Peer-Reviewed Journal Details
Mandatory Fields
Melnikau, D,Elcoroaristizabal, S,Ryder, AG
2018
October
Methods And Applications In Fluorescence
An excitation emission fluorescence lifetime spectrometer using a frequency doubled supercontinuum laser source
Published
WOS: 2 ()
Optional Fields
fluorescence supercontinuum lifetime excitation emission matrix frequency doubled CELL-CULTURE MEDIA TIME-RESOLVED FLUORESCENCE HETEROGENEOUS FLUORESCENCE QUANTITATIVE-ANALYSIS TRYPTOPHAN PROTEINS RESOLUTION SPECTROSCOPY PHASE DECAY
6
4
045007
The accurate fluorescence analysis of complex, multi-fluorophore containing proteins requires the use of multi-dimensional measurement techniques. For the measurement of intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) one needs tuneable UV excitation and for steady-state measurements like Excitation Emission Matrix (EEM) simple pulsed Xe lamps are commonly used. Unfortunately, simultaneous multi-dimensional wavelength and time resolved measurement of intrinsic protein fluorescence in the 260 to 400 nm spectral range are challenging and typically required the use of very complex tuneable laser systems or multiple single excitation wavelength sources. Here we have assembled and validated a novel Excitation Emission Fluorescence Lifetime Spectrometer (EEFLS) using a pulsed, frequency doubled, Super-Continuum Laser (SCL) source coupled with a 16 channel multi-anode Time Correlated Single Photon Counting (TCSPC) measurement system. This EEFLS enabled the collection of near complete lifetime and intensity maps over the most important intrinsic protein fluorescence spectral range (lambda(ex) = 260-350/lambda(em) = 300-500 nm). The 4-dimensional (lambda(ex)/lambda(em)/I-(t)/tau) Excitation Emission Fluorescence Lifetime Matrix (EEFLM) data produced can be used to better characterize the complex intrinsic emission from proteins. The system was capable of measuring fluorescence emission data with high spectral (1-2 nm) resolution and had an Instrument Response Function (IRF) of similar to 650 ps for accurate measurement of nanosecond lifetimes. UV power output was stable after a warm up period, with variations of <2% over 9 hours and reproducible (relative standard deviation RSD < 1.5%). This enabled the collection of accurate EEFLM data at low resolution (similar to 12 nm in excitation and emission) in 1-2 hours or high resolution (4 nm) in similar to 17 hours. EEFLS performance in the UV was compared with a conventional commercial TCSPC system using pulsed LED excitation and validated using solutions of p-terphenyl and tryptophan.
10.1088/2050-6120/aad9ae
Grant Details
This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grand number (14/IA/2282, Advanced Analytics for Biological Therapeutic Manufacture, to AGR).
Publication Themes