A broad and tunable 250- to 430-nm source for microscopy and lifetime measurements by frequency doubling of a 78-MHz-picosecond white-light laser

Bradler, Maximilian; Nielsen, Frederik Donbæk; Eckert, Carl Elias; Riedle, Eberhard

doi:10.1007/s00340-014-5774-5

Cited by 4 publications

(1 citation statement)

References 21 publications

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“…Owen et al [16], demonstrated the use of a UV extended SCL for FLIM, however, this source only generated light at wavelengths >350 nm and thus was incapable of exciting Trp or Tyr. More recently in 2014, frequency doubling of SCL visible output to generate a tuneable UV output between 250 and 430 nm source was demonstrated for microscopy [17]. In this example, a 78 MHz-picosecond pulsed white-light laser was used and output powers in the 1-70 μW range were generated.…”

Section: Introductionmentioning

confidence: 99%

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

Melnikau

Elcoroaristizabal

Ryder

2018

Methods Appl. Fluoresc.

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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.

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Section: Introductionmentioning

confidence: 99%