A Raman instrument was assembled and tested that rejects typically 98–99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm−1 in the blue and 15 cm−1 in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (λexc = 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (λexc = 405 nm). Advantages and disadvantages of this approach for RRS are discussed.
Vol. 2, Iss. 7 Virtual Journal for Biomedical Optics
Evtim V. Efremov, Joost B. Buijs, Cees Gooijer, and Freek Ariese, "Fluorescence Rejection in Resonance Raman Spectroscopy Using a Picosecond-Gated Intensified Charge-Coupled Device Camera," Appl. Spectrosc. 61, 571-578 (2007)