IN RECENT times, fluorescence imaging has evolved as one of the most prevailing modus operandi for observing biomolecules in living systems. The essential molecular tools used here are fluorescent sensors in bio-imaging. A large number of fluorogenic molecules have been developed for this purpose and a host of chemistry has been developed as the fluorogenic systems are also needed for a few other cutting edge technological applications like labels, logic gates, light-emitting materials, and light-harvesting systems
Though several organic molecules with intrinsic fluorescence are available in the literature for use in such areas the properties required of these molecules in bio-imaging are:
a. high emission intensities
b. reasonable photo-stability
c. appreciable good fluorescence quantum yield
d. large Stokes shift
e. absorption in the red and NIR region
f. emission extending in red to NIR region
g. aqueous compatibility/solubility
Inherent high emission intensities are desirable because the intensity is usually impaired in a biological environment. Photo-stability ensures harmful photo-degradation products are not formed during the study. Intrinsically higher quantum yield is preferred because on bio-conjugation the quantum yield is usually lowered. Large Stokes shift is necessary to have a thorough clarity between the light used for excitation and the fluorescence output signal. Absorption extending in the red and NIR region helps in two aspects – non-interference from auto-fluorescence of biomolecules and better penetration enabling deep imaging. Emission extending in the red and NIR region makes the optics simpler. Aqueous solubility/compatibility is crucial because the studies are mainly under aqueous environment. It may be quite challenging to have all the requirements satisfied in a single fluorophore. For instance, many bright organic dyes including rhodamine, fluorescein, boron BODIPY, and cyanine derivatives have the serious disadvantage of very small Stokes shifts (typically less than 25 nm), which can lead to serious self-quenching and fluorescence detection errors because of excitation backscattering effects. Mostly small fluorophores of synthetic origin are preferred are preferred. Main advantages of synthetic fluorophores over other fluorophore types is the ability to employ chemistry to dictate the properties and position of a fluorescent dye in a biological experiment.
Synthetic organic chemistry has played important role in generating several new fluorescent probe molecules for diverse biological applications.
Selective activation of fluorophores by enzymatic catalysis or light enables advanced imaging experimentations where a distinctive subset of fluorophores can be seen against a larger inactivated fluorophore population. There are also approaches that can be applied in a cellular environment allowing the specific labelling of defined molecules and regions inside living cells. Some of the commonly occurring fluorophoric units are shown in the Chart.
Most of the dyes have both the absorption and emission in the visible range (400−650 nm). Fluorescent molecules with absorption and emission in the near-infrared (NIR) region (650−900 nm) are relatively few, although they are very much desired due to their useful applications in chemical biology. As against visible light, NIR light offer advantageous to be employed in biological imaging on account of minimum photodamage to biological samples, deep tissue penetration, and minimum interference from background autofluorescence by biomolecules in the living systems.
Rhodamine dyes occupy a special position as fluorescent and fluorogenic molecules owing to high brightness, excellent photostability, and the facility to modify the properties of the dye by having different functional groups. Another interesting property of these dyes is the equilibrium existing between an open, coloured, fluorescent quinoid form and a closed, colourless, non-fluorescent lactone, which can be tuned by appropriate substitution or by placing in a proper environment. This arises from the ability of the carboxyl group to undergo intramolecular cyclisation reaction. The open form is highly emissive, while the spirocyclic form is fundamentally non-emissive. At physiological pH, the fluorescent open form is predominated. In other words, the fluorescence of rhodamine dyes is on at physiological pH. On the contrary, the fluorescence of rhodamine amide derivatives is off at physiological pH, as the non-fluorescent spirolactam form is predominant. Some of the traditional Rhodamines are: Rhodamine 6G, Rhodamine B, and Rhodamine 101. Several Rhodamine-Styryl, Rhodamine-Cyanine, Rhodamine-Coumarin, and similar hybrids with superior properties like NIR absorption and emission are available, and some of them are commercialised also. Carborhodmines and silicon containing Rhodamines are also widely investigated.
Among the fluorophores the cyanine dyes are one type that are widely employed as NIR fluorophores. Since cyanine dyes have relatively high-lying occupied molecular orbital (HOMO) energy levels there are some restrictions. However, several cyanine fluorophores have been developed for use in imaging.
Several fundamental questions in biology are increasingly answered using fluorescence which has opened an extensive research methodology to access synthetically tailor-made fluorophores of desired properties. Most expensive ab initio computational studies have also taken a front seat in the development programs. Still we need a host fluorescent dyes with superior properties.
Prof. N. Sekar
Dyestuff Technology Department, Institute of Chemical Technology, Matunga, Mumbai - 400 019.