Quenching of Fluorescence with Temperature
In this technical note we show the quenching of fluorescence with temperature using the FS5 Spectrofluorometer.
By using the extended capabilities of FS5-TCSPC we also record, for the same set of temperatures, the fluorescence lifetimes. We can show that temperature dependence of Rhodamine B is exclusively caused by dynamic quenching.
Figure 1: Emission spectra of Rhodamine-B in H2O for temperatures 5°C – 80°C. The parameters of the measurements were λexc=525 nm, Δλexc=Δλem=1 nm, step=1 nm, dwell=1 s, tstab=5 min.
Reliable fluorescence standards and stable fluorescent probes for bio-analytics and chemistry are as important as sensitive indicator fluorophores that utilise the outstanding property of fluorescence of being highly sensitive to the fluorophore’s micro-environment. One of the observable fluorescence parameters, the fluorescence intensity, can be affected by two distinctively different quenching processes: dynamic or static quenching. The fact that a fluorescent molecule can show either dynamic, or static quenching, or a combination of both, limits the chances for this molecule to be used as a standard, where often insensitivity is required over a large range of parameters such like temperature, pH, polarity, concentration, etc.
On the other hand, the property of quenching, in particular that of dynamic quenching, can be used as a sensitive indicator in biological or chemical experiments and assays. The xanthene dye Rhodamine, has been extensively used for both fluorescence standard and probe purposes. However, there is a variety of different forms of Rhodamines, and these variants can be distinctively different in their response to temperature, polarity and pH.
Methods and Materials for the Quenching of Fluorescence
Spectral temperature quenching experiments were run with the FS5 Spectrofluorometer in standard configuration with a 150 W xenon lamp and a single photon counting photomultiplier (PMT) detector (Hamamatsu, R928P). Time resolved fluorescence measurements were made by Time-Correlated Single Photon Counting (TCSPC) with 478 nm excitation provided by a pulsed picosecond diode laser (EPL-485). For these fluorescence decay measurements an additional 515 nm long-pass filter was used to block residual excitation from being detected.
Figure 2: FS5 Spectrofluorometer
A thermoelectrically cooled/heated cuvette holder cassette (SC-25) with integrated controller (TC-125) was used to set and monitor the temperature of the sample. The temperature was monitored for 5 min before each spectral and time-resolved measurement to allow the sample to thermally stabilise. The entire measurement sequence from 5oC to 70oC in intervals of 5oC (with stabilisation times and automated measurement stop conditions) is fully computer controlled by means of the spectrometer’s operating software, Fluoracle®. This applies for both spectral and lifetime measurements. Fluoracle® also contains standard reconvolution fit analysis software. Fluorescence lifetimes T were obtained by fitting to a single exponential model equation I(t)=A+Be(−t/T). Solutions of Rhodamine-B (Sigma-Aldrich, 234141) were prepared in water (Sigma-Aldrich, 95283) in 1 cm quartz cuvettes to an OD525nm=0.2 to keep aggregation effects to a minimum.
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FS5 Spectrofluorometer (For measuring Fluorescence Intensity and the Quenching of Fluorescence)
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