Fluorescent Dye or Marker Characteristics

Fluor350 is an excellent blue fluorescent dye, a modified dye based on the coumarin dye 7-AMCA skeleton. It can serve as an excellent alternative to AMCA, commonly used in fluorescence imaging and flow cytometry analysis, with good water solubility and pH insensitivity. The excitation and emission spectra of Fluor350 are close to those of DAPI, and it can be directly imaged using DAPI filter sets.

Figure 1: Schematic diagram of Fluor350SE chemical structure, showing its molecular composition and bonding method

Figure 2: Fluor350 excitation and emission spectra, showing its characteristics of excitation at 346nm and emission at 445nm

Figure 3: Comparison of Fluor350 and approximate dye spectra (AMCA, DAPI)

Figure 4: Comparison of relative fluorescence intensity between Fluor350 and AMCA, showing the excellent performance of Fluor350 after protein labeling

The excitation/emission maximum of Fluor405 blue fluorescent dye is 400/424nm, which almost perfectly matches the spectral lines of the 405nm violet laser widely used in fluorescence microscopy and flow cytometry. The excitation and emission spectra of Fluor405 highly overlap with those of BV421 dye. Fluor405 is a derivative of Cascade Blue dye, but with optimized fluorescence properties after structural modification. The spectral overlap between Fluor405 dye and green fluorophores is minimal, making it an ideal choice for multicolor applications.

Figure 1: Schematic diagram of Fluor405SE chemical structure, showing its molecular composition and bonding method

Figure 2: Fluor405 excitation and emission spectra

Figure 3: Comparison of Fluor405 and approximate dye spectra (DyLight 405, BV421)

Among active dyes that absorb light in the wavelength range of 400-450nm, significant fluorescence is usually not produced above 500nm. The Fluor430 dye fills this gap in the wavelength range. When excited near its absorption peak at 430nm, this dye exhibits a large Stokes shift and intense yellow-green fluorescence.

Figure 1: Schematic diagram of Fluor430SE chemical structure, showing its molecular composition and bonding method

Figure 2: Fluor430 excitation and emission spectra

Fluor488 is sulfonated rhodamine green. By introducing two sulfonate ions, Fluor488 has significantly enhanced water solubility, pH stability, and photostability. Unlike fluorescein dyes, the fluorescence of Fluor488 remains unchanged over a wide pH range (4-10). Due to its multiple negative charges, Fluor488 is less prone to intermolecular aggregation, and when multiple dye molecules label the same protein, it is less prone to fluorescence self-quenching or causing protein aggregation. Fluor488 is an excellent substitute for fluorescein (FITC or FAM) and CY2, performing extremely well in single-molecule detection of bioconjugates, fluorescence correlation spectroscopy, and fluorescence polarization measurements. It is also widely used in fluorescence microscopy and flow cytometry. Fluor488 is currently one of the best water-soluble small-molecule green fluorescent dyes.

Figure 1: Schematic diagram of Fluor488SE chemical structure, showing its molecular composition and bonding method

Figure 2: Fluor488 excitation and emission spectra

Figure 3: Comparison of Fluor488 and approximate dye spectra (Cy2, FITC)

Figure 4: Comparison of relative fluorescence intensity between goat anti-mouse IgG conjugates prepared with Fluor 488 dye and fluorescein isothiocyanate (FITC). Using the same amount of antibody, the results show that Fluor 488-labeled antibodies have higher fluorescence brightness.

Figure 5: Brightness comparison between Fluor488-labeled rabbit anti-mouse IgG antibodies and Cy2-labeled rabbit anti-mouse IgG antibodies. Human blood cell samples were first blocked with normal rabbit serum, then incubated with anti-CD3 mouse monoclonal antibodies, cells were washed, resuspended, and then incubated with the same concentration of Fluor 488 or Cy2-labeled rabbit anti-mouse IgG secondary antibodies, respectively. After red blood cell lysis, flow cytometry detection showed that Fluor488-labeled secondary antibodies were more effective.

Figure 6: Using Fluor488 and FITC to directly label anti-Mouse CD4 monoclonal antibodies, using the same amount of antibody, flow cytometry detected signal intensity, Fluor488-labeled anti-Mouse CD4 had stronger fluorescence signal intensity.

Fluor555 is an orange-orange-red fluorescent dye, an analog of Cyanine3, commonly used to replace Cyanine3 and TAMRA dyes. Fluor555 dye has high polarity, strong water solubility, excellent fluorescence brightness and fluorescence stability, making it a good dye for protein/antibody labeling. From a chemical structure perspective, the parent nucleus of Fluor555 is cyanine Cyanine3, but the parent nucleus has up to 4 sulfonic acid groups. The tetrasulfonic acid structure makes it extremely hydrophilic, and when the dye is labeled on the protein surface, it does not change the protein surface polarity, reducing the risk of protein hydrophobic aggregation and precipitation, and greatly reducing the probability of fluorescence quenching due to local proximity of the dye. Therefore, proteins labeled with Fluor555 often emit stronger fluorescence. However, because most flow cytometers currently on the market are traditional flow cytometers (not spectral flow cytometers), the laser compatibility is not optimal, so Fluor555 dye is more used in the field of fluorescence imaging.

Figure 2: Fluor555 excitation and emission spectra

Figure 3: Comparison of Fluor555 and approximate dyes (Cy3, TRITC)

Figure 4: By continuously illuminating equimolar concentrations of free Fluor 555 and Cy3, data points were collected every five seconds. Fluorescence intensity has been normalized to the same initial intensity. The results show that Fluor 555 has more excellent resistance to photobleaching.

Figure 5: Comparison of relative fluorescence intensity between Fluor555 and Cy3 two dye-conjugated goat anti-rabbit IgG antibodies at different dye-to-protein binding ratios. It shows that Fluor555 has higher relative fluorescence intensity, and the signal does not decay when the degree of labeling (DOL) is as high as 8 or above.

Fluor568 fluorescent dye perfectly matches 561nm lasers. The maximum excitation/emission of this red-orange fluorescent dye is approximately 578/602nm, which can achieve optimal excitation effect in the 561nm diode laser used in many confocal laser scanning microscopes. Fluor568 has a parent nucleus of rhodamine compounds, and two sulfonate ions are introduced to optimize the water solubility and optical properties of the dye. When excited, it emits bright orange-red fluorescence, which is very sensitive and stable, and can be used to detect low-abundance targets. It is an excellent water-soluble small-molecule fluorescent labeling dye.

Figure 2: Fluor568 excitation and emission spectra

Figure 3: Comparison of Fluor568 and approximate dye spectra (Rhodamine Red)

Fluor594 is a rhodamine compound methylated on the basis of Fluor568 structure. Two sulfonate ions also play a role in optimizing the properties of the dye. When excited, it emits bright red fluorescence, which is very sensitive and stable, and can be used to detect low-abundance targets. Fluor594 is an excellent water-soluble small-molecule fluorescent labeling dye, suitable for labeling various target molecules, and widely used in various biological experiments. Conjugates prepared with Fluor594 dye emit fluorescence in the red region of the spectrum, so they are particularly suitable for multiplex labeling experiments combined with green fluorescent probes. The spectral characteristics of Fluor594 are similar to those of Cyanine3.5 and Texas Red, but the fluorescence intensity is stronger, making it an excellent substitute for Texas Red.

Figure 1: Schematic diagram of Fluor594SE chemical structure, showing its molecular composition and bonding method

Figure 2: Fluor594 excitation and emission spectra

Figure 3: Comparison of Fluor594 and approximate dye spectra (DyLight 594, Texas Red)

Figure 4: Comparison of relative fluorescence intensity between Fluor 594 and Texas Red-X labeled rabbit anti-mouse IgG antibodies at different dye-to-protein ratios. It shows that Fluor 594 has stronger fluorescence intensity.

The spectrum of Fluor647 fluorescent dye almost completely matches that of Cyanine5, so the compatibility with optical filters designed for Cyanine5 can also be optimal. However, the total fluorescence intensity of Fluor647 antibody conjugates is usually significantly higher than that of Cyanine5 conjugates. Moreover, when Fluor647 is conjugated with most proteins, oligonucleotides, and nucleic acids, its absorbance or fluorescence spectrum shows almost no change, so under the same degree of substitution, the total fluorescence intensity is higher, making it an excellent substitute for Cyanine5.

Figure 1: Fluor647 excitation and emission spectra

Figure 2: Comparison of Fluor647 and approximate dye spectra (APC, Cy5)

Figure 3: Comparison of the brightness of Fluor647 and Cy5 fluorescent dye antibody conjugates. It shows that the brightness of Fluor 647 conjugates is much higher than that of conjugates using Cy5.

The peak excitation wavelength of Fluor680 dye is 680nm, and the maximum emission wavelength is 702nm. Its spectral characteristics are similar to Cyanine5.5 dye. The fluorescence of Fluor680 dye is clearly separated from the emission signals of other commonly used red fluorescent dyes (such as tetramethylrhodamine, R-phycoerythrin, Fluor594, and Fluor647), so it is very suitable for multicolor labeling.

Figure 1: Fluor680 excitation and emission spectra

Figure 2: Comparison of fluorescence spectra between Fluor680 and Cy5.5. It shows that their spectra are basically consistent.

Fluor750 is spectrally very similar to Cyanine7 dye, and is an excellent substitute for Cyanine7. The maximum fluorescence emission wavelength is 770nm, which is far from the maximum emission wavelength of commonly used far-red fluorescent substances (Fluor647 or APC), making it convenient for multicolor analysis.

Figure 1: Fluor750 excitation and emission spectra

Figure 2: Comparison of Fluor750 and approximate dye spectra (Cy7)