An excitation emission fluorescence lifetime spectrometer using a frequency doubled supercontinuum laser source

Abstract

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 (λ = 260-350/λ = 300-500 nm). The 4-dimensional (λ /λ/I/τ) 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 ∼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 (∼12 nm in excitation and emission) in 1-2 hours or high resolution (4 nm) in ∼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.

Stokes shift microscopy by excitation and emission imaging

“…While measuring spatially resolved emission spectra has become relativity standard in many laboratories, measuring excitation spectra is more challenging since it requires a tunable excitation source or an interferometer in the excitation path [22,23]. Examples of combining emission and excitation spectroscopy simultaneously are even scarcer [24][25][26]. Here we demonstrate that emission-side interferometry, in combination with a tunable excitation light source, offers an opportunity to achieve spatially resolved excitation and emission information in wide field or TIRF configuration [6].…”

Multidimensional fluorescence spectroscopy of wine using synchronous excitation/emission matrices and time-resolved fluorescence interferometric detection

“…Alternatively, multi-channel TCSPC detectors are now available (e.g. the 16 channel detector from Becker & Hickl, PML-16-C) [28,29] permitting simultaneous acquisition of decay profiles at multiple emission wavelengths, potentially shortening the total acquisition time in some cases. Single photon avalanche photodiode (SPAD) arrays are also becoming available which provide a higher-density of TCSPC detectors (ie.…”

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

“…The fluorescence lifetimes were recorded with a spectrometer assembled to measure intrinsic protein fluorescence in the 260 to 400 nm range (for a precise description, see [20]). Briefly, the excitation pulses were generated by a high-power super-continuum laser (SMHP-60.4 from Leukos, Limoges, France), coupled to a frequency doubling unit (BOX-UVgen2, Leukos, Limoges, France).…”

“…More advanced methods such as ARMES (Anisotropy Resolved Multi-Dimensional Emission Spectroscopy) or EEFL (Excitation Emission Fluorescence Lifetime) can be useful in fluorescence analysis of complex, multi-fluorophore proteins, as they span the complete emission spectra [17][18][19][20]. These techniques are particularly useful in the case of tryptophan (Trp), which exhibits strong dependence of the emission properties on its environment; however, interpretation of its fluorescence spectrum and lifetime is not straightforward [21][22][23].…”