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一、Fluorescent Protein
1.The structure of proteins encodes fluorescence. These fluorescent proteins include Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), and various derivatives.
（1）The Ancestor of Fluorescent Proteins——Green Fluorescent Protein
Green Fluorescent Protein (GFP). Based on the crystal structure of GFP, site-directed or random mutagenesis is used to identify GFP mutants with enhanced functions. The most widely used ones include various GFP derivatives such as Enhanced Green Fluorescent Protein (EGFP), Emerald, destabilized Enhanced Green Fluorescent Protein (dEGFP), Enhanced Yellow Fluorescent Protein (EYFP), Enhanced Blue Fluorescent Protein (EBFP), and Enhanced Cyan Fluorescent Protein (ECFP), each emitting fluorescence of a distinct color.
（2）Red Fluorescent Protein
Although Green Fluorescent Protein (GFP) has a wide range of applications, its emission spectrum is limited to 440-529 nm. This limitation leads to relatively high background interference during intracellular imaging and prevents it from achieving deeper fluorescent labeling in the subcutaneous tissues of living organisms. In contrast, Red Fluorescent Protein (RFP) has longer excitation and emission wavelengths, resulting in lower background during intracellular imaging. Additionally, RFP can be used for co-labeling alongside GFP.
The first red fluorescent protein (RFP) used in research is DsRed, which has an emission peak at 583 nm. Derived from corals, DsRed exhibits strong fluorescence and good stability. However, it tends to form oligomers and has a slow maturation rate. Mutants of DsRed include mBanana, mOrange, dTomato, mTangerine, mStrawberry, and mCherry. Among these, mStrawberry and mCherry have emission peaks at 596 nm and 610 nm, respectively, with brightness levels approximately 75% and 50% that of Enhanced Green Fluorescent Protein (EGFP).
2.Derived from phycobiliproteins found in algae and plants
These proteins use phycobiliprotein cofactors to absorb light energy, including phycoerythrin (PE), allophycocyanin (APC), and peridinin-chlorophyll (PerCP).
Features:
They have a large Stokes shift (75-200 nm) and a stable emission spectrum. Due to the large size of phycobiliproteins, they typically have a 1:1 protein-to-fluorescent dye ratio during conjugation, making them highly useful for quantitative flow cytometry. However, they are susceptible to photobleaching, so prolonged or repeated exposure to excitation light sources is not recommended.
二、Organic Small Molecules
Such as FITC (molecular weight: 389 Da), Alexa Fluor 488 (an FITC analog, molecular weight: 643 Da), Texas Red (also known as TMRed, molecular weight: 625 Da), Alexa Fluor 647 (molecular weight: 1155 Da), Pacific Blue (molecular weight: 242 Da), and Cy5 (molecular weight: 625 Da).
Features:
They have a consistent emission spectrum and a relatively small Stokes shift (the difference between the excitation wavelength and the emission wavelength, approximately 50-100 nm), exhibit good photostability, can be easily conjugated with antibodies, and have a wide range of applications.
三、Quantum Dots
QDot is a type of semiconductor, whose emission wavelength can be adjusted according to the size of its particles.
Five different quantum dot probe solutions are shown being excited by the same long-wavelength UV lamp; the size of the probes determines their color. Qdot probes are nanoscale (approximately the size of proteins) atomic clusters, consisting of semiconductor materials (cadmium mixed with selenium or arsenic) that contain hundreds to thousands of atoms. This material is coated with an additional semiconductor shell (zinc sulfide) to improve the optical properties of the material. These particles emit fluorescence in a manner completely different from traditional fluorophores, without the involvement of electronic transitions.
Features:
High brightness and good photostability.
QDs are typically excited by violet lasers. Due to the difficulty in resolving these compensation issues and the challenge of conjugating QDs to antibodies, these reagents are mostly replaced by macromolecular dyes in multiparametric protocols.
四、Polymer Dyes
Brilliant Violet(BV)、Brilliant Ultraviolet(BUV)和Brilliant Blue (BB)。
Features:
The dye is composed of polymer chains that collect light signals, and the absorption and emission of light at specific wavelengths can be “tuned” according to the length of the polymer chains and the attached molecular subunits. These dyes are highly stable, have quantum efficiency similar to that of phycobiliproteins, and exhibit significantly improved photostability.
They only absorb light of specific wavelengths, thus avoiding the cross-wavelength excitation issue that exists with Qdots.
五、Heavy Metal Ions
It is suitable for mass cytometry analysis. Instead of being conjugated with fluorochromes, it binds to single-isotope heavy metal ions from the lanthanide series.
Currently, there are 35 types of lanthanide metals commercially available for antibody conjugation.
六、Tandem Dyes
Tandem dyes are a special class of fluorescent molecules that utilize the principle of F?rster resonance energy transfer (referred to as FRET or fluorescence resonance energy transfer). A tandem dye (or fluorophore) consists of two covalently linked fluorescent molecules. One serves as the donor molecule, while the other acts as the acceptor. This results in a unique fluorophore with the excitation properties of the donor molecule and the emission properties of the acceptor.
Features:
Tandem dyes are extremely bright and have large Stokes shift values (150-300 nm), making them highly useful for detecting low-density antigens. However, the stability of tandem dyes is not as good as that of donor fluorochromes, and the efficiency of energy transfer varies between batches, which complicates compensation.
Note: Tandem dyes are highly sensitive and prone to degradation. This leads to a loss of acceptor emission and an enhancement of donor emission, especially if the fluorophores are excited by the same laser. For example, if PE and PE-Cy7 are used and the PE-Cy7 fluorophore begins to degrade, the light it emits will be detected in the PE channel, resulting in erroneous data. One of the main causes of tandem dye degradation (in addition to oxygen free radicals) is exposure to light – this is completely preventable! Therefore, when staining cells, avoid exposure to light or extreme temperature fluctuations. The reason for the latter recommendation is that some tandem dyes may be susceptible to cell-mediated decoupling, so slowing down cellular metabolism at low temperatures can help maintain stability.
七、Nucleic Acid Dyes
The nucleic acid dye DAPI can intercalate into the DNA double helix.
After nucleic acid fluorescent dyes stain cell nuclei, quantitative measurement of the fluorescence intensity emitted by the cells allows determination of the content of DNA and RNA in the nuclei. Additionally, this method enables analysis of the cell cycle (using dyes such as PI, 7-AAD, Dyecycle Violet, and DAPI) and cell proliferation status, sorting of chromosomes (using dyes such as Hoechst 33342 and Chromomycin A3), sorting of stem cells via side population analysis (using dyes such as Hoechst 33342), differentiation between live and dead cells, and sorting of bacteria. There are various fluorescent dyes capable of staining DNA or RNA in cells: commonly used DNA dyes include propidium iodide (PI), DAPI, and Hoechst 33342, while RNA dyes include thiazole orange and acridine orange.
八、Cell Proliferation Dyes
Cells are incubated with BrdU (bromodeoxyuridine) to allow its incorporation into the cellular DNA synthesis process. Then，the cells are stained with a BrdU-specific antibody and a DNA dye, enabling the measurement of cell proliferation. However, this method is not suitable for long-term proliferation studies.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to track multiple divisions of proliferating cells. At an appropriate concentration, it does not affect cell growth or morphology, making it suitable for long-term proliferation studies.
九、Cell Viability Dyes
Cell viability can be determined using exclusion dyes (such as propidium iodide, DAPI). These dyes cannot be fixed and are only suitable for non-infectious cells that require immediate analysis. Cell viability can also be detected through the binding of dyes to intracellular amines; amine-based dyes include Live/Dead (ThermoFisher), Zombie (Biolegend), or Fixable Viability (BD Biosciences). These dyes are suitable for fixed cells, including infectious cells, cells stained for intracellular antigens, and cells fixed with paraformaldehyde.
十、Calcium Indicator Dyes
Calcium indicator dyes undergo a color shift upon binding to calcium, which can be used to indicate cell activation and signal transduction.
The commonly used dye remains Indo-1, which is an ultraviolet ratiometric calcium probe. Additionally, there are blue-green calcium probes such as Fluo-3.
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