Fluorometer

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Fluorometer designed to measure chlorophyll fluorescence in plants OS1p modulated fluorometer measuring photosynthetic yield Y(II) in the field..jpg
Fluorometer designed to measure chlorophyll fluorescence in plants

A fluorometer, fluorimeter or fluormeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. [1] These parameters are used to identify the presence and the amount of specific molecules in a medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.

Contents

Fluorescence analysis can be orders of magnitude more sensitive than other techniques. Applications include chemistry/biochemistry, medicine, environmental monitoring. For instance, they are used to measure chlorophyll fluorescence to investigate plant physiology.

Components and design

A simplistic design of the components of a fluorometer Fluorimeter.svg
A simplistic design of the components of a fluorometer

Typically fluorometers utilize a double beam. These two beams work in tandem to decrease the noise created from radiant power fluctuations. The upper beam is passed through a filter or monochromator and passes through the sample. The lower beam is passed through an attenuator and adjusted to try and match the fluorescent power given off from the sample. Light from the fluorescence of the sample and the lower, attenuated beam are detected by separate transducers and converted to an electrical signal that is interpreted by a computer system.

Within the machine the transducer that detects fluorescence created from the upper beam is located a distance away from the sample and at a 90-degree angle from the incident, upper beam. The machine is constructed like this to decrease the stray light from the upper beam that may strike the detector. The optimal angle is 90 degrees. There are two different approaches to handling the selection of incident light that gives way to different types fluorometers. If filters are used to select wavelengths of light, the machine is called a fluorometer. While a spectrofluorometer will typically use two monochromators, some spectrofluorometers may use one filter and one monochromator. Where, in this case, the broad band filter acts to reduce stray light, including from unwanted diffraction orders of the diffraction grating in the monochromator.

Light sources for fluorometers are often dependent on the type of sample being tested. Among the most common light source for fluorometers is the low-pressure mercury lamp. This provides many excitation wavelengths, making it the most versatile. However, this lamp is not a continuous source of radiation. The xenon arc lamp is used when a continuous source of radiation is needed. Both of these sources provide a suitable spectrum of ultraviolet light that induces chemiluminescence. These are just two of the many possible light sources. [ citation needed ]

Glass and silica cuvettes are often the vessels in which the sample is placed. Care must be taken to not leave fingerprints or any other sort of mark on the outside of the cuvette, because this can produce unwanted fluorescence. "Spectro grade" solvents such as methanol are sometimes used to clean the vessel surfaces to minimize these problems.

Uses

Dairy industry

Fluorimetry is widely used by the dairy industry to verify whether pasteurization has been successful. This is done using a reagent which is hydrolysed to a fluorophore and phosphoric acid by alkaline phosphatase in milk. [2] If pasteurization has been successful then alkaline phosphatase will be entirely denatured and the sample will not fluoresce. This works because pathogens in milk are killed by any heat treatment which denatures alkaline phosphatase. [3] [4]

Fluorescence assays are required by milk producers in the UK to prove successful pasteurization has occurred, [5] so all UK dairies contain fluorimetry equipment.

Protein aggregation and TSE detection

Thioflavins are dyes used for histology staining and biophysical studies of protein aggregation. [6] For example, thioflavin T is used in the RT-QuIC technique to detect transmissible spongiform encephalopathy-causing misfolded prions.

Oceanography

Photosynthetic phytoplankton from the Pacific Ocean observed using epifluorescence microscopy (blue exciting light). Picoplancton fluorescence Pacific.jpg
Photosynthetic phytoplankton from the Pacific Ocean observed using epifluorescence microscopy (blue exciting light).
Filter after a water sample has been filtered through it to isolate the phytoplankton on the filter before benchtop chlorophyll fluorometry. August 6, 2011 Chlorophyll filtered from our water samples (6016326962).jpg
Filter after a water sample has been filtered through it to isolate the phytoplankton on the filter before benchtop chlorophyll fluorometry.

Fluorometers are widely used in oceanography to measure chlorophyll concentrations based on chlorophyll fluorescence by phytoplankton cell pigments. Chlorophyll fluorescence is a widely-used proxy for the quantity (biomass) of microscopic algae in the water. In the lab after water sampling, researchers extract the pigments out of a filter that has phytoplankton cells on it, then measure the fluorescence of the extract in a benchtop fluorometer in a dark room. [7] To directly measure chlorophyll fluorescence "in situ" (in the water), researchers use instruments designed to measure fluorescence optically (for example, sondes with extra electronic optical sensors attached). The optical sensors emit blue light to excite phytoplankton pigments and make them fluoresce or emit red light. The sensor measures this induced fluorescence by measuring the red light as a voltage, and the instrument saves it to a data file. The voltage signal of the sensor gets converted to a concentration with a calibration curve in the lab, using either red-colored dyes like Rhodamine, standards like Fluorescein, or live phytoplankton cultures. [8]

Ocean chlorophyll fluorescence is measured on research vessels, small boats, buoys, docks, and piers all over the world. Fluorometry measurements are used to map chlorophyll concentrations in support of ocean color remote sensing. Special fluorometers for ocean waters can measure properties beyond the total amount of fluorescence, such as the quantum yield of photochemistry, the timing of the fluorescence, and the fluorescence of cells when subjected to increasing amounts of light. [9] Aquaculture operations such as fish farms us fluorometers to measure food availability for filter feeding animals like mussels [10] and to detect the onset of Harmful Algal Blooms (HABs) and/or "red tides" (not necessarily the same thing). [11]

Molecular biology

Fluorometers can be used to determine the nucleic acid concentration in a sample. [12]

Fluorometer types

There are two basic types of fluorometers: the filter fluorometers and spectrofluorometer. The difference between them is the way they select the wavelengths of incident light; filter fluorometers use filters while spectrofluorometers use grating monochromators. Filter fluorometers are often purchased or built at a lower cost but are less sensitive and have less resolution than spectrofluorometers. Filter fluorometers are also capable of operation only at the wavelengths of the available filters, whereas monochromators are generally freely tunable over a relatively wide range. The potential disadvantage of monochromators arises from that same property, because the monochromator is capable of miscalibration or misadjustment, whereas the wavelength of filters are fixed when manufactured.

See also

Related Research Articles

<span class="mw-page-title-main">Fluorescence</span> Emission of light by a substance that has absorbed light

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

<span class="mw-page-title-main">Ultraviolet–visible spectroscopy</span> Range of spectroscopic analysis

UV spectroscopy or UV–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.

<span class="mw-page-title-main">X-ray fluorescence</span> Emission of secondary X-rays from a material excited by high-energy X-rays

X-ray fluorescence (XRF) is the emission of characteristic "secondary" X-rays from a material that has been excited by being bombarded with high-energy X-rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis, particularly in the investigation of metals, glass, ceramics and building materials, and for research in geochemistry, forensic science, archaeology and art objects such as paintings.

<span class="mw-page-title-main">Spectrophotometry</span> Branch of spectroscopy

Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spectrophotometers, that can measure the intensity of a light beam at different wavelengths. Although spectrophotometry is most commonly applied to ultraviolet, visible, and infrared radiation, modern spectrophotometers can interrogate wide swaths of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, and/or microwave wavelengths.

<span class="mw-page-title-main">Flow cytometry</span> Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

<span class="mw-page-title-main">Fluorescence spectroscopy</span> Type of electromagnetic spectroscopy

Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.

<span class="mw-page-title-main">Monochromator</span> Optical device

A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. The name is from the Greek roots mono-, "single", and chroma, "colour", and the Latin suffix -ator, denoting an agent.

A filter fluorometer is a type of fluorometer that may be employed in fluorescence spectroscopy.

<span class="mw-page-title-main">Optical filter</span> Filters which selectively transmit specific colors

An optical filter is a device that selectively transmits light of different wavelengths, usually implemented as a glass plane or plastic device in the optical path, which are either dyed in the bulk or have interference coatings. The optical properties of filters are completely described by their frequency response, which specifies how the magnitude and phase of each frequency component of an incoming signal is modified by the filter.

<span class="mw-page-title-main">Photometer</span> Instrument to measure light intensity

A photometer is an instrument that measures the strength of electromagnetic radiation in the range from ultraviolet to infrared and including the visible spectrum. Most photometers convert light into an electric current using a photoresistor, photodiode, or photomultiplier.

Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

A spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, and the emission is observed either at a single wavelength, or a scan is performed to record the intensity versus wavelength, also called an emission spectrum. The instrument is used in fluorescence spectroscopy.

Dye tracing is a method of tracking and tracing various flows using dye as a flow tracer when added to a liquid. Dye tracing may be used to analyse the flow of the liquid or the transport of objects within the liquid. Dye tracking may be either qualitative, showing the presence of a particular flow, or quantitative, when the amount of the traced dye is measured by special instruments.

<span class="mw-page-title-main">Colored dissolved organic matter</span> Optically measurable component of the dissolved organic matter in water

Colored dissolved organic matter (CDOM) is the optically measurable component of dissolved organic matter in water. Also known as chromophoric dissolved organic matter, yellow substance, and gelbstoff, CDOM occurs naturally in aquatic environments and is a complex mixture of many hundreds to thousands of individual, unique organic matter molecules, which are primarily leached from decaying detritus and organic matter. CDOM most strongly absorbs short wavelength light ranging from blue to ultraviolet, whereas pure water absorbs longer wavelength red light. Therefore, water with little or no CDOM, such as the open ocean, appears blue. Waters containing high amounts of CDOM can range from brown, as in many rivers, to yellow and yellow-brown in coastal waters. In general, CDOM concentrations are much higher in fresh waters and estuaries than in the open ocean, though concentrations are highly variable, as is the estimated contribution of CDOM to the total dissolved organic matter pool.

<span class="mw-page-title-main">Ocean color</span> Explanation of the color of oceans and ocean color remote sensing

Ocean color is the branch of ocean optics that specifically studies the color of the water and information that can be gained from looking at variations in color. The color of the ocean, while mainly blue, actually varies from blue to green or even yellow, brown or red in some cases. This field of study developed alongside water remote sensing, so it is focused mainly on how color is measured by instruments.

<span class="mw-page-title-main">Chlorophyll fluorescence</span> Light re-emitted by chlorophyll molecules during return from excited to non-excited states

Chlorophyll fluorescence is light re-emitted by chlorophyll molecules during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis, as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectrum of applications.

The nutrient content of a plant can be assessed by testing a sample of tissue from that plant. These tests are important in agriculture since fertilizer application can be fine-tuned if the plants nutrient status is known. Nitrogen most commonly limits plant growth and is the most managed nutrient.

Piezospectroscopy (also known as photoluminescence piezospectroscopy) is an analytical technique that reveals internal stresses in alumina-containing materials(Al2O3, also known as aluminum oxide), particularly thermal barrier coatings (TBCs). A typical procedure involves illuminating the sample with laser light of a known wavelength, causing the material to release its own radiation in response(see fluorescence). By measuring the emitted radiation and comparing the location of the peaks to a stress-free sample, stresses in the material can be revealed without any destructive interaction.

<span class="mw-page-title-main">Ocean optics</span> The study of light interaction with water and submerged materials

Ocean optics is the study of how light interacts with water and the materials in water. Although research often focuses on the sea, the field broadly includes rivers, lakes, inland waters, coastal waters, and large ocean basins. How light acts in water is critical to how ecosystems function underwater. Knowledge of ocean optics is needed in aquatic remote sensing research in order to understand what information can be extracted from the color of the water as it appears from satellite sensors in space. The color of the water as seen by satellites is known as ocean color. While ocean color is a key theme of ocean optics, optics is a broader term that also includes the development of underwater sensors using optical methods to study much more than just color, including ocean chemistry, particle size, imaging of microscopic plants and animals, and more.

References

  1. ↑ "Fluorescence Spectrophotometry". Encyclopedia of Life Sciences . Macmillan Publishers Ltd. 2002.
  2. ↑ Langridge, E W. The Determination of Phosphatase Activity. Quality Management Ltd. Retrieved 2013-12-20.
  3. ↑ Kay, H. (1935). "Some Results of the Application of a Simple Test for Efficiency of Pasteurisation". The Lancet. 225 (5835): 1516–1518. doi:10.1016/S0140-6736(01)12532-8.
  4. ↑ Hoy, W. A.; Neave, F. K. (1937). "The Phosphatase Test for Efficient Pasteurisation". The Lancet. 230 (5949): 595. doi:10.1016/S0140-6736(00)83378-4.
  5. ↑ BS EN ISO 11816-1:2013
  6. ↑ Biancalana M, Koide S (July 2010). "Molecular mechanism of Thioflavin-T binding to amyloid fibrils". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1804 (7): 1405–12. doi:10.1016/j.bbapap.2010.04.001. PMC   2880406 . PMID   20399286.
  7. ↑ Holm-Hansen, Osmund; Lorenzen, Carl J.; Holmes, Robert W.; Strickland, John D. H. (1965). "Fluorometric Determination of Chlorophyll". ICES Journal of Marine Science. 30 (1): 3–15. doi:10.1093/icesjms/30.1.3.
  8. ↑ Earp, Alan (2011). "Review of fluorescent standards for calibration of in situ fluorometers: Recommendations applied in coastal and ocean observing programs". Optics Express. 19 (27): 26768–26782. doi:10.1364/OE.19.026768. hdl: 10453/18163 . PMID   22274260.
  9. ↑ Falkowski, Paul G.; Lin, Hanzhi; Gorbunov, Maxim Y. (14 August 2017). "What limits photosynthetic energy conversion efficiency in nature? Lessons from the oceans". Philosophical Transactions of the Royal Society B: Biological Sciences. The Royal Society. 372 (1730): 20160376. doi:10.1098/rstb.2016.0376. ISSN   0962-8436. PMC   5566876 . PMID   28808095.
  10. ↑ Ogilvie, Shaun C.; Ross, Alex H.; Schiel, David R. (2000). "Phytoplankton biomass associated with mussel farms in Beatrix Bay, New Zealand". Aquaculture. 181 (1–2): 71–80. doi:10.1016/S0044-8486(99)00219-7.
  11. ↑ Anderson, Donald M.; Anderson, Per; Bricelj, V. Monica; Cullen, John J.; Rensel, J. E. Jack (2001). Monitoring and Management Strategies for Harmful Algal Blooms in Coastal Waters, APEC #201-MR-01.1 (PDF). Paris: Asia Pacific Economic Program, Singapore, and Intergovernmental Oceanographic Commission Technical Series No. 59.
  12. ↑ Mészáros, Éva (2021). "Determine the concentration and purity of nucleic acids". INTEGRA Biosciences.
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