Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of cells by using microscopy to image cell components tagged with fluorescent 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. [1]
Microfluorimetry has uses for many different fields including cell biology, microbiology, immunology, cell cycle analysis and "flow karyotyping" of cells. [2] 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. [3] 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. [2] It allows for the accurate measure of an antibody reacting with an antigen. [1] 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. [4] For example, microfluorimetry is used in neurons to compare the effects of neurotoxins on both calcium ion concentration and mitochondrial membrane potential in individual cells. [5] 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. [6] 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. [7]
Another use of microfluorometry is flow cytometry which uses the emission of fluorochrome molecules and usually a laser as a light source to create data from particles and cells. [8] 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. [9] 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. [10]
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. [11]
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. [12] 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. [13] 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. [12]
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. [1]
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.
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.
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry. This technique primarily makes use of fluorophores to visualise the location of the antibodies.
Comparative genomic hybridization(CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33,342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation–emission spectra.
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.
Acridine orange is an organic compound. It is used as a nucleic acid-selective fluorescent dye, that has cationic properties useful for cell cycle determination. Being cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic attractions, respectively. When bound to DNA, it is very similar spectrally to fluorescein, with a maximum excitation at 502 nm and 525 nm (green). When acridine orange associates with RNA, the maximum excitation shifts to 460 nm (blue), and the maximum emission shifts to 650 nm (red). Acridine orange will also enter acidic compartments such as, lysosomes where it becomes protonated and sequestered. Within these low pH vesicles, the dye emits red fluorescence when excited by blue light. Thus, acridine orange can be used to visualize primary lysosomes and phagolysosomes that may include products of phagocytosis of apoptotic cells. The dye is often used in epifluorescence microscopy and flow cytometry. The cell-permeable characteristic of both acridine orange and fluorescein, allows the stain to differentiate between various types of cells, especially bacterial cells and low pH. The shift in maximum excitation and emissions provides a foundation to predict the wavelength at which cells will stain.
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analyzation of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800's. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980's, FISH uses probes with complimentary base sequences to locate the presence or absence of the specific DNA regions you are looking for. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.
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.
Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are brought to the same phase. Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows population-wide data to be collected rather than relying solely on single-cell experiments. The types of synchronization are broadly categorized into two groups; physical fractionization and chemical blockade.
Flow-FISH is a cytogenetic technique to quantify the copy number of specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols. Flow-FISH is most commonly used to quantify the length of telomeres, which are stretches of repetitious DNA at the distal ends of chromosomes in human white blood cells, and a semi-automated method for doing so was published in Nature Protocols. Telomere length in white blood cells has been a subject of interest because telomere length in these cell types declines gradually over the human lifespan, resulting in cell senescence, apoptosis, or transformation. This decline has been shown to be a surrogate marker for the concomitant decline in the telomere length of the hematopoietic stem cell pool, with the granulocyte lineage giving the best indication, presumably due to the absence of a long lived memory subtype and comparatively rapid turnover of these cells.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
The Qubit fluorometer is a lab instrument developed and distributed by Invitrogen that, among other applications, is used for the quantification of DNA, RNA, and protein.
Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labelled synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software. Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as genome instability. To this day, the Q-FISH method continues to be utilized in the field of telomere research.
Cytometry is the measurement of the characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology, cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. Cytometry is used to characterize and count blood cells in common blood tests such as the complete blood count. In a similar fashion, cytometry is also used in cell biology research and in medical diagnostics to characterize cells in a wide range of applications associated with diseases such as cancer and AIDS.
Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains DNA quantitatively, such as propidium iodide (PI) or 4,6-diamidino-2-phenylindole (DAPI). The fluorescence intensity of the stained cells correlates with the amount of DNA they contain. As the DNA content doubles during the S phase, the DNA content (and thereby intensity of fluorescence) of cells in the G0 phase and G1 phase (before S), in the S phase, and in the G2 phase and M phase (after S) identifies the cell cycle phase position in the major phases (G0/G1 versus S versus G2/M phase) of the cell cycle. The cellular DNA content of individual cells is often plotted as their frequency histogram to provide information about relative frequency (percentage) of cells in the major phases of the cell cycle.
Bacterioplankton counting is the estimation of the abundance of bacterioplankton in a specific body of water, which is useful information to marine microbiologists. Various counting methodologies have been developed over the years to determine the number present in the water being observed. Methods used for counting bacterioplankton include epifluorescence microscopy, flow cytometry, measures of productivity through frequency of dividing cells (FDC), thymine incorporation, and leucine incorporation.