Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of

cells Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a d ...

by using

microscopy Microscopy is the technical field of using microscopes to view subjects too small to be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical mic ...

to image cell components tagged with

fluorescent Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with color ...

molecules. It is a type of microphotometry that gives a quantitative measure of the qualitative nature of fluorescent measurement and therefore, allows for definitive results that would have been previously indiscernible to the naked eye.

Uses

Microfluorimetry has uses for many different fields including

cell biology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...

microbiology Microbiology () is the branches of science, scientific study of microorganisms, those being of unicellular organism, unicellular (single-celled), multicellular organism, multicellular (consisting of complex cells), or non-cellular life, acellula ...

immunology Immunology is a branch of biology and medicine that covers the study of Immune system, immune systems in all Organism, organisms. Immunology charts, measures, and contextualizes the Physiology, physiological functioning of the immune system in ...

, cell cycle analysis and "flow

karyotyping A karyotype is the general appearance of the complete set of chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is discerned by de ...

" of cells. In flow karotyping, isolated metaphase chromosomes are stained and measured in a flow microfluorometer. Fluorescent staining of chromosomes can also give distribution about the relative frequency of occurrence and the chromosomal DNA content of the measured chromosomes. This technique allows for karyotyping at higher speeds than with previous methods and was shown to be accurate using Chinese hamster chromosomes. Flow microfluorimetry (FMF) can also be used to determine different populations of cells using fluorescent markers with small cell samples. The markers used for measurement in flow microfluorimetry are made up of fluorescent antigens or DNA binding agents. It allows for the accurate measure of an antibody reacting with an antigen. Flow microfluorimetry is also used in pharmaceutical research to determine cell type, protein and DNA expression, cell cycle, and other properties of a cell during drug treatment. For example, microfluorimetry is used in

neurons A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

to compare the effects of

neurotoxins Neurotoxins are toxins that are destructive to nerve tissue (causing neurotoxicity). Neurotoxins are an extensive class of exogenous chemical neurological insultsSpencer 2000 that can adversely affect function in both developing and mature n ...

on both calcium ion concentration and mitochondrial membrane potential in individual cells. Microfluorimetry can also be used as a method to distinguish different microorganisms from one another by analyzing and comparing the DNA content of each cell. This same concept can also be applied to distinguish between cell types using a suitable fluorescent dye which varies depending on purpose and is a critical technique in modern cell biology and genomics. Another use of microfluorometry is

flow cytometry Flow cytometry (FC) is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the ...

which uses the emission of fluorochrome molecules and usually a laser as a light source to create data from particles and cells. It can be used to separate chromosomes at a very high rate and used easily with next-gen sequencing. This technique can simply results by separating only the relevant chromosomes at a very fast rate. For example, E. coli bacteriophages lambda and T4 were able to be separated by flow cytometry which allowed for genomic analysis which was previously difficult.

Concept

Microfluorimetry is building upon the established method of fluorimetric measurement. Using a dye that fluoresces in the presence of a target compound, fluorimetry can detect the presence of the compound by determining the presence and intensity of fluorescence. Differences in the intensity can be used to determine concentration of the compound. Additionally, if the dye undergoes a spectral shift then you can determine the absolute concentration of the target regardless of knowledge of the concentration of the dye. Fura-2 is an example of a fluorescent dye used to measure calcium. Microfluorimetry expands on fluorimetry by adding a microscopic component to measurements to allow analysis of single cells and other microscopic interests.

Microfluorometer

A microfluorometer is a fluorescence spectrophotometer combined with a microscope, designed to measure fluorescence spectra of microscopic samples or areas or can be configured to measure the transmission and reflectance spectra of microscopic sample areas. It can either be a complete microfluorometer built exclusively for fluorescence microspectroscopy or the fluorescence spectrometer unit which attaches to the optical port of a microscope. A microfluorometer can be used to estimate amounts and distributions of chemical components in individual cells or in chromosomes. In order to estimate the amount of chemical components, its fluorescent intensity is measured by photoelectrical photometry while distribution is found by measuring the intensities of photos of negative chromosomes' metaphase plates. A microspectrophotometer can measure transmission, absorbance, reflectance and emission spectra then using built in algorithms a spectra is produced that can be compared against previous data in order to determine composition, concentration, etc.

Limitations

There are many sources of error in the process but biological errors such as an inability to prepare homogenous samples are more likely to be a limitation than technical errors.

References

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External links