Secondary Structure of Bovine Albumin as Studied by Polarization-Sensitive Multiplex CARS Spectroscopy

Abstract

The first application of polarization-sensitive multiplex coherent antiStokes Raman spectroscopy (MCARS) in the absence of resonance enhancement to the resolution of the secondary structure of a protein in solution is reported. Polarization MCARS spectra of bovine albumin in D20 were obtained in the range 1370 to 1730 cm -I with the aid of the background suppression technique. The spectra were fitted simultaneously with a single set of parameters (band positions, bandwidths, amplitudes, and depolarization ratios). Polarized Raman spectra simulated with these parameters revealed a good correspondence with the spontaneous Raman spectra measured. The broad amide I' band was decomposed assuming the three major secondary conformations of protein, of which the contribution of B-sheet structure was found to be negligible. Relative weights of a-helix and random coil conformations agree well with the estimates obtained with Raman and circular dichroism (CD) spectroscopies.

Shot noise limited heterodyne detection of CARS signals

“…The resonant enhancement of the CARS process makes it faster than Raman and compared to IR spectroscopy the short wavelengths involved, ensure a higher spatial resolution. CARS spectroscopy and microscopy have therefore attracted considerable attention [1,2,3,4]. Implementation has been limited due to the complexity of the required systems and the low yield of the highly nonlinear interaction.…”

Ultra-Broadband Time-Resolved Coherent Anti-Stokes Raman Scattering Spectroscopy and Microscopy with Photonic Crystal Fiber Generated Supercontinuum

“…In order to effectively distinguish different molecules, a method for simultaneously obtaining the complete molecular vibrational spectra is required. For this purpose, many methods for extending the simultaneously detectable spectral range of CARS spectroscopy and 171 microscopy are presented [34][35][36][37][38] . With the advent and the progress of supercontinuum (SC) generated by photonic crystal Fibre (PCF) pumping with ultra-short laser pulses [39] , the broadband CARS microscopy based on SC has been developed that provided better feasibilities [40][41][42][43][44] .…”

“…The multiplex molecular vibrational modes of a sample can be resonantly enhanced, the corresponding CARS signals can be detected simultaneously, the energy diagram of M-CARS shown in figure 10. In the previous works, a narrowband and a broadband dye laser was used for the pump/probe and the Stokes beams respectively [36][37][38] . The recent progress in wavelengthtunable ultra-short pulse laser has been giving a powerful momentum to the development of M-CARS.…”

Vibrational phase contrast CARS microscopy

“…Polari- zation CARS [36,37] exploits this difference to remove the non-resonant signal, but unfortunately often also rejects a large part of the resonant signal. An early description of polarization CARS (P-CARS) was given by Ahkmanov in 1977 [38] and is sketched in figure 2.3.…”

“…In multiplex CARS (M-CARS) spectroscopy, spectra are recorded over large parts of the vibrational spectrum [36,42,43]. In the fingerprint region the spectra are highly congested.…”