Hubbry Logo
Fluorescent tagFluorescent tagMain
Open search
Fluorescent tag
Community hub
Fluorescent tag
logo
8 pages, 0 posts
0 subscribers
Be the first to start a discussion here.
Be the first to start a discussion here.
Fluorescent tag
Fluorescent tag
Fluorescent tag
View on Wikipedia
from Wikipedia
S. cerevisiae septins revealed with fluorescent microscopy utilizing fluorescent labeling

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent dye, 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.[1] 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.[2]

History

[edit]
Stokes George G
Osamu Shimomura-press conference Dec 06th, 2008-1

The development of methods to detect and identify biomolecules has been motivated by the ability to improve the study of molecular structure and interactions. Before the advent of fluorescent labeling, radioisotopes were used to detect and identify molecular compounds. Since then, safer methods have been developed that involve the use of fluorescent dyes or fluorescent proteins as tags or probes as a means to label and identify biomolecules.[3] Although fluorescent tagging in this regard has only been recently utilized, the discovery of fluorescence has been around for a much longer time.

Sir George Stokes developed the Stokes Law of Fluorescence in 1852 which states that the wavelength of fluorescence emission is greater than that of the exciting radiation. Richard Meyer then termed fluorophore in 1897 to describe a chemical group associated with fluorescence. Since then, Fluorescein was created as a fluorescent dye by Adolph von Baeyer in 1871 and the method of staining was developed and utilized with the development of fluorescence microscopy in 1911.[4]

Ethidium bromide and variants were developed in the 1950s,[4] and in 1994, fluorescent proteins or FPs were introduced.[5] Green fluorescent protein or GFP was discovered by Osamu Shimomura in the 1960s and was developed as a tracer molecule by Douglas Prasher in 1987.[6] FPs led to a breakthrough of live cell imaging with the ability to selectively tag genetic protein regions and observe protein functions and mechanisms.[5] For this breakthrough, Shimomura was awarded the Nobel Prize in 2008.[7]

New methods for tracking biomolecules have been developed including the use of colorimetric biosensors, photochromic compounds, biomaterials, and electrochemical sensors. Fluorescent labeling is also a common method in which applications have expanded to enzymatic labeling, chemical labeling, protein labeling, and genetic labeling.[1]

Types of biosensors

Methods for tracking biomolecules

[edit]

There are currently several labeling methods for tracking biomolecules. Some of the methods include the following.

Isotope markers

[edit]

Common species that isotope markers are used for include proteins. In this case, amino acids with stable isotopes of either carbon, nitrogen, or hydrogen are incorporated into polypeptide sequences.[8] These polypeptides are then put through mass spectrometry. Because of the exact defined change that these isotopes incur on the peptides, it is possible to tell through the spectrometry graph which peptides contained the isotopes. By doing so, one can extract the protein of interest from several others in a group. Isotopic compounds play an important role as photochromes, described below.

Colorimetric biosensors

[edit]

Biosensors are attached to a substance of interest. Normally, this substance would not be able to absorb light, but with the attached biosensor, light can be absorbed and emitted on a spectrophotometer.[9] Additionally, biosensors that are fluorescent can be viewed with the naked eye. Some fluorescent biosensors also have the ability to change color in changing environments (ex: from blue to red). A researcher would be able to inspect and get data about the surrounding environment based on what color he or she could see visibly from the biosensor-molecule hybrid species.[10]

Colorimetric assays are normally used to determine how much concentration of one species there is relative to another.[9]

Photochromic compounds

[edit]

Photochromic compounds have the ability to switch between a range or variety of colors. Their ability to display different colors lies in how they absorb light. Different isomeric manifestations of the molecule absorbs different wavelengths of light, so that each isomeric species can display a different color based on its absorption. These include photoswitchable compounds, which are proteins that can switch from a non-fluorescent state to that of a fluorescent one given a certain environment.[11]

The most common organic molecule to be used as a photochrome is diarylethene.[12] Other examples of photoswitchable proteins include PADRON-C, rs-FastLIME-s and bs-DRONPA-s, which can be used in plant and mammalian cells alike to watch cells move into different environments.[11]

Biomaterials

[edit]

Fluorescent biomaterials are a possible way of using external factors to observe a pathway more visibly. The method involves fluorescently labeling peptide molecules that would alter an organism's natural pathway. When this peptide is inserted into the organism's cell, it can induce a different reaction. This method can be used, for example to treat a patient and then visibly see the treatment's outcome.[13]

Electrochemical sensors

[edit]

Electrochemical sensors can be used for label-free sensing of biomolecules. They detect changes and measure current between a probed metal electrode and an electrolyte containing the target analyte. A known potential to the electrode is then applied from a feedback current and the resulting current can be measured. For example, one technique using electrochemical sensing includes slowly raising the voltage causing chemical species at the electrode to be oxidized or reduced. Cell current vs voltage is plotted which can ultimately identify the quantity of chemical species consumed or produced at the electrode.[14] Fluorescent tags can be used in conjunction with electrochemical sensors for ease of detection in a biological system.

Fluorescent labels

[edit]
Aequorea victoria
GFP structure

Of the various methods of labeling biomolecules, fluorescent labels are advantageous in that they are highly sensitive even at low concentration and non-destructive to the target molecule folding and function.[1]

Green fluorescent protein is a naturally occurring fluorescent protein from the jellyfish Aequorea victoria that is widely used to tag proteins of interest. GFP emits a photon in the green region of the light spectrum when excited by the absorption of light. The chromophore consists of an oxidized tripeptide -Ser^65-Tyr^66-Gly^67 located within a β barrel. GFP catalyzes the oxidation and only requires molecular oxygen. GFP has been modified by changing the wavelength of light absorbed to include other colors of fluorescence. YFP or yellow fluorescent protein, BFP or blue fluorescent protein, and CFP or cyan fluorescent protein are examples of GFP variants. These variants are produced by the genetic engineering of the GFP gene.[15]

Synthetic fluorescent probes can also be used as fluorescent labels. Advantages of these labels include a smaller size with more variety in color. They can be used to tag proteins of interest more selectively by various methods including chemical recognition-based labeling, such as utilizing metal-chelating peptide tags, and biological recognition-based labeling utilizing enzymatic reactions.[16] However, despite their wide array of excitation and emission wavelengths as well as better stability, synthetic probes tend to be toxic to the cell and so are not generally used in cell imaging studies.[1]

Fluorescent labels can be hybridized to mRNA to help visualize interaction and activity, such as mRNA localization. An antisense strand labeled with the fluorescent probe is attached to a single mRNA strand, and can then be viewed during cell development to see the movement of mRNA within the cell.[17]

Fluorogenic labels

[edit]

A fluorogen is a ligand (fluorogenic ligand) which is not itself fluorescent, but when it is bound by a specific protein or RNA structure becomes fluorescent.[18]

For instance, FAST is a variant of photoactive yellow protein which was engineered to bind chemical mimics of the GFP tripeptide chromophore.[19] Likewise, the spinach aptamer is an engineered RNA sequence which can bind GFP chromophore chemical mimics, thereby conferring conditional and reversible fluorescence on RNA molecules containing the sequence.[20]

Use of tags in fluorescent labeling

[edit]
In a direct fluorescent antibody test, antibodies have been chemically linked to a fluorescent dye
FISH image of bifidobacteria Cy3
FISH analysis di george syndrome

Fluorescent labeling is known for its non-destructive nature and high sensitivity. This has made it one of the most widely used methods for labeling and tracking biomolecules.[1] Several techniques of fluorescent labeling can be utilized depending on the nature of the target.

Enzymatic labeling

[edit]

In enzymatic labeling, a DNA construct is first formed, using a gene and the DNA of a fluorescent protein.[21] After transcription, a hybrid RNA + fluorescent is formed. The object of interest is attached to an enzyme that can recognize this hybrid DNA. Usually fluorescein is used as the fluorophore.

Chemical labeling

[edit]

Chemical labeling or the use of chemical tags utilizes the interaction between a small molecule and a specific genetic amino acid sequence.[22] Chemical labeling is sometimes used as an alternative for GFP. Synthetic proteins that function as fluorescent probes are smaller than GFP's, and therefore can function as probes in a wider variety of situations. Moreover, they offer a wider range of colors and photochemical properties.[23] With recent advancements in chemical labeling, Chemical tags are preferred over fluorescent proteins due to the architectural and size limitations of the fluorescent protein's characteristic β-barrel. Alterations of fluorescent proteins would lead to loss of fluorescent properties.[22]

Protein labeling

[edit]

Protein labeling use a short tag to minimize disruption of protein folding and function. Transition metals are used to link specific residues in the tags to site-specific targets such as the N-termini, C-termini, or internal sites within the protein. Examples of tags used for protein labeling include biarsenical tags, Histidine tags, and FLAG tags.[1]

Genetic labeling

[edit]

Fluorescence in situ hybridization (FISH), is an example of a genetic labeling technique that utilizes probes that are specific for chromosomal sites along the length of a chromosome, also known as chromosome painting. Multiple fluorescent dyes that each have a distinct excitation and emission wavelength are bound to a probe which is then hybridized to chromosomes. A fluorescence microscope can detect the dyes present and send it to a computer that can reveal the karyotype of a cell. This technique allows abnormalities such as deletions and duplications to be revealed.[24]

Cell imaging

[edit]

Chemical tags have been tailored for imaging technologies more so than fluorescent proteins because chemical tags can localize photosensitizers closer to the target proteins.[25] Proteins can then be labeled and detected with imaging such as super-resolution microscopy, Ca2+-imaging, pH sensing, hydrogen peroxide detection, chromophore assisted light inactivation, and multi-photon light microscopy. In vivo imaging studies in live animals have been performed for the first time with the use of a monomeric protein derived from the bacterial haloalkane dehalogenase known as the Halo-tag.[22][26] The Halo-tag covalently links to its ligand and allows for better expression of soluble proteins.[26]

Advantages

[edit]

Although fluorescent dyes may not have the same sensitivity as radioactive probes, they are able to show real-time activity of molecules in action.[27] Moreover, radiation and appropriate handling is no longer a concern.

With the development of fluorescent tagging, fluorescence microscopy has allowed the visualization of specific proteins in both fixed and live cell images. Localization of specific proteins has led to important concepts in cellular biology such as the functions of distinct groups of proteins in cellular membranes and organelles. In live cell imaging, fluorescent tags enable movements of proteins and their interactions to be monitored.[24]

Latest advances in methods involving fluorescent tags have led to the visualization of mRNA and its localization within various organisms. Live cell imaging of RNA can be achieved by introducing synthesized RNA that is chemically coupled with a fluorescent tag into living cells by microinjection. This technique was used to show how the oskar mRNA in the Drosophila embryo localizes to the posterior region of the oocyte.[17]

See also

[edit]

Notes

[edit]
[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Fluorescent tag

View on Grokipedia
from Grokipedia
A fluorescent tag, also known as a or fluorescent label, is a that absorbs at one , becomes electronically excited, and subsequently emits at a longer , a process known as that enables the sensitive detection and visualization of specific biomolecules. These tags are typically covalently attached to target structures such as proteins, nucleic acids, antibodies, or cells, allowing researchers to track their localization, interactions, and dynamics in real-time using techniques like fluorescence microscopy and . Fluorescent tags encompass a diverse array of types, broadly classified into organic dyes, genetically encoded proteins, and inorganic nanoparticles. Organic fluorophores, including fluorescein, , and dyes (e.g., Cy3 and Cy5), are small synthetic molecules prized for their high , photostability, and broad spectral range, making them suitable for labeling purified biomolecules via reactive groups like amines or thiols. Genetically encoded fluorescent proteins, such as (GFP) and its engineered variants like or YFP, consist of β-barrel structures that self-assemble into fluorescent chromophores, enabling labeling through genetic fusion to proteins of interest without external chemical addition. Inorganic options, notably quantum dots—semiconductor nanocrystals—offer superior brightness and resistance to compared to organic dyes, though their larger (15–50 nm) can influence biomolecular function. The foundational advancement in fluorescent tagging came with the isolation of GFP from the Aequorea victoria in 1962 by Osamu Shimomura, whose work elucidated its structure and fluorescence mechanism, leading to its adaptation as a genetic tag in the early 1990s by and Roger Tsien. This innovation, recognized with the 2008 , transformed by enabling non-invasive imaging of cellular processes. Key applications include monitoring protein localization and trafficking in live cells, studying molecular interactions via Förster resonance energy transfer (), single-molecule tracking for enzymatic kinetics, and high-throughput assays in and diagnostics. Recent advances as of 2025 include chemigenetic strategies that combine synthetic dyes with genetic encoding for enhanced imaging fidelity. Despite their utility, challenges such as potential interference with target function and necessitate careful tag selection and experimental design.

Introduction and History

Definition and Basic Principles

Fluorescent tags are chemical or biological entities that absorb light at a specific and re-emit it at a longer , enabling the visualization of tagged biomolecules in biological systems. These tags, also known as fluorophores or fluorescent probes, are designed to bind selectively to target molecules such as proteins, nucleic acids, or cellular structures, allowing their detection through emitted light without the need for additional processing. Fluorescence differs from phosphorescence in that it involves rapid emission of light (on the order of nanoseconds) from an excited back to the , whereas phosphorescence arises from a slower transition involving a after , often lasting milliseconds to seconds. In biological contexts, autofluorescence from endogenous molecules like flavins or can interfere with signal detection, producing background emission typically in the range that must be distinguished from tag-specific . The underlying photophysical processes are depicted in the Jablonski diagram, which illustrates the electronic states of a fluorophore: upon absorption of a photon, an electron is excited from the ground singlet state (S₀) to a higher vibrational level of an excited singlet state (S₁ or higher); rapid vibrational relaxation occurs to the lowest level of S₁, followed by fluorescence emission as the electron returns to S₀, often to a higher vibrational level. This emission results in a Stokes shift, the energy difference between absorption and emission wavelengths (typically 10–100 nm), arising from the loss of vibrational energy and solvent reorganization, which separates excitation and emission spectra for effective detection against minimal background. The efficiency of this process is quantified by the quantum yield (Φ), the ratio of emitted photons to absorbed photons, ranging from 0.05 to 1.0 for most fluorophores, and the fluorescence lifetime (τ), the average time the molecule spends in the excited state before emitting, usually 1–10 ns. The fluorescence intensity IfI_f is given by If=Φ⋅IaI_f = \Phi \cdot I_a, where IaI_a is the intensity of absorbed light, highlighting the direct dependence on excitation efficiency and emission yield. Excitation and emission spectra of fluorescent tags generally show broad, overlapping bands due to vibrational transitions, with excitation peaks often sharper than emission peaks; for instance, a typical tag might absorb maximally around 488 nm and emit around 510 nm, illustrating the red-shifted emission characteristic of the . The phenomenon of was first systematically described in 1852 by George Stokes, who observed it in solutions under UV light.

Historical Development

The phenomenon of was first systematically described in 1852 by George Gabriel Stokes, who observed and coined the term for the emission of longer-wavelength light from excited solutions, laying the foundational observation for later tag development. The synthesis of the first artificial , , by in 1871 marked the advent of synthetic dyes suitable for biological applications, with following shortly thereafter as another early xanthene-based compound. In 1941, Albert Hewett Coons pioneered the use of fluorescent tags in by developing (FITC) to label antibodies, enabling the first assays for detecting antigens in tissues. The mid-20th century saw expanded dye options, with derivatives introduced in the 1970s for their enhanced photostability and red-shifted emission, improving multicolor labeling in . dyes, particularly the Cy series commercialized in the 1980s, further advanced labeling by offering tunable spectra and reduced for and protein probes. A transformative shift occurred with protein-based tags when Osamu Shimomura discovered (GFP) in the Aequorea victoria in 1962, isolating the protein responsible for its green bioluminescence. The GFP was cloned by in 1992, and demonstrated its expression as a in Escherichia coli and Caenorhabditis elegans in 1994, while Roger Tsien engineered brighter, color-variant forms starting in the mid-1990s. Shimomura, Chalfie, and Tsien shared the 2008 for these contributions, which enabled non-invasive tracking of proteins in living organisms. Nanomaterial innovations emerged in 1998 with the independent demonstrations by and Shuming Nie of quantum dots as biocompatible fluorescent labels, prized for their size-tunable emission and high . Self-labeling followed in 2003 with the , engineered by Alice Keppler, Stefan Gendreizig, and Kai Johnsson from human O6-alkylguanine-DNA alkyltransferase, allowing covalent attachment of diverse fluorophores to fusion proteins . Subsequent refinements included mNeonGreen in 2013, a monomeric yellow-green fluorescent protein derived from the lancelet Branchiostoma lanceolatum and optimized by Nathan C. Shaner and colleagues, offering 2-3 times the brightness of enhanced GFP for superior imaging contrast. By 2025, advancements continued with SNAP-tag2, an engineered variant providing faster kinetics and brighter signals for live-cell applications.
YearMilestoneKey Contributors
1852Discovery and naming of fluorescence phenomenonGeorge Gabriel Stokes
1871Synthesis of first artificial fluorophore (fluorescein)Adolf von Baeyer
1941Development of FITC for antibody labelingAlbert Hewett Coons
1970sIntroduction of rhodamine dyes for biological labelingVarious (e.g., refinements for microscopy)
1980sCommercialization of cyanine dyes (Cy series)GE Healthcare developers
1962Isolation of GFP from jellyfishOsamu Shimomura
1992Cloning of GFP geneDouglas Prasher
1994Expression of GFP in bacteria and wormsMartin Chalfie
Mid-1990sEngineering of GFP variantsRoger Tsien
1998Introduction of quantum dots as tagsA. Paul Alivisatos, Shuming Nie
2003Development of SNAP-tagAlice Keppler, Stefan Gendreizig, Kai Johnsson
2008Nobel Prize for GFP discovery and developmentShimomura, Chalfie, Tsien
2013Creation of mNeonGreenNathan C. Shaner et al.
2025Engineering of SNAP-tag2 for improved labelingKai Johnsson et al.

Types of Fluorescent Tags

Small-Molecule Dyes

Small-molecule fluorescent dyes are organic compounds with molecular weights typically below 1,000 Da, designed to absorb at specific wavelengths and emit at longer wavelengths through , enabling their use in biological labeling. These dyes are synthesized from various chemical scaffolds, allowing precise control over their spectral properties, such as excitation and emission wavelengths, quantum yields, and Stokes shifts. Unlike larger biomolecular tags, small-molecule dyes minimize steric interference when conjugated to targets, facilitating high-resolution in techniques like . Key chemical classes include xanthene-based dyes like fluoresceins and , polymethine-based cyanines, boron-dipyrromethene (BODIPY) dyes, and coumarins. Fluorescein derivatives, such as (FITC), exhibit green with an excitation maximum at 495 nm and emission at 519 nm, and a high of approximately 0.95 in basic conditions, though they are susceptible to and pH sensitivity, with intensity decreasing below 7 due to of the ring. derivatives, like tetramethylrhodamine isothiocyanate (TRITC), provide orange-red emission with excitation at 550 nm and emission at 573 nm, offering a around 0.7 and greater photostability than fluoresceins, making them suitable for longer sessions. Cyanine dyes, such as and , feature conjugated polymethine chains that enable tunable near-infrared emission; has excitation/emission at 550/570 nm, while shifts to 649/670 nm for deeper tissue penetration, with quantum yields typically 0.1-0.4 due to their extended conjugation but benefiting from sulfonation for aqueous . BODIPY dyes are noted for their exceptional photostability and narrow emission bands, often achieving quantum yields near 1.0 across tunable wavelengths from to , with minimal environmental sensitivity. dyes emit in the blue spectrum (excitation ~370-450 nm, emission ~450-500 nm) and possess large Stokes shifts (>100 nm), though their quantum yields vary (0.5-0.8) and they are prone to in polar environments.
Dye ClassExampleExcitation (nm)Emission (nm)Quantum Yield (Φ)Key Property
FluoresceinFITC495519~0.95pH-sensitive; high brightness but photobleaches easily
RhodamineTRITC550573~0.7Good photostability; orange-red emission
CyanineCy35505700.15-0.3Tunable chain length for NIR shift
CyanineCy56496700.27Low photobleaching in NIR; water-soluble variants
BODIPYBODIPY FL505513~1.0Narrow bandwidth; environment-insensitive
CoumarinCoumarin 343442499~0.6Large ; blue emission
These dyes are functionalized with reactive groups like (NHS) esters for attachment or maleimides for coupling, enabling covalent labeling of biomolecules. Their small size (~0.5-1 nm) reduces perturbation to target function compared to larger tags, and synthetic modifications allow with distinct spectra for multicolor applications, such as simultaneous detection of multiple antigens in assays. Small-molecule dyes provide superior brightness and spectral versatility over genetically encoded alternatives for fixed-sample labeling, though the latter excel in live-cell dynamics.

Fluorescent Proteins

Fluorescent proteins (FPs) are genetically encoded biomolecules that emit light upon excitation, primarily derived from marine organisms. The (GFP) was first isolated from the Aequorea victoria, where it serves as an energy-transfer acceptor in the of the photoprotein. The intrinsic of GFP forms through a post-translational autocatalytic process involving the cyclization and oxidation of the sequence Ser65-Tyr66-Gly67, resulting in a p-hydroxybenzylideneimidazolinone that enables fluorescence. Another early natural FP, DsRed, was cloned from the coral Discosoma sp., providing red fluorescence through a similar chromophore maturation from a Gln-Tyr-Gly , expanding the spectral palette beyond . Engineering efforts have produced a diverse array of GFP variants by targeted mutations to shift excitation and emission spectra. Blue fluorescent protein (EBFP) and cyan fluorescent protein (CFP) were developed through mutations like Y66H and Y66W in GFP, respectively, enabling shorter-wavelength emission for multicolor imaging. , achieved via substitutions such as T203Y and S65G, emits at longer wavelengths around 527 nm, facilitating pairs with CFP. To address oligomerization issues in early red FPs like tetrameric DsRed, monomeric variants such as were engineered in 2004 through iterative , yielding a bright red emitter with excitation at 587 nm and emission at 610 nm. Similarly, mNeonGreen, derived from a lancelet () FP in 2013, represents the brightest monomeric green FP to date, with excitation at 506 nm and emission at 517 nm, surpassing EGFP in and extinction coefficient. Far-red options like mKate2, a 2010 monomeric variant, offer emission at 633 nm for deeper tissue penetration, with brightness nearly threefold higher than earlier far-red FPs. Key properties of FPs include their spectral characteristics, maturation kinetics, and oligomeric state, which impact their utility in biological applications. Wild-type GFP exhibits major excitation at 395 nm (minor at 475 nm) and emission at 509 nm, with chromophore maturation requiring about 30 minutes at 28°C under aerobic conditions. DsRed, in contrast, is obligately tetrameric, which can disrupt localization, though monomeric mutants mitigate this while retaining excitation at 558 nm and emission at 583 nm. Engineered FPs like and mNeonGreen mature faster (under 10 minutes) and remain monomeric, enhancing their performance in live-cell imaging. Recent advances continue to optimize FP performance, particularly for demanding imaging scenarios. In April 2025, a suite of new monomeric FPs—spanning cyan, green, yellow, and red spectra—was reported, featuring enhanced brightness (up to twofold over mNeonGreen in the green channel) and photostability (retaining >80% fluorescence after prolonged illumination), achieved through directed evolution to reduce aggregation and improve folding efficiency. These variants build on prior engineering strategies, prioritizing minimal perturbation when fused to target proteins.

Nanomaterial-Based Tags

Nanomaterial-based fluorescent tags encompass inorganic nanoparticles that exhibit fluorescence through quantum confinement or energy transfer mechanisms, offering distinct advantages over traditional organic fluorophores due to their tunable optical properties and enhanced stability. These tags include quantum dots (QDs), carbon dots, and upconversion nanoparticles (UCNPs), each leveraging nanoscale dimensions to achieve size-dependent emission spectra suitable for biological labeling. QDs, typically composed of semiconductor materials like CdSe cores overcoated with ZnS shells, provide size-tunable emission from 400 to 800 nm, enabling multicolor imaging with a single excitation source. Carbon dots, derived from carbon-based precursors, exhibit broad excitation and emission in the visible range, while UCNPs, often lanthanide-doped such as NaYF4:Yb/Er, enable near-infrared (NIR) excitation with visible emission, minimizing autofluorescence in biological samples. The optical properties of these are characterized by high quantum yields, broad absorption spectra, and narrow emission bands, which facilitate efficient signal detection. For instance, CdSe/ZnS QDs achieve quantum yields exceeding 50%, with full-width at half-maximum (FWHM) emission peaks as narrow as 30-40 nm, contrasting with the broader spectra of organic dyes. They also demonstrate superior resistance to compared to small-molecule dyes, allowing prolonged observation in live-cell , though single-particle QDs may exhibit intermittent due to charge . Carbon dots offer quantum yields up to 80% with excitation-dependent emission, providing tunable colors from to , and excellent photostability. UCNPs feature sharp emission lines (<10 nm FWHM) and long luminescence lifetimes (>100 μs), enabling time-gated detection to further reduce background noise. These properties make nanomaterial tags ideal for high-sensitivity applications, often used alongside organic dyes in multiplexed setups. Synthesis of these tags typically involves colloidal methods to control size and composition, followed by surface functionalization for . The hot-injection technique, introduced in 1993 for CdSe QDs, rapidly injects organometallic precursors into a hot coordinating solvent to yield monodisperse nanocrystals with precise size control. Carbon dots are commonly synthesized via of organic precursors like , producing water-soluble particles in a one-pot process. UCNPs are prepared through or co-precipitation of rare-earth salts, resulting in core-shell structures that enhance upconversion efficiency. is achieved by coating with polymers such as (PEG) for stability or linking to for specific biomolecular targeting, ensuring aqueous dispersibility without compromising . Recent advances focus on developing non-toxic alternatives to cadmium-based QDs to improve for applications. (InP) QDs, synthesized via similar colloidal routes but with shells like ZnSe/ZnS, exhibit comparable emission tunability (450-700 nm) and quantum yields (>60%) while reducing heavy-metal , with 2024 studies demonstrating their efficacy in cellular without significant . quantum dots (GQDs), produced by oxidative cutting of graphene or hydrothermal methods, offer edge-state emission in the 400-600 nm range, high , and low , with recent 2024 optimizations through doping for bioimaging and sensing. These eco-friendly address regulatory concerns and expand the scope of fluorescent tagging in biomedical research.

Labeling Methods

Chemical Labeling

Chemical labeling involves the covalent attachment of fluorescent tags to biomolecules through reactive functional groups, enabling visualization without altering the of the target. This non-genetic approach is widely used for labeling purified proteins, antibodies, and nucleic acids, offering flexibility in selecting dyes with desired spectral properties. Techniques rely on the chemical reactivity of specific side chains or introduced functional groups, ensuring stable conjugation under controlled conditions. Amine-reactive chemistries, such as (NHS) esters and , target primary amines on residues or N-terminal α-amino groups of proteins. NHS esters form stable amide bonds with lysines, which have a higher pKa (10-11) compared to N-terminal amines (pKa ~7-9), allowing selective labeling at neutral by exploiting reactivity differences. For example, (FITC), an dye, is commonly conjugated to by reacting with solvent-accessible amines in (PBS) at 7.4-8.0 and , typically achieving a fluorophore-to-protein ratio of 1-5 while preserving ~94% avidity. Thiol-reactive reagents, like maleimides, selectively bind cysteine sulfhydryl groups (-SH) to form thioether linkages, with reactions proceeding rapidly (10 minutes to 2 hours) in buffers at 6.5-7.5 to minimize disulfide formation. Site-specificity is enhanced by engineering unique cysteines via , reducing off-target effects from endogenous thiols. Click chemistry provides bioorthogonal conjugation via copper-catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC), linking azides and terminal or strained alkynes to form stable triazole rings. CuAAC, introduced in 2002, accelerates the Huisgen cycloaddition by 10^6-10^7-fold using Cu(I) catalysts like sodium ascorbate and ligands (e.g., THPTA) in aqueous buffers at pH 7-8, but requires post-reaction copper removal to avoid cellular toxicity. SPAAC, developed in 2004, is catalyst-free and biocompatible, employing cyclooctynes for live-cell applications, though it reacts ~100-fold slower and lacks regioselectivity. These methods are applied to DNA by incorporating azides or alkynes via phosphoramidite synthesis, followed by dye conjugation. Direct labeling targets purified biomolecules, where reactive dyes are incubated with proteins or DNA under optimized conditions, followed by purification (e.g., gel filtration) to remove unbound fluorophores. Indirect labeling uses haptens like biotin, which binds streptavidin-conjugated dyes with high affinity (K_d ~10^{-15} M), allowing modular attachment without direct covalent modification of the target. For instance, biotinylated antibodies are detected with streptavidin-FITC, enabling multivalent amplification for enhanced signal intensity. Bioorthogonal labeling extends these to live cells by introducing non-native groups (e.g., azides via metabolic incorporation) for selective tagging, as in SPAAC-mediated in vivo conjugation of fluorophores to azide-modified cell surfaces. An example is FITC conjugation to anti-HA antibodies for immunofluorescence, where excess dye is quenched to achieve uniform labeling. Strain-promoted click chemistry has been used for in vivo tagging of transplanted chondrocytes with azido-sugars and cyclooctyne-fluorophores, enabling long-term tracking without toxicity. Key considerations include reaction conditions to maximize yield and minimize artifacts: amine reactions favor pH 8-9 in amine-free buffers (e.g., ), while thiol couplings use pH 7-7.5 with reducing agents like DTT to expose cysteines. Specificity is critical to avoid off-target labeling; for proteins with multiple lysines (~20-50 per ), partial occupancy follows , with higher dye ratios risking functional impairment (e.g., 20% loss at ratios >3). Click reactions demand bioorthogonal handles to prevent interference from cellular nucleophiles, and CuAAC requires chelators (e.g., EDTA) for detoxification. Overall, these methods balance efficiency and , though they necessitate purified samples or orthogonal groups for use.

Genetic Labeling

Genetic labeling involves the incorporation of fluorescent tags into proteins through , enabling their expression in living cells and organisms. This approach relies on fusing the coding sequence of a fluorescent protein (FP) to that of a target protein, allowing the resulting to be visualized in real time without the need for exogenous labeling agents. One primary technique is the creation of constructs, where the FP is genetically linked to the target protein via flexible linker peptides, such as glycine-serine repeats, to preserve the functionality and localization of both components. For more precise endogenous labeling, /Cas9-mediated knock-in strategies insert FP-coding sequences directly into the genomic locus of the target gene, minimizing overexpression artifacts and enabling study of native protein dynamics. Delivery of these genetic constructs is achieved using various vectors tailored to the model system. In mammalian cells, plasmids are commonly used for transient , while viral vectors like (AAV) and provide stable, long-term expression due to their ability to transduce both dividing and non-dividing cells. vectors are particularly favored for applications owing to their low and for specific tissues, whereas lentiviral vectors excel in integrating transgenes into the host for heritable expression. In prokaryotic systems like , simple plasmid-based expression under inducible promoters facilitates high-yield production, and in , integrative vectors enable stable chromosomal insertion for eukaryotic folding studies. Notable examples include transgenic animals engineered to express FP fusions, such as mice with neuron-specific GFP-histone fusions for tracking cellular processes during development. Another application is (BiFC), where split-FP fragments are fused to interacting proteins; upon association, the fragments reassemble to form a functional , visualizing protein-protein interactions . To optimize these constructs, linker design is critical: short, rigid alpha-helical linkers reduce steric hindrance for periplasmic proteins, while longer flexible ones accommodate domain movements in cytoplasmic fusions. Additionally, codon optimization adjusts the nucleotide sequence to match the host organism's tRNA preferences, enhancing efficiency; for instance, humanized codons in FPs can increase expression levels by up to 100-fold in mammalian cells. These optimizations ensure minimal interference with protein function and maximal fluorescence output.

Enzymatic and Self-Labeling Techniques

Enzymatic labeling techniques utilize biocatalysts to achieve site-specific attachment of fluorescent tags, offering enhanced specificity and amplification compared to non-enzymatic methods. Horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) is a prominent example, where HRP catalyzes the deposition of fluorophore-conjugated tyramide radicals onto nearby tyrosine residues in the presence of hydrogen peroxide, enabling high-density labeling of targets such as proteins or nucleic acids. This process amplifies signals up to 100-fold, making it particularly useful for detecting low-abundance biomolecules in fixed tissues or cells. Another enzymatic approach involves DNA polymerases incorporating fluorescent nucleotide analogs, such as Cy3- or Cy5-labeled dUTPs, during DNA synthesis to label newly synthesized strands or probes. These analogs maintain polymerase fidelity while providing bright, stable fluorescence for applications like in situ hybridization. Self-labeling techniques rely on engineered proteins that covalently bind specific synthetic substrates, allowing modular and post-translational fluorescent tagging without external enzymes. The , derived from human O6-alkylguanine-DNA alkyltransferase (AGT), reacts irreversibly with O6-benzylguanine derivatives conjugated to fluorophores, enabling rapid and specific labeling of fusion proteins expressed in cells. Introduced in 2003, this system exhibits high substrate specificity, with minimal to endogenous AGT. The CLIP-tag, a SNAP-tag variant mutated to recognize O2-benzylcytosine substrates (e.g., derivatives), facilitates orthogonal labeling alongside SNAP-tag for multicolor imaging. Similarly, the , based on a bacterial dehalogenase, forms a with alkyl halide ligands bearing fluorophores, offering robust labeling in diverse cellular environments due to its chloroalkane reactivity. These techniques leverage substrate specificity to ensure precise targeting; for instance, SNAP- and CLIP-tags distinguish between and derivatives, while 's halide chemistry avoids interference from cellular nucleophiles. A recent advancement, SNAP-tag2, engineered in 2025, improves upon the original by achieving 100-fold faster labeling kinetics and fivefold brighter fluorescence when paired with fluorogenic dyes, enhancing in live cells. Live-cell applications often involve pulse labeling with cell-permeable substrates, such as ligands, which allow temporal tracking of protein dynamics by sequential addition and washout. This modularity complements genetic fusions by enabling probe exchange post-expression for varied experimental needs.

Applications

Microscopy and Imaging

Fluorescent tags enable the visualization of specific cellular structures and dynamic processes in various microscopy techniques by converting molecular recognition events into detectable light signals. In widefield epifluorescence microscopy, these tags facilitate basic localization studies by uniformly illuminating the sample with broadband excitation light, allowing real-time observation of tagged proteins in thin specimens or surface features. This approach is particularly effective for live-cell imaging due to its simplicity and speed, though it suffers from out-of-focus blur in thicker samples. Confocal microscopy enhances resolution through optical sectioning, employing a pinhole to reject stray light and generate sharp z-stack images of fluorescently labeled structures, which is essential for three-dimensional reconstructions of complex cellular architectures. Super-resolution methods push beyond the diffraction limit of approximately 200 nm; stimulated emission depletion (STED) microscopy uses a doughnut-shaped depletion laser to inhibit fluorescence outside a central spot, achieving lateral resolutions of 20-50 nm with tags that withstand high-intensity light. Photoactivated localization microscopy (PALM), on the other hand, exploits photo-switchable fluorescent proteins to stochastically activate and localize single molecules, reconstructing super-resolved images from thousands of frames with precisions down to 10-20 nm. Key applications include tracking protein trafficking, such as with (GFP)-tagged Rab , which reveal vesicle dynamics during endocytic and secretory pathways in live cells. Organelle-specific labeling, exemplified by mitochondrial-targeted GFP (mito-GFP), permits detailed of fission, fusion, and transport events within the mitochondrial network. Multicolor leverages spectrally distinct tags to simultaneously monitor multiple targets, with spectral unmixing algorithms deconvolving overlapping emission spectra for accurate separation and quantification. Time-lapse of using such tags captures dynamic events like spindle assembly and over hours, providing insights into temporal without fixation artifacts. Förster resonance energy transfer (FRET) pairs like cyan fluorescent protein (CFP) and (YFP) enable nanoscale distance measurements (1-10 nm) between tagged molecules, quantifying conformational changes or interactions in contexts. The FRET efficiency EE follows the equation: E=11+(rR0)6E = \frac{1}{1 + \left( \frac{r}{R_0} \right)^6} where rr is the donor-acceptor separation and R0R_0 is the Förster distance specific to the pair (typically 4-6 nm for CFP-YFP). Instrumentation relies on excitation sources like lasers tuned to tag absorption maxima (e.g., 488 nm for GFP) and filter sets comprising bandpass excitation filters, dichroic mirrors, and emission filters to isolate signals while minimizing crosstalk.

Biosensing and Molecular Interactions

Fluorescent tags play a crucial role in biosensing by enabling the detection of biochemical events such as molecular interactions and environmental changes within cells. These tags are incorporated into probes that report dynamic processes, including protein-protein interactions (PPIs) and variations in ion concentrations or , through changes in properties like intensity, wavelength, or lifetime. For instance, (FRET) is a widely used method where energy transfers from a donor to an acceptor when they are in close proximity (typically 1-10 nm), signaling molecular binding events. In studies of PPIs, FRET has been applied to monitor -substrate interactions, such as those involving p38 kinase, by fusing fluorescent proteins to the interacting partners and measuring energy transfer efficiency to quantify activity spatiotemporal profiles. Environment-sensitive dyes further expand biosensing capabilities by altering their in response to local conditions like . The aminonaphthylethenylpyridinium (ANEP) dyes, such as di-4-ANEPPS, exhibit voltage-dependent shifts in their excitation spectra, allowing ratiometric measurements where the of at two wavelengths (e.g., 440 nm and 505 nm excitation) reports changes in transmembrane potential with high suitable for tracking millisecond-scale events in excitable cells. Specific biosensors leverage these principles for targeted detection; for example, the cameleon indicators use between cyan fluorescent protein (CFP) as donor and (YFP) as acceptor, flanking a calmodulin-M13 domain that undergoes conformational change upon calcium binding, increasing efficiency and enabling ratiometric imaging of intracellular Ca²⁺ dynamics. Similarly, pH sensors based on fluorescein derivatives employ dual-excitation ratiometry (e.g., at 490 nm and 440 nm), where the emission varies with state, providing quantitative readout of subcellular fluctuations while minimizing artifacts from dye concentration or illumination variations. Advanced imaging techniques enhance the precision of these biosensors. Fluorescence lifetime imaging microscopy (FLIM) measures the decay time of fluorescence, which shortens in the presence of , allowing quantification of binding kinetics for PPIs without spectral overlap issues; for example, FLIM-FRET has been used to screen binding partners among protein families by analyzing donor lifetime reductions indicative of interaction affinity. resonance energy transfer (BRET) complements FRET by using as a donor paired with fluorescent proteins like YFP, enabling low-background detection of PPIs in deep tissues or low-expression systems, as demonstrated in assays fusing luciferase to one protein and a fluorescent tag to its partner to monitor energy transfer upon co-expression. in these systems often relies on ratiometric approaches, such as emission or excitation intensity ratios, to correct for experimental artifacts like , motion, or uneven illumination, ensuring reliable quantification of molecular events.

In Vivo and Therapeutic Uses

Fluorescent tags enable non-invasive imaging in whole organisms, particularly through transgenic models expressing (GFP) to track . For instance, GFP-expressing tumor cells in mice allow real-time visualization of metastatic spread, such as in orthotopic models where primary tumors and distant metastases are monitored without surgical intervention, revealing dynamics of tumor growth and over weeks. Similarly, transgenic GFP-Met mice facilitate detection of early tumorigenesis and circulating metastatic cells in blood, providing insights into cancer progression at the single-cell level. These models have revolutionized by enabling longitudinal studies in live animals, as highlighted in reviews emphasizing GFP's role in non-invasive optical imaging. Two-photon microscopy enhances deep-tissue imaging with fluorescent tags by using near-infrared excitation to minimize and achieve penetration depths of up to 1 mm in living tissues, far surpassing conventional fluorescence methods. This technique, often paired with GFP or other , allows high-resolution imaging of neural activity or vascular structures in intact brains or organs, reducing and enabling repeated observations. Recent advancements, such as fast-scanning two-photon systems developed in 2025, further extend imaging into previously inaccessible regions like deep cortical layers, supporting studies of disease progression in models of neurodegeneration or cancer. In therapeutic applications, photoactivatable fluorescent tags fused to enable optogenetic control of cellular activity , where light activation of these fusions restores sensory functions or silences neural circuits with high precision. For example, variants tagged with fluorescent proteins allow simultaneous visualization and manipulation of targeted neurons in models of blindness or , achieving millisecond temporal resolution. Quantum dots (QDs) track by conjugating to therapeutic agents, enabling real-time monitoring of distribution in tumors; anti-HER2 antibody-QD conjugates, for instance, reveal and accumulation in xenografts within hours post-injection. Targeted tumor imaging employs Cy5.5-conjugated dyes for near-infrared detection, where affibody molecules linked to Cy5.5 specifically bind EGFR-overexpressing tumors in mice, providing high-contrast images for surgical guidance. Similarly, Cy5.5-deoxyglucose analogues accumulate in glucose-avid tumors, enabling non-invasive tracking of metabolic activity . For RNA trafficking, fluorescent light-up aptamers (FLAPs) such as Mango variants label endogenous genetically, allowing live-cell and observation of localization and transport; advances from 2019–2025 include optimized FLAPs for minimal perturbation during viral studies or neuronal mRNA dynamics. Despite these advances, challenges persist in applications, including limited tissue penetration due to photon absorption and scattering by hemoglobin and lipids, which confines imaging to superficial depths unless mitigated by clearing techniques or longer wavelengths. Clearance of tags also poses issues, as non-degradable probes like QDs can accumulate in organs, leading to and signal persistence that complicates repeated dosing. Recent developments in 2024–2025 include peptide-based probes targeting dipeptidylpeptidase-4 (DPP4) for senescence detection, where activatable fluorophores conjugated to peptides visualize senescent cells in obese models, showing elevated signals in liver and via whole-body . These probes offer specificity for senescence-associated enzymes, aiding therapeutic targeting in aging-related diseases.

Advantages and Limitations

Key Benefits

Fluorescent tags offer exceptional specificity and sensitivity in biological research, enabling the precise labeling and detection of individual molecules within complex cellular environments. By genetically fusing tags like (GFP) to proteins of interest, researchers can achieve targeted visualization at submicrometer spatial resolution, facilitating single-molecule detection without significant interference from background noise. This high specificity arises from the covalent attachment of fluorophores to biomolecules, ensuring minimal off-target labeling, while the sensitivity is enhanced by the high brightness of modern tags, such as mNeonGreen, which supports detection in low-abundance scenarios. Furthermore, capabilities allow simultaneous of multiple targets using spectrally distinct fluorophores, with systems supporting 5-10 colors and low crosstalk, as demonstrated in advanced FRET-based labels that distinguish up to 27 unique signatures through combined spectroscopic parameters like lifetime and . A key advantage of fluorescent tags is their non-invasiveness, permitting real-time tracking of dynamic processes in living cells and organisms without the hazards associated with radioactive alternatives. Genetic encoding ensures that tags are expressed under physiological conditions, maintaining cellular viability and function during extended imaging sessions, often at subsecond . Unlike radioisotopes, which require handling precautions and produce decaying signals, fluorescent tags provide stable, visual readouts via optical excitation, enabling safe, repeated observations of protein localization and interactions . The versatility of fluorescent tags extends their utility across a wide array of experimental techniques, from microscopy to high-throughput assays. They integrate seamlessly with methods like flow cytometry for quantifying protein expression in thousands to millions of cells and enzyme-linked immunosorbent assays (ELISA) for molecular detection, while fusion tags like GFP also serve as affinity tools for protein purification. Available in diverse spectral ranges from blue to near-infrared, these tags support multicolor labeling and are adaptable to both chemical and genetic incorporation strategies, broadening their applicability in diverse biological contexts. Quantitatively, fluorescent tags deliver high signal-to-noise ratios due to their tunable properties, outperforming isotopic methods by avoiding signal decay and offering direct visual quantification without specialized detection equipment. This enables accurate measurement of biomolecular concentrations and dynamics, with photostable variants maintaining signal integrity over prolonged observations, thus providing reliable data for quantitative analyses in live systems.

Challenges and Considerations

One major challenge in using fluorescent tags is , where prolonged exposure to excitation leads to irreversible loss of intensity. To mitigate this, anti-fade mounting media, such as glycerol-based formulations containing antioxidants like or , are commonly applied to samples, reducing formation and significantly extending signal duration, often by factors of several-fold, during imaging. Recent efforts have produced highly photostable monomeric FP variants, such as mGold2 and mScarlet3-H, which exhibit 25- to 29-fold improved resistance to bleaching compared to predecessors like mVenus, enabling longer live-cell observations without significant signal loss. Fluorescent tags can interfere with biological processes by altering the structure or function of the labeled molecule, particularly when tag size disrupts , localization, or interactions. For instance, early (GFP) variants tend to dimerize via hydrophobic interfaces, which can artificially oligomerize fusion partners and perturb their native behavior, such as in cytoskeletal dynamics or enzymatic activity. A single-point like A206K has been introduced to generate monomeric GFP, minimizing these artifacts while preserving brightness. applications face additional hurdles from tag toxicity; quantum dots (QDs) often incorporate like , leading to , organ accumulation (e.g., in liver and ), and upon shell degradation, limiting their use in long-term animal studies. Technical limitations further complicate multiplexed imaging with fluorescent tags, including spectral overlap where emission spectra of different tags bleed into adjacent channels, reducing signal-to-noise ratios and complicating unmixing algorithms. This issue is exacerbated in high-throughput setups, necessitating advanced spectral imaging techniques like hyperspectral detection to deconvolute signals from up to 10 or more tags. Low labeling efficiency poses another barrier, especially for sparse or low-abundance targets in bioimaging, where incomplete tag incorporation for chemical methods results in weak signals and false negatives, as highlighted in recent reviews on molecular labeling strategies. Mislocalization artifacts arise when tags influence protein partitioning into biomolecular condensates, such as phase-separated organelles like , potentially altering condensate formation, dynamics, or composition. A 2025 study demonstrated that common FP tags, including GFP and , enhance or inhibit condensation of proteins like Dhh1, leading to ectopic localization and skewed interpretations of cellular organization. To address such perturbations, minimal tags incorporating unnatural (UAAs) via expansion offer a solution, enabling site-specific attachment of small fluorescent moieties (e.g., 1-2 residues) without bulky domains, as shown in systems achieving near-quantitative labeling in mammalian cells.

References

  1. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/fluorescent-tag
  2. https://pmc.ncbi.nlm.nih.gov/articles/PMC3691395/
  3. https://pmc.ncbi.nlm.nih.gov/articles/PMC3037093/
  4. https://pubmed.ncbi.nlm.nih.gov/19771329/
  5. https://pmc.ncbi.nlm.nih.gov/articles/PMC12407358/
  6. https://pmc.ncbi.nlm.nih.gov/articles/PMC5385933/
  7. https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/introduction-to-fluorescence-techniques.html
  8. https://probes.bocsci.com/resources/fluorescent-labeling-definition-principles-types-and-applications.html
  9. https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_%28Physical_and_Theoretical_Chemistry%29/Spectroscopy/Electronic_Spectroscopy/Fluorescence_and_Phosphorescence
  10. https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_%28Physical_and_Theoretical_Chemistry%29/Spectroscopy/Electronic_Spectroscopy/Radiative_Decay/Fluorescence
  11. https://www.nature.com/articles/ncb1009-1165
  12. https://biotium.com/blog/cf-dyes-what-started-it-all-part-1-a-history-of-fluorescence/
  13. https://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem-061516-044839
  14. https://www.sciencedirect.com/science/article/abs/pii/S136759312300073X
  15. https://www.nobelprize.org/prizes/chemistry/2008/summary/
  16. https://www.nature.com/articles/s41589-025-01942-z
  17. https://pubs.acs.org/doi/10.1021/cb500078u
  18. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/fluorescein.html
  19. https://pmc.ncbi.nlm.nih.gov/articles/PMC7729466/
  20. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/tritc-dye.html
  21. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/cy3-dye.html
  22. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/cy5-dye.html
  23. https://blog.addgene.org/better-dyeing-through-chemistry-small-molecule-fluorophores
  24. https://pubmed.ncbi.nlm.nih.gov/13911999/
  25. https://web.math.princeton.edu/~sswang/GECI/GFP_Tsien_1998_annual_reviews.pdf
  26. https://pmc.ncbi.nlm.nih.gov/articles/PMC17281/
  27. https://einsteinmed.edu/uploadedFiles/research/facilities/Fluorescent/Oleych2006.pdf
  28. https://pubmed.ncbi.nlm.nih.gov/23524392/
  29. https://pmc.ncbi.nlm.nih.gov/articles/PMC2893397/
  30. https://www.science.org/doi/10.1126/sciadv.ads6201
  31. https://pmc.ncbi.nlm.nih.gov/articles/PMC8228846/
  32. https://pubs.acs.org/doi/10.1021/cr400425h
  33. https://pubs.acs.org/doi/10.1021/jp971091y
  34. https://www.sciencedirect.com/science/article/abs/pii/S0006291X12021754
  35. https://www.nature.com/articles/s41377-022-00764-1
  36. https://pubs.rsc.org/en/content/articlelanding/2017/ra/c7ra07573a
  37. https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5b00221
  38. https://pmc.ncbi.nlm.nih.gov/articles/PMC10887166/
  39. https://pubs.rsc.org/en/content/articlehtml/2024/ra/d4ra01431f
  40. https://link.springer.com/article/10.1007/s11814-024-00279-y
  41. https://pmc.ncbi.nlm.nih.gov/articles/PMC2876214/
  42. https://www.abcam.com/ps/products/102/ab102884/documents/FITC%20Conjugation%20Kit_protocol_booklet_ab102884%20%28website%29.pdf
  43. https://doi.org/10.1002/anie.200201089
  44. https://pmc.ncbi.nlm.nih.gov/articles/PMC6310217/
  45. https://doi.org/10.1021/acs.bioconjchem.6b00010
  46. https://pmc.ncbi.nlm.nih.gov/articles/PMC2875081/
  47. https://www.pnas.org/doi/10.1073/pnas.1606731113
  48. https://www.nature.com/articles/s41392-021-00487-6
  49. https://www.thermofisher.com/blog/life-in-the-lab/aav-vs-lv/
  50. https://www.sciencedirect.com/science/article/pii/S2211124722006180
  51. https://www.sciencedirect.com/science/article/pii/S1097276502004963
  52. https://pmc.ncbi.nlm.nih.gov/articles/PMC3726540/
  53. https://pmc.ncbi.nlm.nih.gov/articles/PMC2663184/
  54. https://pubmed.ncbi.nlm.nih.gov/9792422/
  55. https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/tyramide-signal-amplification-technology.html
  56. https://www.tandfonline.com/doi/full/10.2144/05382RR02
  57. https://academic.oup.com/nar/article/46/12/e73/4965821
  58. https://pmc.ncbi.nlm.nih.gov/articles/PMC3920298/
  59. https://www.sciencedirect.com/science/article/pii/S1074552108000410
  60. https://pubs.acs.org/doi/10.1021/cb800025k
  61. https://pubs.acs.org/doi/10.1021/acs.biochem.1c00258
  62. https://www.sciencedirect.com/science/article/pii/S0021925820304713
  63. https://pmc.ncbi.nlm.nih.gov/articles/PMC3483677/
  64. https://pubmed.ncbi.nlm.nih.gov/19844443/
  65. https://www.science.org/doi/10.1126/science.1127344
  66. https://www.science.org/doi/10.1126/science.8303295
  67. https://rupress.org/jcb/article/156/3/511/32500/Visualization-of-Rab9-mediated-vesicle-transport
  68. https://onlinelibrary.wiley.com/doi/abs/10.1046/j.1365-313X.1997.11030613.x
  69. https://www.sciencedirect.com/science/article/pii/S0014579303005210
  70. https://pmc.ncbi.nlm.nih.gov/articles/PMC3137897/
  71. https://pmc.ncbi.nlm.nih.gov/articles/PMC5038762/
  72. https://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html
  73. https://www.nature.com/articles/srep28186
  74. https://www.nature.com/articles/s41598-023-33953-y
  75. https://www.pnas.org/doi/10.1073/pnas.92.8.3156
  76. https://www.pnas.org/doi/10.1073/pnas.96.1.151
  77. https://www.nature.com/articles/s41467-020-15687-x
  78. https://www.sciencedirect.com/science/article/abs/pii/S1470204502008483
  79. https://pmc.ncbi.nlm.nih.gov/articles/PMC1592452/
  80. https://academic.oup.com/carcin/article/25/12/2285/2475875
  81. https://pmc.ncbi.nlm.nih.gov/articles/PMC5766275/
  82. https://www.ucdavis.edu/blog/new-two-photon-microscopy-system-aims-see-impossible-spaces
  83. https://pmc.ncbi.nlm.nih.gov/articles/PMC10111946/
  84. https://aacrjournals.org/cancerres/article/67/3/1138/533986/In-vivo-Real-time-Tracking-of-Single-Quantum-Dots
  85. https://pubmed.ncbi.nlm.nih.gov/20615009/
  86. https://pubs.acs.org/doi/10.1021/bc050345c
  87. https://www.tandfonline.com/doi/full/10.4155/fmc-2019-0207
  88. https://pmc.ncbi.nlm.nih.gov/articles/PMC6391945/
  89. https://www.sciencedirect.com/science/article/abs/pii/S095656632400976X
  90. https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-022-01648-7
  91. https://royalsocietypublishing.org/doi/10.1098/rsfs.2013.0001
  92. https://onlinelibrary.wiley.com/doi/10.1111/cas.16229
  93. https://www.molbiolcell.org/doi/10.1091/mbc.e16-07-0504
  94. https://www.nature.com/articles/s41565-024-01672-8
  95. https://pmc.ncbi.nlm.nih.gov/articles/PMC2729480/
  96. https://www.nature.com/articles/s41592-022-01660-7
  97. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades.html
  98. https://www.nature.com/articles/s41467-025-58223-5
  99. https://pubmed.ncbi.nlm.nih.gov/40247125/
  100. https://pmc.ncbi.nlm.nih.gov/articles/PMC6471090/
  101. https://pmc.ncbi.nlm.nih.gov/articles/PMC10759915/
  102. https://www.nature.com/articles/s41377-021-00536-3
  103. https://www.sciencedirect.com/science/article/pii/S2667074725000059
  104. https://www.molbiolcell.org/doi/full/10.1091/mbc.E24-11-0521
  105. https://www.nature.com/articles/s41467-022-27956-y
Add your contribution
Related Hubs
User Avatar
No comments yet.