Low MolecularWeight Fluorophores

One of the major benefits ofHCS is the ability to perform multiplexed assays where different cellular characteristics are simultaneously measured using multicolor fluorophores containing narrow emission spectra.

These fluorophores should have the optical properties of a bright fluorescence signal, combined with a large Stoke's shift and a high molar absorption [84]. Additional practical considerations when selecting fluorophores are their desired insensitivity to fixation procedures in end-point assays, their photostability, resistance against photobleaching [85], and solubility in aqueous buffers. One practical issue is the chemical labeling of the protein with dyes, as conjugation at high molar ratios of the fluorophore with the protein can lead to fluorescence quenching, presumably due to dye-dye interactions [86].

In general, the fluorophores are chemically conjugated to the protein of interest, or the protein is indirectly visualized via a fluorophore tagged antibody. Such fluorescent dyes with emission maxima ranging over a broad range are assembled by the indocyanine dyes Cy- (Amersham Biosciences, Piscataway, NJ, USA) and the Alexa fluor dyes (Molecular Probes, Eugene, OR, USA).

The long-wavelength dyes (> 550 nm excitation wavelength) are especially favored in multicolor assays because they fluoresce at wavelengths longer than the usual sources of cellular autofluorescence, and the background fluorescence of the dyes is generally low [87]. Both dyes are similar with respect to absorption and emission maxima, Stoke's shift, and extinction coefficient. However, the Cy-dyes were significantly less resistant to photobleaching than the Alexa dyes, and formed aggregates when coupled to proteins at high degrees of labeling [88].

A further step with regard to the use ofoptimal fluorophores for cellular imaging was taken with the development of fluorescent semiconductor nanocrystals - so-called Quantum dots - for biological applications. These have a broadband absorption spectrum, which makes them ideal for multicolor detection because only a single excitation source is needed. Quantum dots tend to be brighter than commonly used dyes because of the compounded effects of extinction coefficients, which are a magnitude larger than those of most other dyes. The Quantum dots are also more resistant to photobleaching than other dyes [89].

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