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Most quantitative fluorescence measurements report emission quantum yields by referring the integrated fluorescence profile to that of a well-known standard compound measured under carefully controlled conditions. This simple protocol works well provided an appropriate standard fluorophore is available and that the experimental conditions used for reference and unknown are closely comparable. Commercial fluorescence spectrophotometers tend to perform very well at wavelengths between 250 and 650 nm but are less responsive at longer wavelengths. There are no recognized emission standards for the far-red region. We now report fluorescence quantum yields for a series of commercially available cyanine dyes in methanol solution at room temperature. The compounds are selected to span the wavelength region from 600 to 850 nm, with absolute emission quantum yields being determined by thermal blooming spectrometry. Calibration of the instrument is made by reference to aluminium(iii) phthalocyanine tetrasulfonate and aza-BODIPY in methanol. [ABSTRACT FROM AUTHOR]

pH-dependent protein adsorption on mesoporous silica nanoparticle (MSN) was examined as a unique means for pH monitoring. Assuming that the degree of protein adsorption determines the distance separating protein molecules, we examined the feasibility of nanoscale pH probes based on fluorescence resonance energy transfer (FRET) between two fluorescent proteins (mTurquoise2 and mNeonGreen, as donor and acceptor, respectively). Since protein adsorption on MSN is pH-sensitive, both fluorescent proteins were modified to make their isoelectric points (pIs) identical, thus achieving comparable adsorption between the proteins and enhancing FRET signals. The adsorption behaviors of such modified fluorescent proteins were examined along with ratiometric FRET signal generation. Results demonstrated that the pH probes could be manipulated to show feasible sensitivity and selectivity for pH changes in hosting solutions, with a good linearity observed in the pH range of 5.5-8.0. In a demonstration test, the pH probes were successfully applied to monitor progress of enzymatic reactions. Such an 'in situ-assembling' pH sensor demonstrates a promising strategy in developing nanoscale fluorescent protein probes. [ABSTRACT FROM AUTHOR]