be used in technology development for:

  1. The removal of metals from aqueous industrial effluents,
  2. Metal recovery from industrial process streams,
  3. Bioremediationof contaminated surface waters and groundwater.

Among the existing microorganisms in nature, bacteriafungiyeastsalgae , bacteria are used in most cases for metal and metalloid sequestering from water streams.


Biosorption can be defined as the selective sequestering of metal soluble species that result in the immobilization of the metals by microbial cells. It refers to physicochemical mechanisms of inactive (i.e. non-metabolic) metal uptake by microbial biomass. Metal sequestering by different parts of the cell can occur via various processes .

  1. Complexation
  2. Chelation
  3. Coordination
  4. Ion exchange
  5. Precipitation
  6. Reduction

Immobilization may be the result of more than one mechanism, for example, metal complexation may be followed by metal reduction or metal precipitation.

Metabolically active and inactive cells behave in different ways. Thus inactive microbial cells can only immobilize metals by biosorption, whereas active microbial cells may immobilize soluble metal species both by biosorption and by other mechanisms that are part of and/or are due to the microbial metabolism. The cell wall is usually the first cellular structure in contact with the soluble species of the metals if we exclude the possibility of metal species interactions and retention by extracellular excretions produced by some microbial cells.

The functional groups available in the biopolymers, constituents of the cell wall and the other parts of the cell, have a significant potential for metal binding. These biopolymers, constituents of the cell wall and the other parts of the cell possess functional groups that have a significant potential for metal binding.Furthermore, intracellular biopolymers such as proteins and DNA may also contribute to metal immobilisation. In many cases, extracellular polymeric substances such as Exo-PolySaccharides (EPS) that are closely related to the cell membrane can also participate in metal immobilisation.


How bacteria influence speciation of mercury in the environment


Mercury is a contaminant of global concern, as bioaccumulation of methylmercury poses significant risk to aquatic ecosystems and human health. Controlling the transport of mercury in the environment is challenging due to deposition of airborne mercury at locations far from point sources. Mobility of mercury is strongly dependent on its chemical form, with the elemental mercury being volatile and hence mobile in the environment, while oxidized forms are much less mobile (though more toxic). This study, led by Dr. Mishra along with follow researchers of molecular environmental science group at Argonne National Laboratory, has provided improved understanding of the role of bacteria in controlling the chemical form of mercury in subsurface environments. Using X-ray absorption spectroscopy experiments at the Advanced Photon Source to study the sorption of oxidized HgII to Bacillus subtilis, a gram positive soil bacterium, they determined that HgII sorbs to bacterial cells via high and low affinity sulfhydryl and carboxyl binding groups on the cell surfaces. Additionally, they found that HgII that is sorbed to cells via high affinity sulfhydryl groups remains unavailable for reduction by magnetite, a reactive iron-containing mineral often found in sediments, even after two months of reaction time. This is in sharp contrast to their observation of complete reduction of HgII to Hg0 within two hours when HgII is sorbed to cells via the lower affinity carboxyl groups. Since binding of HgII to high-affinity sulfhydryl groups on bacteria could have important implications for the overall mobility of Hg in subsurface environments, these results identify a mechanism by which mercury might be immobilized in the environment.






Phytoremediation is a bioremediation process that uses various types of plants to remove, transfer, stabilize, and/or destroy contaminants in the soil and groundwater. There are several different types of phytoremediation mechanisms. These are:

  1. Rhizosphere biodegradation.In this process, the plant releases natural substances through its roots, supplying nutrients to microorganisms in the soil. The microorganisms enhance biological degradation.
  2. Phyto-stabilization.In this process, chemical compounds produced by the plant immobilize contaminants, rather than degrade them.
  3. Phyto-accumulation (also called phyto-extraction).In this process, plant roots sorb the contaminants along with other nutrients and water. The contaminant mass is not destroyed but ends up in the plant shoots and leaves. This method is used primarily for wastes containing metals. At one demonstration site, water-soluble metals are taken up by plant species selected for their ability to take up large quantities of lead (Pb). The metals are stored in the plantÍs aerial shoots, which are harvested and either smelted for potential metal recycling/recovery or are disposed of as a hazardous waste. As a general rule, readily bioavailable metals for plant uptake include cadmium, nickel, zinc, arsenic, selenium, and copper. Moderately bioavailable metals are cobalt, manganese, and iron. Lead, chromium, and uranium are not very bioavailable. Lead can be made much more bioavailable by the addition of chelating agents to soils. Similarly, the availability of uranium and radio-cesium 137 can be enhanced using citric acid and ammonium nitrate, respectively.
  4. Hydroponic Systems for Treating Water Streams (Rhizofiltration).Rhizofiltration is similar to phyto-accumulation, but the plants used for cleanup are raised in greenhouses with their roots in water. This system can be used for ex-situ groundwater treatment. That is, groundwater is pumped to the surface to irrigate these plants. Typically hydroponic systems utilize an artificial soil medium, such as sand mixed with perlite or vermiculite. As the roots become saturated with contaminants, they are harvested and disposed of.
  5. Phyto-volatilization.In this process, plants take up water containing organic contaminants and release the contaminants into the air through their leaves.
  6. Phyto-degradation.In this process, plants actually metabolize and destroy contaminants within plant tissues.
  7. Hydraulic Control.In this process, trees indirectly remediate by controlling groundwater movement. Trees act as natural pumps when their roots reach down towards the water table and establish a dense root mass that takes up large quantities of water. A poplar tree, for example, pulls out of the ground 30 gallons of water per day, and a cottonwood can absorb up to 350 gallons per day.

The plants most used and studied are poplar trees. The U.S. Air Force has used poplar trees to contain trichloroethylene (TCE) in groundwater. In Iowa,EPA demonstrated that poplar trees acted as natural pumps to keep toxic herbicides, pesticides, and fertilizers out of the streams and groundwater. The US Army Corps of Engineers has experimented with wetland plants to destroy explosive compounds in the soil and groundwater. Submersed and floating-leafed species (coontail and pondweed, and arrowhead, respectively) decreased trinitrotoluene (TNT) to 5% of original concentration. Submersed plants were able to decrease Royal Demolition Explosive (RDX) levels by 40%, and when microbial degradation was added, RDX decreased by 80%. Sunflowers, using rhizofiltration, were used successfully to remove radioactive contaminants from pond water in a test at Chernobyl, Ukraine.

Limitations and Concerns

The toxicity and bioavailability of biodegradation products is not always known.

Degradation by-products may be mobilized in groundwater or bio-accumulated in animals. Additional research is needed to determine the fate of various compounds in the plant metabolic cycle to ensure that plant droppings and products do not contribute toxic or harmful chemicals into the food chain.

Scientists need to establish whether contaminants that collect in the leaves and wood of trees are released when the leaves fall in the autumn or when firewood or mulch from the trees is used.

Disposal of harvested plants can be a problem if they contain high levels of heavy metals.

The depth of the contaminants limits treatment. The treatment zone is determined by plant root depth. In most cases, it is limited to shallow soils, streams, and groundwater. Pumping the water out of the ground and using it to irrigate plantations of trees may treat contaminated groundwater that is too deep to be reached by plant roots. Where practical, deep tilling, to bring heavy metals that may have moved downward in the soil closer to the roots, may be necessary.

Generally, the use of phytoremediation is limited to sites with lower contaminant concentrations and contamination in shallow soils, streams, and groundwater. However, researchers are finding that the use of trees (rather than smaller plants) allows them to treat deeper contamination because tree roots penetrate more deeply into the ground.

The success of phytoremediation may be seasonal, depending on location. Other climatic factors will also influence its effectiveness.

The success of remediation depends in establishing a selected plant community. Introducing new plant species can have widespread ecological ramifications. It should be studied beforehand and monitored. Additionally, the establishment of the plants may require several seasons of irrigation. It is important to consider extra mobilization of contaminants in the soil and groundwater during this start-up period.

If contaminant concentrations are too high, plants may die.

Some phytoremediation transfers contamination across media, (e.g., from soil to air).

Phytoremediation is not effective for strongly sorbed contaminants such as polychlorinated biphenyls (PCBs).

Phytoremediation requires a large surface area of land for remediation.


Phytoremediation is used for the remediation of metals, radionuclidespesticides, explosives, fuels, volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs). Research is underway to understand the role of phytoremediation to remediate perchlorate, a contaminant that has been shown to be persistent in surface and groundwater systems. It may be used to cleanup contaminants found in soil and groundwater. For radioactive substances, chelating agents are sometimes used to make the contaminants amenable to plant uptake.




Contaminants in the environment pose a global problem for wildlife and human health. Phytoremediation is a recently developed technology that offers a cost-effective solution by using plants, and associated soil microbes, to reduce the content, or toxic effects, of contaminants in the environment. Phytoremediation technologies include:

(a) phytostabilization, where contaminants are retained in the soil,

(b) phytodegradation, where organic contaminants are converted to less harmful substances,

(c) phytovolatilization, where contaminants are converted inside plants to a gaseous state and released into the atmosphere via the evapotranspiration process, and,

(d) phytoextraction, where plants are used to accumulate contaminants in the aboveground, harvestable biomass.







Bioremediation means to use a biological remedy to abate or clean up contamination. This makes it different from remedies where contaminated soil or water is removed for chemical treatment or decontamination, incineration, or burial in a landfill. Microbes are often used to remedy environmental problems found in soil, water, and sediments. Plants have also been used to assist bioremediation processes. This is called phytoremediation. Biological processes have been used for some inorganic materials, like metals, to lower radioactivity and to remediate organic contaminants. With metal contamination the usual challenge is to accumulate the metal into harvestable plant parts, which must then be disposed of in a hazardous waste landfill before or after incineration to reduce the plant to ash. Two exceptions are mercury and selenium, which can be released as volatile elements directly from plants to atmosphere. The concept and practice of using plants and microorganisms to remediate contaminated soil have developed over the past thirty years.

The idea of bioremediation has become popular with the onset of the twenty-first century. In principle, genetically engineered plants and microorganisms


Essential Factors for Microbial Bioremediation


Factor Desired Conditions
Microbial population Suitable kinds of organisms that can biodegrade all of the contaminants
Oxygen Enough to support aerobic biodegradation (about 2% oxygen in the gas phase or 0.4 mg/liter in the soil water)
Water Soil moisture should be from 50–70% of the water holding capacity of the soil
Nutrients Nitrogen, phosphorus, sulfur, and other nutrients to support good microbial growth
Temperature Appropriate temperatures for microbial growth (0–40˚C)
pH Best range is from 6.5 to 7.5


can greatly enhance the potential range of bioremediation. For example, bacterial enzymes engineered into plants can speed up the breakdown of TNT and other explosives. With transgenic poplar trees carrying a bacterial gene, methyl mercury may be converted to elemental mercury, which is released to the atmosphere at extreme dilution. However, concern about release of such organisms into the environment has limited actual field applications.

Natural Bioremediation

Natural bioremediation has been occurring for millions of years. Biodegradation of dead vegetation and dead animals is a kind of bioremediation. It is a natural part of the carbon, nitrogen, and sulfur cycles. Chemical energy present in waste materials is used by microorganisms to grow while they convert organic carbon and hydrogen to carbon dioxide and water.

Managed Bioremediation

When bioremediation is applied by people, microbial biodegradation processes are said to be managed. However, bioremediation takes place naturally and often it occurs prior to efforts to manage the process. One of the first examples of managed bioremediation was land farming (refers to the managed biodegradation of organic compounds that are distributed onto the soil surface, fertilized, and then tilled). Many petroleum companies have used it. High-molecular-weight organic compounds (i.e., oil sludges and wastes) are spread onto soil and then tilled into the ground with fertilizer, as part of the managed bioremediation process. Good conditions for microbial biodegradation are maintained by controlling soil moisture and soil nutrients. In 1974 R.L. Raymond was awarded a patent for the bioremediation of gasoline. This was one of the first patents granted for a bioremediation process.

Since about 1980, prepared bed systems have been used for bioremediation. In this approach, contaminated soil is excavated and deposited with appropriate fertilizers into a shallow layer over an impermeable base. Conditions are managed to obtain biodegradation of the contaminants of concern.


Composting has been used as a bioremediation process for many different organic compounds. It is widely employed to recycle nutrients in garden and yard waste. A finished compost can be used as a soil conditioner. Extending composting technology to new bioremediation applications requires experiments. The biodegradation process must be effective within the context of existing environmental conditions, and odors and gases that are generated by the process have to be strictly controlled.

In Situ Bioremediation

In situ processes (degrading the contaminants in place) are often recommended because less material has to be moved. These processes can be designed with or without plants. Plants have been used because they take up large quantities of water. This helps to control contaminated water, such as a groundwater contaminant plume, in the soil. Aerobic (oxygen-using) processes may occur in the unsaturated layer of soil, the vadose zone, which is found above the water table. The vadose zone is defined as the layer of soil having continuously connected passages filled with air, while the saturated zone is the deeper part where the pores are filled with water. Oxygen moves in the unsaturated zone by diffusion through pores in the soil. Some plants also provide pathways to move oxygen into the soil. This can be very important to increase the aerobic degradation of organic compounds.

Fate of Various Organic Contaminants

Petroleum-contaminated soil has been remediated in situ with plants added to enhance the degradation processes. The biodegradation of phenol, oil, gasoline, jet fuel, and other petroleum hydrocarbons occurs in soil. When plants are present, soil erosion is reduced and more microbes are present in the plant root zone. Methyl tertiary butyl ether (MTBE), used in gasoline to enhance the octane rating of the fuel, is difficult to remediate because it is very soluble in water and is hard to break down using microbes normally present in soil. In vegetation-based bioremediation, MTBE is moved from the soil to the atmosphere along with the water that plants take up from soil and release to the air. The MTBE breaks down rapidly in the atmosphere. Benzotriazoles, used as corrosion inhibitors in antifreeze and aircraft deicer fluids, are treated by plant-based bioremediation. The benzotriazole adsorbs or sticks to the plant roots and ends up as part of the plant biomass. Trichloroethylene (TCE) is a common chlorinated solvent that is biotransformed in the soil. It can be taken up by plants along with water. Then the TCE diffuses into the atmosphere where it is destroyed by atmospheric processes.


Bioremediation requires good nutrient and environmental conditions for biodegradation. When oxygen is needed for oxidation of the organic contaminants, bioventing (pumping air into the soil) is often used. Sometimes, fertilizers are added to the soil. In certain places irrigation is necessary so that plants or microbes can grow.


There are a number of cost/efficiency advantages to bioremediation, which can be employed in areas that are inaccessible without excavation. For example, hydrocarbon spills (specifically, petrol spills) or certain chlorinated solvents may contaminate groundwater, and introducing the appropriate electron acceptor or electron donor amendment, as appropriate, may significantly reduce contaminant concentrations after a long time allowing for acclimation. This is typically much less expensive than excavation followed by disposal elsewhere, incineration or other ex situ treatment strategies, and reduces or eliminates the need for “pump and treat”, a practice common at sites where hydrocarbons have contaminated clean groundwater. Using archaea for bioremediation of hydrocarbons also has the advantage of breaking down contaminants at the molecular level, as opposed to simply chemically dispersing the contaminant.

Read more:


Alexander, Martin. (1994). Biodegradation and Bioremediation. New York: Academic Press.

Davis, Lawrence C.; Castro-Diaz, Sigifredo; Zhange, Qizhi; and Erickson, Larry E. (2002). “Benefits of Vegetation for Soils with Organic Contaminants.” Critical Reviews in Plant Sciences. 21 (5):457–491.

Eweis, Juana B.; Ergas, Sarina J.; Chang, Daniel P.Y.; and Schroeder, Edward D. (1998). Bioremediation Principles. New York: McGraw-Hill.

Hannink, Nerissa K.; Rosser, Susan J.; and Bruce, Neil C. (2002). “Phytoremediation of Explosives.” Critical Reviews in Plant Sciences. 21(5):511–538.

McCutcheon, Steven C.; Schnoor, Jerald L., eds. (2003). Phytoremediation: Managing Contamination by Organic Compounds. New York: Wiley-Interscience.

Pilon-Smits, Elizabeth, and Pilon, Marinus. (2002). “Phytoremediation of Metals Using Transgenic Plants.” Critical Reviews in Plant Sciences. 21(5):439–456.

Rittmann, Bruce E. (1993). In Situ Bioremediation: When Does It Work? Washington, DC: National Academy Press.

Thomas, J.M.; Ward, C.H.; Raymond, R.L.; Wilson, J.T.; and Loehr, R.C. (1992). “Bioremediation.” In Encyclopedia of Microbiology, Vol. 1, edited by Joshua Lederberg, pp. 369–385. New York: Academic Press.


Internet Resources

Bioremediation Discussion Group. Available from .

Natural and Accelerated Bioremediation Research Web site. Available from .

Larry Eugene Erickson and Lawrence C. Davis

Read more:

Fluorescence Microscopy

Introduction to Fluorescence Microscopy

The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence orphosphorescence. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.Fl-1


British scientist Sir George G. Stokes first described fluorescence in 1852 and was responsible for coining the term when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. Stokes noted that fluorescence emission always occurred at a longer wavelength than that of the excitation light. Early investigations in the 19th century showed that many specimens (including minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, and inorganic compounds) fluoresce when irradiated with ultraviolet light. However, it was not until the 1930s that the use of fluorochromes was initiated in biological investigations to stain tissue components, bacteria, and other pathogens. Several of these stains were highly specific and stimulated the development of the fluorescence microscope.


The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can simultaneously identify several target molecules simultaneously. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved.


Fundamentals of Excitation and Emission

The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.

Illustrated in Figure 1 is a cutaway diagram of a modern epi-fluorescence microscope equipped for both transmitted and reflected fluorescence microscopy. The vertical illuminator in the center of the diagram has the light source positioned at one end (labeled the episcopic lamphouse) and the filter cube turret at the other. The design consists of a basic reflected light microscope in which the wavelength of the reflected light is longer than that of the excitation. Johan S. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. In a fluorescence vertical illuminator, light of a specific wavelength (or defined band of wavelengths), often in the ultraviolet, blue or green regions of the visible spectrum, is produced by passing multispectral light from an arc-discharge lamp or other source through a wavelength selective excitation filter. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also termed a dichroic) mirror or beamsplitter, through the microscope objective to bath the specimen with intense light. If the specimen fluoresces, the emission light gathered by the objective passes back through the dichromatic mirror and is subsequently filtered by abarrier (or emission) filter, which blocks the unwanted excitation wavelengths. It is important to note that fluorescence is the only mode in optical microscopy where the specimen, subsequent to excitation, produces its own light. The emitted light re-radiates spherically in all directions, regardless of the excitation light source direction.


As presented in Figure 1, the reflected light vertical illuminator comprises an arc-discharge lamphouse at the rear end (usually a mercury or xenon burner). Excitation light travels along the illuminator perpendicular to the optical axis of the microscope, passes through collector lenses and a variable, centerable aperture diaphragm, and then through a variable, centerable field diaphragm (see Figure 1). The light then impinges upon the excitation filter where selection of the desired band and blockage of unwanted wavelength occurs. The selected wavelengths, after passing through the excitation filter, reach the dichromatic beamsplitting mirror, which is a specialized interference filter that efficiently reflects shorter wavelength light and efficiently passes longer wavelength light. The dichromatic beamsplitter is tilted at a 45-degree angle with respect to the incoming excitation light and reflects this illumination at a 90-degree angle directly through the objective optical system and onto the specimen. Fluorescence emission produced by the illuminated specimen is gathered by the objective, now serving in its usual image-forming function. Because the emitted light consists of longer wavelengths than the excitation illumination, it is able to pass through the dichromatic mirror and upward to the observation tubes or electronic detector.

Stokes’ Shift

Vibrational energy is lost when electrons relax from the excited state back to the ground state. As a result of the energy loss, the emission spectrum of an excited fluorophore is usually shifted to longer wavelengths when compared to the absorption or excitation spectrum (note that wavelength varies inversely to radiation energy). This well-documented phenomenon is known as Stokes’ Law or Stokes’ shift. As Stokes’ shift values increase, it becomes easier to separate excitation from emission light through the use of fluorescence filter combinations.



Fading, Quenching, and Photobleaching

A wide spectrum of conditions often come into play that ultimately affect the re-radiation of fluorescence emission and thus reduce the intensity. The general term for a reduction of fluorescence emission intensity is fading, a catch-all category that is usually further subdivided into quenching and photobleaching phenomena for more precise descriptions. Photobleaching is the irreversible decomposition of the fluorescent molecules in the excited state because of their interaction with molecular oxygen before emission. The occurrence of photobleaching is exploited in a technique known as fluorescence recovery after photobleaching (FRAP), a very useful mechanism for investigating the diffusion and motion of biological macromolecules. The method is based upon photobleaching a sharply defined region of the specimen by an intense burst of laser light, accompanied by the subsequent observation of the rates and pattern of fluorescence recovery in the photobleached area.


Presented in Figure 4 is a typical example of photobleaching (fading) observed in a series of digital images captured at different time points for a multiply-stained culture of Indian Muntjac deer epidermis fibroblast cells.

Fluorescence Light Sources

The mercury burners do not provide even intensity across the spectrum from ultraviolet to infrared, and much of the intensity of the lamp is expended in the near ultraviolet. Prominent peaks of intensity occur at 313, 334, 365, 406, 435, 546, and 578 nanometers. At other wavelengths in the visible light region, the intensity is steady although not nearly so bright .


In the past few years, optical microscopy has experienced an increase in the application of laser light sources, particularly the argon-ion and argon-krypton (ion) lasers. These lasers have the virtues of small source size, low divergence, near-monochromicity, and high mean luminance.


Luminous Density of Selected Light Sources

Lamp Current
Luminous Flux
Mean Luminous
Density (cd/mm2)
Arc Size
(H x W)
Mercury Arc
(100 Watt)
5 2200 1700 0.25 x 0.25
Xenon Arc
(75 Watt)
5.4 850 400 0.25 x 0.50
Xenon Arc
(500 Watt)
30 9000 3500 0.30 x 0.30
8 2800 45 4.2 x 2.3





Table 1

The efficiency of detection is a function of the optical collection efficiency and the detector quantum efficiency. A 1.4-numerical aperture objective with 100-percent transmission (an unrealistic condition) has a maximum collection efficiency, limited by the acceptance angle of about 30 percent. The transmission efficiency of the dichromatic mirror is 85 percent and that of the barrier filter is 80 percent. The overall collection efficiency is then about 20 percent or 140 billion photons per second. If the detector is a conventional charge-coupled device (CCD), the quantum efficiency is about 50 percent for the green fluorescein emission (at 525 nanometers), so the detected signal would be 70 billion photons per second or about 10 percent of the emitted fluorescence. Even with a perfect detector (100 percent quantum efficiency), only about 20 percent of the fluorescence emission photons can be detected.


Detecting Single Molecules

Under ideal conditions, it is often possible to detect the fluorescence emission from a single molecule, provided that the optical background and detector noise are sufficiently low. As discussed above, a single fluorescein molecule could emit as many as 300,000 photons before it is destroyed by photobleaching. Assuming a 20-percent collection and detection efficiency, about 60,000 photons would be detected. Using avalanche photodiode or electron multiplying CCD detectors for these experiments, investigators have been able to monitor the behavior of single molecules for many seconds and even minutes. The major problem is adequate suppression of the optical background noise. Because many of the materials utilized in construction of microscope lenses and filters display some level of autofluorescence, efforts were initially directed toward the manufacture of very low fluorescence components. However, it soon became evident that fluorescence microscopy techniques utilizing total internal reflection (TIR) provided the desired combination of low background and high excitation light flux.



Total internal reflection fluorescence microscopy can also be conducted through a modification of the epi-illumination approached utilized in widefield techniques (as illustrated in Figure 7(b)). This method requires a very high numerical aperture objective (at least 1.4, but preferably 1.45 to 1.6) and partial illumination of the microscope field from one side by a small sport or more uniform illumination by a thin annulus. High refractive index lens immersion medium and microscope cover glass are required to achieve the illumination angle resulting in total internal reflection. As presented in Figure 7(b), light rays exiting the objective front lens element at an angle less than the critical angle (denoted asA(1)) in figure 7(b)) are transmitted away from the microscope. When the angle is increased to or beyond the critical angle (indicated a angle A(2) in Figure 7(b)), total internal reflection results.



  • The era when optical microscopy was purely a descriptive instrument or an intellectual toy is past. At present, optical image formation is only the first step toward data analysis.
  • The microscope accomplishes this first step in conjunction with electronic detectors, image processors, and display devices that can be viewed as extensions of the imaging system. Computerized control of focus, stage position, optical components, shutters, filters, and detectors is in widespread use and enables experimental manipulations that were not humanly possible with mechanical microscopes.
  • The increasing application of electro-optics in fluorescence microscopy has led to the development of optical tweezers capable of manipulating sub-cellular structures or particles, the imaging of single molecules, and a wide range of sophisticated spectroscopic applications.



Flow Cytometry

Flow Cytometry: Principles and Clinical Applications in Hematology

General Principles

Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or “colors”), allows several cell properties to be measured simultaneously. Commonly used dyes include propidium iodide, phycoerythrin, and fluorescein, although many other dyes are available. Tandem dyes with internal fluorescence resonance energy transfer can create even longer wavelengths and more colors. The  list of clinical applications and cellular characteristics that are commonly measured. Several excellent texts and reviews are available.




Common clinical uses of flow cytometry.

Field Clinical application Common characteristic measured
Immunology Histocompatibility cross-matching IgG, IgM
Transplantation rejection CD3, circulating OKT3
HLA-B27 detection HLA-B27
Immunodeficiency studies CD4, CD8
Oncology DNA content and S phase of tumors DNA
Measurement of proliferation markers Ki-67, PCNA1
Hematology Leukemia and lymphoma phenotyping Leukocyte surface antigens
Identification of prognostically important subgroups TdT, MPO
Hematopoietic progenitor cell enumeration CD34
Diagnosis of systemic mastocytosis CD25, CD69
Reticulocyte enumeration RNA
Autoimmune and alloimmune disorders
Anti-platelet antibodies IgG, IgM
Anti-neutrophil antibodies IgG
Immune complexes Complement, IgG
Feto-maternal hemorrhage quantification Hemoglobin F, rhesus D
Blood banking Immunohematology Erythrocyte surface antigens
Assessment of leukocyte contamination of blood products Forward and side scatter, leukocyte surface antigens
Genetic disorders PNH CD55, CD59
Leukocyte adhesion deficiency CD11/CD18 complex


Common clinical uses of flow cytometry.

Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. At the interrogation point, a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is collected via optics that direct the light to a series of filters and dichroic mirrors that isolate particular wavelength bands. The light signals are detected by photomultiplier tubes and digitized for computer analysis. Fig. 1 is a schematic diagram of the fluidic and optical components of a flow cytometer. The resulting information usually is displayed in histogram or two-dimensional dot-plot formats.


Figure 1.

Schematic of a flow cytometer.

A single cell suspension is hydrodynamically focused with sheath fluid to intersect an argon-ion laser. Signals are collected by a forward angle light scatter detector, a side-scatter detector (1), and multiple fluorescence emission detectors (2–4). The signals are amplified and converted to digital form for analysis and display on a computer screen.

DNA Content Analysis

The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Abnormal DNA content, also known as “DNA content aneuploidy”, can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy; however, certain benign conditions may appear aneuploid. DNA aneuploidy correlates with a worse prognosis in many types of cancer but is associated with improved survival in rhabdomyosarcoma, neuroblastoma, multiple myeloma, and childhood acute lymphoblastic leukemia (ALL). In multiple myeloma, ALL, and myelodysplastic syndromes, hypodiploid tumors cells portend a poor prognosis. In contrast, hyperdiploid cells in ALL have a better prognosis. For many hematologic malignancies, there are conflicting reports regarding the independent prognostic value of DNA content analysis. Although conventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells.

Immunophenotyping Applications in Hematology

The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and permit analysis of an increasing number of intracellular markers.

erythrocyte analysis

The use of flow cytometry for the detection and quantification of fetal red cells in maternal blood has increased in recent years. Currently in the United States, rhesus D-negative women receive prophylactic Rh-immune globulin at 28 weeks and also within 72 h of delivery. The standard single dose is enough to prevent alloimmunization from ∼15 mL of fetal rhesus D+ red cells. If feto-maternal hemorrhage is suspected, the mother’s blood is tested for the presence and quantity of fetal red cells, and an appropriate amount of Rh-immune globulin is administered. The quantitative test most frequently used in clinical laboratories is the Kleihauer-Betke acid-elution test. This test is fraught with interobserver and interlaboratory variability, and is tedious and time-consuming. The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test. Fluorescently labeled antibodies to the rhesus (D) antigen can be used, or more recently, antibodies directed against hemoglobin F. This intracellular approach, which uses permeabilization of the red cell membrane and an antibody to the γ chain of human hemoglobin, is precise and sensitive. This method has the ability to distinguish fetal cells from F-cells (adult red cells with small amounts of hemoglobin F). Fig. 2 is a histogram of a positive test for feto-maternal hemorrhage. Although the flow cytometry method is technically superior to the Kleihauer-Betke test, cost, instrument availability, and stat access may limit its practical utility.


Figure 2.

Hemoglobin F test for feto-maternal hemorrhage.

Most adult RBCs do not have any hemoglobin F and are included in the large peak on the left. A few adult red cells have a small amount of hemoglobin F and are called F cells. Higher quantities of hemoglobin F in fetal cells yield a higher fluorescence signal and allow discrimination between fetal cells and adult F cells.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. PNH can arise in the setting of aplastic anemia and may be followed by acute leukemia. The disease is caused by deficient biosynthesis of a glycosylphosphatidylinositol linker that anchors several complement and immunoregulatory surface proteins on erythrocytes, monocytes, neutrophils, lymphocytes, and platelets. On erythrocytes, deficiencies of decay-accelerating factor and membrane-inhibitor of reactive lysis render red cells susceptible to complement-mediated lysis. Conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham’s acid hemolysis test. Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH. In affected patients, two or more populations of erythrocytes can be readily identified, with different degrees of expression of CD55 and CD59 (Fig. 3 )


Figure 3.

Diagnosis of PNH.

Control individuals (A) show high expression of CD55 and CD59 on all red cells. In PNH (B), some stem cell clones produce RBCs with decreased expression of CD55 and CD59. In the PNH patient (B), two distinct populations are present: normal red cells with high CD55 and CD59 expression and a second population with low CD55 and CD59 expression.

leukocyte analysis

Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper/inducer T lymphocytes via the CD4 antigen. Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. As HIV disease progresses, CD4-positive T lymphocytes decrease in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals. Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts. However, CD4 counts directly assess the patient’s immune status and not just the amount of virus. It is likely that both CD4 T-cell enumeration and HIV viral load will continue to be used for diagnosis, prognosis, and therapeutic management of HIV-infected persons.

Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas. Immunophenotyping as part of the diagnostic work-up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. Diagnostic interpretations depend on a combination of antigen patterns and fluorescence intensity. Several recent review articles are available. Flow cytometry is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias. Additionally, CD45/side scatter gating often can better isolate the blast population for more definitive phenotyping than is possible with forward scatter/side scatter gating.

Quantification of Soluble Molecules

Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. For example, OKT3 is a mouse anti-human antibody useful in treating transplant rejection. Circulating concentrations of OKT3 can be quantified by incubating with normal CD3-positive lymphocytes, followed by a fluorescently labeled anti-mouse antibody. Fluorescence values are compared to a calibration curve generated with known amounts of OKT3. Recently, multiplex assays for several antigens have become possible by the use of beads indexed by incorporating two different dyes.


Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Applications in hematology include DNA content analysis, leukemia and lymphoma phenotyping, immunologic monitoring of HIV-infected individuals, and assessment of structural and functional properties of erythrocytes, leukocytes, and platelets. Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease.

flow pic