Alexa Fluor is a series of fluorescent dyes developed by American Molecular Probes, which can be used in the labeling and localization of tissues, cells and biomolecules in biomedical research. Its excitation and emission spectra cover most of the visible and part of the infrared spectral region and are suitable for most fluorescence microscopes.
The Alexa series of dyes are named after Alex Haugland, son of Dick Haugland, one of the founders of Molecular Probes. Another fluorescent dye product of the company, Marina Blue, is named after its daughter Marina.
Each dye in this series is named mainly according to the maximum wavelength (nm) of its excitation light.
Alexa fluor dyes introduce sulfonate groups into coumarin, xanthene (such as fluorescein and rhodamine), cyanine and other dyes through chemical synthesis, making Alexa dyes negatively charged and hydrophilic, making Alexa have Better optical stability, luminous intensity and pH adaptability. American Life Technologies has related patents after acquiring Molecular Probes.
A third-party organization has compared DNA staining with Alexa Fluor 647 and Cy5 (which have similar wavelengths) and found that Cy5 is brighter but less optically stable than Alexa 647. Another study found that the dyeing brightness of Alexa dyes had better photostability (eg comparison of Alexa Fluor 488 with Dylight 488 and Fluo probes 488.
AlexaFluor series dye features:
Alexa Fluor 350 – brilliant blue and UV excitation
The blue fluorescence of Alexa Fluor 350 is 50% brighter than that of AMCA, and its maximum emission wavelength is slightly shorter (442 nm vs. 448 nm for AMCA), making it easier to distinguish from existing green fluorophores.
Alexa Fluor 405 – a near-perfect match for blue diode lasers
The excitation/emission maximum wavelength of Alexa Fluor 405 is 402/421nm, which is a near-perfect match with blue diode lasers with the 405nm spectral line for fluorescence microscopy and flow cytometry. Alexa Fluor 405 succinyl ester is an amino-reactive derivative of Cascade Blue, which is an acetyl azide. Alexa Fluor 405 succinyl ester is more pure and has a 4-piperidine carboxylic acid spacer to minimize reactivity between the fluorophore and its conjugated biomolecule.
Alexa Fluor 430 – 430nm absorption with high Stokes shift
A few dyes that absorb at 400nm-450nm have measurable fluorescence deviating from 500nm in aqueous solution. Alexa Fluor 430 makes up for this deficiency, emitting around 540nm when excited at 430nm.
Alexa Fluor 488 – the best green fluorophore
Alexa Fluor 488 conjugated proteins outperform other fluoresceins and have been shown experimentally to be better than other green fluorophores including Cy2. Not only the brightness is higher, but also the light stability is better. Stability in the pH range of 4-10 is another advantage.
Alexa Fluor 532 – the best dye for 532nm excitation light source
The excitation and emission wavelengths are between the green fluorescent Alexa Fluor 488 and the orange fluorescent Alexa Fluor 546, and are suitable for 532 nm excitation light sources including dual-frequency Nd:YAG lasers.
Alexa Fluor 546 – an alternative to Cy3 and tetramethylrhodamine
Alexa Fluor 546-labeled conjugates work well in the orange range, are more fluorescent than tetramethylrhodamine (TRITC, TAMRA) and Cy3, are easily excited by mercury arc lamps, and emit at 546 nm.
Alexa Fluor 555 – an alternative to Cy3
Alexa Fluor 555 matches the spectrum of Cy3 conjugates and is therefore suitable for filters for Cy3. Alexa Fluor 555 has higher total fluorescence and better photostability, providing researchers with longer image capture times.
Alexa Fluor 568 – Best for 568 nm excitation light source
The orange-red fluorescence of Alexa Fluor 568 is best suited for the Ar–Kr mixed gas laser for confocal microscopy, which emits a spectral line at 568 nm. The conjugate of Alexa Fluor 568 is brighter than Lissamine Rhodamine B and Rhodamine Red-X, which have similar excitation/emission maxima.
Alexa Fluor 594 – a better alternative to Texas Red
Alexa Fluor 594 conjugates emit red light and can be used with green fluorescent probes for multicolor labeling experiments. Higher brightness than Texas Red conjugate.
Alexa Fluor 610 – the best red dye for Alexa Fluor
Alexa Fluor 610 is bright, photostable, and can be detected with optics that detect Texas Red and Alexa Fluor 594. The predecessor is the acceptor dye of FRET-based Alexa Fluor–phycobiliprotein. The new Alexa Fluor 610 excitation/emission wavelength of 612/628nm is easily distinguishable from green fluorophores and is suitable for multicolor labeling. And Alexa Fluor 610 is still visible to the human eye, outperforming Alexa Fluor 633 and other longer wavelength fluorophores.
Alexa Fluor 633 – more photostable
Far-red dyes are popular because their spectral range lies outside the autofluorescence of most samples. The widespread use of the 633nm spectral line of He–Ne lasers and the 635nm spectral lines of red diode lasers has accelerated the development of compatible dyes. Alexa Fluor 633 conjugates are bright and photostable with absorption peaks at 632nm and emission peaks at 650nm.
Alexa Fluor 635 – the best dye for the 633nm line of H-Ne lasers
The new Alexa Fluor 635 dye, its conjugate is brighter than Alexa Fluor 633 because its absorption spectrum does not split into two peaks like Alexa Fluor 633, Cy5 and tetramethylrhodamine. Alexa Fluor 635 is also suitable for the 633 nm spectral line of the He-Ne laser and the 635 nm spectral line of the red diode laser, with an absorption peak at 633 nm and an emission peak at 647 nm.
Alexa Fluor 647 – an alternative to Cy5
Alexa Fluor 647 is spectrally and filter matched to Cy5 conjugates. The overall fluorescence brightness of Alexa Fluor 647 secondary antibody conjugates is higher than that of Cy5 conjugates from other companies. When combined with most proteins, oligonucleotides, and nucleic acids, Alexa Fluor 647 exhibits less fluctuation in the fluorescence absorption spectrum than Cy5, and therefore fluoresces more strongly with the same degree of substitution.
Alexa Fluor 680 – Alternative to Cy5.5
The excitation peak of Alexa Fluor 680 is at 679nm, and the maximum emission is 702nm, which is similar to Cy5.5. Alexa Fluor 680 is well differentiated from other red fluorophores, such as Texas Red, R-phycoerythrin, Alexa Fluor 594 and Alexa Fluor 647, and is an ideal dye for tri- and quad-color labeling.
Alexa Fluor 700 – the optimal dye for far-red diode lasers
Alexa Fluor 700 has an absorption maximum at 696nm and can be excited by xenon arc lamps, far-infrared diode lasers or dye-pumped lasers in the 675-700nm range. The emitted light is in the infrared region with a peak at 719 nm.
Alexa Fluor 750 – Alexa Fluor’s longest wavelength product
Alexa Fluor 750 is the longest wavelength in the current Alexa Fluor family and has a similar spectrum to Cy7. The maximum wavelength of its emission is 779nm, which can be distinguished from Alexa Fluor 647 and allophycocyanin (APC), and is suitable for multicolor analysis. The 752nm excitation maximum of the Alexa Fluor 700 conjugate makes it suitable for use with xenon arc lamps or dye-pumped lasers in the 720–750nm range.