Fluorescence imaging

Last updated April 27, 2024

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

Fluorescence imaging photographs fluorescent dyes and fluorescent proteins to mark molecular mechanisms and structures. It allows one to experimentally observe the dynamics of gene expression, protein expression, and molecular interactions in a living cell.^[3] It essentially serves as a precise, quantitative tool regarding biochemical applications.

A common misconception, fluorescence differs from bioluminescence by how the proteins from each process produce light. Bioluminescence is a chemical process that involves enzymes breaking down a substrate to produce light. Fluorescence is the physical excitation of an electron, and subsequent return to emit light.

Attributes

Fluorescence mechanism

When a certain molecule absorbs light, the energy of the molecule is briefly raised to a higher excited state. The subsequent return to ground state results in emission of fluorescent light that can be detected and measured. The emitted light, resulting from the absorbed photon of energy hv, has a specific wavelength. It is important to know this wavelength beforehand so that when an experiment is running, the measuring device knows what wavelength to be set at to detect light production. This wavelength is determined by the equation:

$\lambda _{emission}=\ {\frac {hc}{{Energy}_{emission}}}$

Where h = Planck's constant, and c = the speed of light. Typically a large scanning device or CCD is used here to measure the intensity and digitally photograph an image.^[1]

Fluorescent dyes versus proteins

Fluorescent dyes, with no maturation time, offer higher photo stability and brightness in comparison to fluorescent proteins. In terms of brightness, luminosity is dependent on the fluorophores’ extinction coefficient or ability to absorb light, and its quantum efficiency or effectiveness at transforming absorbed light into fluorescently emitting luminescence. The dyes themselves are not very fluorescent, but when they bind to proteins, they become more easily detectable. One example, NanoOrange, binds to the coating and hydrophobic regions of a protein while being immune to reducing agents. Regarding proteins, these molecules themselves will fluorescence when they absorb a specific incident light wavelength. One example of this, green fluorescent protein (GFP), fluoresces green when exposed to light in the blue to UV range. Fluorescent proteins are excellent reporter molecules that can aid in localizing proteins, observing protein binding, and quantifying gene expression.^[1]

Imaging range

Since some wavelengths of fluorescence are beyond the range of the human eye, charged-coupled devices (CCD) are used to accurately detect light and image the emission. This typically occurs in the 300 – 800 nm range. One of the advantages of fluorescent signaling is that intensity of emitted light behaves rather linearly in regards to the quantity of fluorescent molecules provided. This is obviously contingent that the absorbed light intensity and wavelength are constant. In terms of the actual image itself, it is usually in a 12-bit or 16-bit data format.^[1]

Imaging systems

The main components of fluorescence imaging systems are:

Excitation Source: a device that produces either a broad-wavelength source like UV light, or a narrow wavelength source like a laser.
Light display optics: the mechanism of which light illuminates the sample. This is typically done through direct illumination of the sample.
Light assortment optics: the collection method of the light itself. This typically constitutes lenses, mirrors, and filters.
Filtration of emitted light: optical filters ensure that reflected and scattered light are not included with the fluorescence. The three classes of emission filters are long-pass, short-pass, and band-pass.
Detection, amplification, and visualization: either photomultiplier (PMT) or charge-couple device (CCD) is used to detect and quantify emitted photons

Applications

In PCR (agarose gel electrophoresis): SYBR Green is a very common dye that binds to DNA and is used to visualize DNA bands in an agarose gel. The dye absorbs blue light and fluoresces green for an imaging system to capture.
Blotting (Western, Northern, and Southern): fluorochromes can bind to antibodies, RNA, and DNA to fluoresce and quantify data
DNA Sequencing: Sanger Sequencing is a common form of nucleic acid detection that may use fluorescently labeled ddNTPs to image fluorescence peaks
Fluorescence image guided surgery: is a medical imaging approach that fluorescently labels a mass to aid in navigation. For example, indocyanine green can be used to detect lymph nodes in cancer patients.^[4]
Fluorescence imaging with one nanometer accuracy (FIONA): utilizes total internal reflection illumination to reduce noise and increase brightness of fluorophores
Calcium imaging: technique that utilizes fluorescent molecules called calcium indicators that change in fluorescence when bound to Ca²⁺ ions. This is a key part in seeing when cells are active in the nervous system.^[5]
Pegulicianine (Lumisight) is indicated for fluorescence imaging in adults with breast cancer as an adjunct for the intraoperative detection of cancerous tissue within the resection cavity following removal of the primary specimen during lumpectomy surgery.^[6]

Types of microscopy

A different array of microscope techniques can be employed to change the visualization and contrast of an image. Each method comes with pros and cons, but all utilize the same mechanism of fluorescence to observe a biological process.

Total internal reflection fluorescence microscopy: A microscopy technique that uses evanescent waves to selectively observe the fluorescence of a single molecule.^[7]
Light sheet fluorescence microscopy: A fluorescence microscopy technique that illuminates a thin slice of a sample at a perpendicular angle of examination.^[8]
Fluorescence-lifetime imaging microscopy: An imaging technique that records changes in fluorescence over time

Advantages

Non-invasive: imaging in vivo can take place without having to puncture the skin
Sensitive: probes are designed to be extremely sensitive to detecting biological molecules like DNA, RNA, and proteins.^[1]
Multi-labeling: multiple fluorochromes can be detected within the samples allowing for an easy way to integrate standards and a control.
Stability of labeled molecules: fluorescently labeled molecules used in imaging can be stored for months while other molecules like ones that are radiolabeled, will decay over a few days.^[8]
Relatively safe to handle: most fluorophores can be safely and sufficiently handled with gloves, while for example, radioisotopes may require lead shields or other radiation protection.^[8]
Simple disposal: many fluorophores require minimal disposal methods while radioactive wastes require regulated disposal and long-term handling. This also aids in lowering the cost needed to utilize these products.

Disadvantages

Photobleaching: A prevalent issue with fluorophores where the constant cycling between ground state and excited state damages the molecule and reduces its intensity.^[8]
Environmental susceptibility: environmental factors like temperature, ion concentration, and pH can affect the efficiency and emission of fluorochromes
Toxicity: Aome fluorochromes can be toxic to cells, to tissues, in vivo, or by producing mutations.^[9]
Limited resolving power: Fluorescence microscopes are limited in their ability to distinguish close objects at the macroscopic level. In comparison, electron microscopes for example, have the capacity to resolve at a much smaller range.
Limited initial luminosity range: the incidence light source intensity has a limit and beyond this point can result in photodestruction of molecules.^[1]

Overall, this form of imaging is extremely useful in cutting-edge research, with its ability to monitor biological processes. The progression from 2D fluorescent images to 3D ones has allowed scientists to better study spatial precision and resolution. In addition, with concentrated efforts towards 4D analysis, scientists are now able to monitor a cell in real time, enabling them to monitor fast acting processes.

Future directions

Developing more effective fluorescent proteins is a task that many scientists have taken up in order to improve imaging probe capabilities. Often, mutations in certain residues can significantly change the protein's fluorescent properties. For example, by mutating the F64L gene in jellyfish GFP, the protein is able to more efficiently fluoresce at 37 °C, an important attribute to have when growing cultures in a laboratory.^[10] In addition to this, genetic engineering can produce a protein that emits light at a better wavelength or frequency.^[10] In addition, the environment itself can play a crucial role. Fluorescence lifetime can be stabilized in a polar environment.

Mechanisms that have been well described but not necessarily incorporated into practical applications hold promising potential for fluorescence imaging. Fluorescence resonance energy transfer (FRET) is an extremely sensitive mechanism that produce signaling molecules in the range of 1-10 nm.^[7]

Improvements in the techniques that constitute fluorescence processes is also crucial towards more efficient designs. Fluorescence correlation spectroscopy (FCS) is an analysis technique that observes the fluctuation of fluorescence intensity. This analysis is a component of many fluorescence imaging machines and improvements in spatial resolution could improve the sensitivity and range.^[7]

Development of more sensitive probes and analytical techniques for laser induced fluorescence can allow for more accurate, up-to-date experimental data.

Related Research Articles

Fluorescence is one of two kinds of emission of light by a substance that has absorbed light or other electromagnetic radiation. Fluorescence involves no change in electron spin multiplicity and generally it immediately follows absorption; phosphorescence involves spin change and is delayed. Thus fluorescent materials generally cease to glow nearly immediately when the radiation source stops, while phosphorescent materials, which continue to emit light for some time after.

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<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 the physical and chemical characteristics of a population of cells or particles.

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.

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

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 fluorescence microscope is an optical microscope that uses fluorescence 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 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.

<span class="mw-page-title-main">Two-photon excitation microscopy</span> Fluorescence imaging technique

Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

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.

<span class="mw-page-title-main">Photobleaching</span> Loss of colour by a pigment when illuminated

In optics, photobleaching is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.

In fluorescence microscopy, colocalization refers to observation of the spatial overlap between two different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another. The definition can be split into two different phenomena, co-occurrence, which refers to the presence of two fluorophores in the same pixel, and correlation, a much more significant statistical relationship between the fluorophores indicative of a biological interaction. This technique is important to many cell biological and physiological studies during the demonstration of a relationship between pairs of bio-molecules.

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

RESOLFT, an acronym for REversible Saturable OpticaLFluorescence Transitions, denotes a group of optical fluorescence microscopy techniques with very high resolution. Using standard far field visible light optics a resolution far below the diffraction limit down to molecular scales can be obtained.

Vertico spatially modulated illumination (Vertico-SMI) is the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI and SPDM. The effective optical resolution of this optical nanoscope has reached the vicinity of 5 nm in 2D and 40 nm in 3D, greatly surpassing the λ/2 resolution limit applying to standard microscopy using transmission or reflection of natural light according to the Abbe resolution limit That limit had been determined by Ernst Abbe in 1873 and governs the achievable resolution limit of microscopes using conventional techniques.

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. 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.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

Chromatin bridge is a mitotic occurrence that forms when telomeres of sister chromatids fuse together and fail to completely segregate into their respective daughter cells. Because this event is most prevalent during anaphase, the term anaphase bridge is often used as a substitute. After the formation of individual daughter cells, the DNA bridge connecting homologous chromosomes remains fixed. As the daughter cells exit mitosis and re-enter interphase, the chromatin bridge becomes known as an interphase bridge. These phenomena are usually visualized using the laboratory techniques of staining and fluorescence microscopy.

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

References

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