Fluorescence Microscopy/Imaging Flow Cytometry Organic Dyes Quantum Dots Fluorogens
There are a variety of organic dyes availble for fluorescence microscopy, but MBIC is particularly famous for it's development and subsequent commercialization of the CyDyes. These polymethine dyes have been accepted as the next generation of fluorescent probes for imaging, microscopy and flow cytometry. The cyanines are readily synthesized and are easily adaptable for use as labels and physiological indicators. These dyes possess high absorbencies, high fluorescence quantum yields, and low triplet-state yields.
All the cyanine dyes are variations on a single theme. The basic structure of cyanine dyes is shown in Figure 1. Heterocyclic groups at either end are bridged by a polymethine chain of (2m +1) CH groups, forming a conjugated unsaturated system containing a delocalized positive charge. When m=1, the dye is classified as a Cy3; when m=2, the dye is classified as a Cy5, and so on. A feature of the cyanine dyes is their exceptionally high extinction coefficients. Certain cyanines can exhibit high quantum yields, producing very bright signals. This brightness also requires minimal self-association (aggregation) of the water-soluble cyanine dyes.
The spectral properties of cyanine dyes (as illustrated in Figure 2) show little influence by changes in their environment, such as solvent polarity or pH. While fluorescein fluorescence drops significantly as the pH decreases from 8 to 4, Cy3 fluorescence is quite constant in the pH range of 2 to 11. Absorption and emission maxima of the cyanines shift less than 10 nm between ethanol and water.
