Fluorescent probes

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

1. Fluorescent Probes

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.
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.

2. 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.

3. 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.
Reference:
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.