Fluorescent Probes

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Fluorescence, a form of luminescence, is the emission of light produced by a substance that has absorbed light or electromagnetic radiation. Fluorescent molecules re-emit light at longer wavelength and lower energy in response to the absorbed radiation. On account of its properties, fluorescence technology is widely used in fluorescent imaging and spectroscopy, utilizing fluorescent probes, dyes and other bioactive reagents.

Fluorophores are fluorescent compounds that are used alone or bonded to molecules to create fluorescent probes. Fluorophores are generally classified as four types: organic dyes, fluorescent proteins, quantum dots (luminescent nanocrystals), and biological structures suitable for label-free imaging. Most fluorophores are small-molecule dyes with low molecular weight (0.2-1 kDa), and some are relatively large proteins, such as GFP (green), YFP (yellow) and RFP (red). The broad selection of fluorophores offers a wide range of applications in life science and fundamental research, depending on their physical properties as size, biocompatibility, excitation and emission wavelength, intensity, quantum yield, fluorescence lifetime, and the interaction with substances.

Figure 1. Leukemia cells labeled with fluorescent molecules

Fluorescent probes are single fluorophores or fluorophores covalently conjugated with biological molecules. Several types of fluorescent probes are provided on BOC Sciences website as below:

Cell and Organelle Stains: Fluorescence brought about a new approach for visualization of cell and organelle structure, as well as cell tracking. The Green fluorescent protein, a 238-amino acid protein, was first isolated from the jellyfish Aequorea Victoria in 1961, boosting the applications of fluorescent proteins in cell biology. Soon afterwards, more fluorescent proteins from other species have been discovered and isolated. Upon the rapid development of fluorescent protein technology, the utilization of genetically encoded fluorophore for a wide spectrum of applications beyond the simple tracking of tagged biomolecules in live cells has recently become fully appreciated.

Other non-protein fluorescent probes can stain membranes, organelles, nucleic acid and proteins in live or fixed cells for the investigation of cells and cell components. Cell staining technique is utilized in microscopy, flow cytometry, and the application in disease diagnosis has been advancing, ranging from cancers to bacterial infections. Epicocconone, a long stokes’ shift fluorescent dye metabolized from the Fungus Epicoccum nigrum, was reported for staining human colon cancer cell line HCT-116, showing a maximum emission peak at 590 nm. Epicocconone is suitable for live cell imaging in that it does not affect growth of mammalian cells at concentrations similar to those used for staining.

DNA Stains: DNA stains are ultrasensitive dyes enabling researchers to visualize DNA fragment and amount. Cyanine dyes, phenanthridines and acridines, indoles and imidazoles, and some other nucleic acid dyes are generally included in DNA stains. In flow cytometry and microscopy, Cy3 and Cy5 are most popular cyanine dyes commonly with an N-hydroxysuccinimidyl ester (NHS-ester) reactive group attached for labeling DNA. In addition, the TOTO family, the TO-PRO family, the SYTO family, and the SYBR family dyes are cyanine dyes optimized for different purposes. SYBR Green I preferentially binds to double-stranded DNA to form a complex emitting over 1000-fold fluorescence. SYBR Green I has been utilized for DNA detection in PCR, gel electrophoresis, flow cytometry and microscopy.

Hoechst dyes are bisbenzimides that are excited by UV light at 350 nm and fluoresce blue light at 460 nm. The relatively large Stokes’ shift enables Hoechst dyes suitable for multicolor labeling experiments. Hoechst dyes stain DNA via selectively binding to the minor groove of AT-rich double-stranded DNA. Hoechst 33258, Hoechst 33342, and Hoechst 34580 are related stains in this family.

Fluorescent Enzyme Substrates: Fluorogenic substrates for multiple enzymes are used in enzymatic activity assays. They are generally composed of a fluorescent compound and a specific enzyme substrate. Upon the enzymatic cleavage, the fluorescent substrate releases the fluorescent moiety and yields fluorescence, of which the ratios or the intensity can be used to quantify the enzymatic activity. Some substrates are developed for live cell enzyme assays, enabling to investigate the physiological functions of various enzymes in situ.

Ion Indicators and Sensors: The concentrations of calcium, sodium, potassium, zinc and other metal ions in humans should be maintained within a suitable range to guarantee their normal biological functions. Ion indicators are generally available in both the membrane-impermeant salt forms and the membrane-permeant AM ester forms. Upon entrance to cells, the ion indicators are released via hydrolysis, and binds to ions. Calcium indicators are classified as chemical indicators and genetically encoded calcium indicators. Chemical indicators are fluorophores coupled to the calcium chelator BAPTA structure, such as indo-1, fura-2, fluo-3, fluo-4. The fluorescent quantum yield, excitation/emission wavelength, and spectral shift generated from binding of calcium ions are used for their quantification. Genetically encoded calcium indicators are fluorescent proteins derived from green fluorescent protein.

Porphyrin analogs are designed as metal ion or anion chemosensors for detecting environmental systems and biological processes. The framework of sensors can be classified into 2 types: Type 1 is composed of a reporter and a recognition unit. Once the recognition unit is incorporated to interact with the target analyte, a photophysical signal will be reported from the reporter. Type 2 only consists of one component, acting simultaneously as the recognition and the reporting unit. Different types of chemosensors are developed depending on various photophysical processes.

pH Indicators: Intracellular pH is of great importance for cell, tissue and enzyme activities, and abnormal pH values are commonly involved in inappropriate cell function and growth, which can be observed in some diseases such as cancers. Measurement of intracellular pH provides critical information for studying physiological processes in cells. As the qualitative measurement is easily influenced by optical path length, temperature, altered excitation intensities, and varied emission collection efficiencies, ratiometric spectroscopy is employed to detect pH. The measurement method requires fluorescent probes that are differentially sensitive to the analyte for at least two excitation or emission wavelengths. BCECF is the most widely used ratiometric excitation pH indicator in live cells.


  1. H.-Y. Choi, D.A. Veal, & P. Karuso. (2006). Epicocconone, A New Cell-Permeable Long Stokes’ Shift Fluorescent Stain for Live Cell Imaging and Multiplexing. Journal of Fluorescence, 4(16), 475-482.
  2. Dragan, A. I., Pavlovic, R., McGivney, J. B., Casas-Finet, J. R., Bishop, E. S., Strouse, R. J., ... & Geddes, C. D. (2012). SYBR Green I: fluorescence properties and interaction with DNA. Journal of fluorescence, 22(4), 1189-1199.
  3. Ding, Y., Zhu, W. H., & Xie, Y. (2016). Development of ion chemosensors based on porphyrin analogues. Chemical reviews, 117(4), 2203-2256.
  4. Han, J., & Burgess, K. (2009). Fluorescent indicators for intracellular pH. Chemical reviews, 110(5), 2709-2728.