Multiplex CARS

Toleutaev B N, Tahara T and Hamaguchi H 1994 Broadband (1000 cm multiplex CARS spectroscopy application to polarization sensitive and time-resolved measurements Appl. Phys. 59 369-75... 

The alternative approach is based on a non-iterative procedure using the maximum entropy model (MEM) to extract the complex dielectric susceptibility from the intensity measurements. This technique was first proposed 15 years ago (Vartiainen 1992), and recently was used for multiplexed CARS measurements (Petrov et al. 2007, Vartiainen et al. 2006). 

Beyond imaging, CARS microscopy offers the possibility for spatially resolved vibrational spectroscopy [16], providing a wealth of chemical and physical structure information of molecular specimens inside a sub-femtoliter probe volume. As such, multiplex CARS microspectroscopy allows the chemical identification of molecules on the basis of their characteristic Raman spectra and the extraction of their physical properties, e.g., their thermodynamic state. In the time domain, time-resolved CARS microscopy allows recording of ultrafast Raman free induction decays (RFIDs). CARS correlation spectroscopy can probe three-dimensional diffusion dynamics with chemical selectivity. We next discuss the basic principles and exemplifying applications of the different CARS microspectroscopies. 

The principal concept of multiplex CARS spectroscopy, which was first demonstrated by Akhmanov et al. [48], is illustrated in Fig. 6.8A. In multiplex CARS,... 

Fig. 6.8. A Principle of frequency-multiplexed CARS microspectroscopy A narrow-bandwidth pump pulse determines the inherent spectral resolution, while a broad-bandwidth Stokes pulse allows simultaneous detection over a wide range of Raman shifts. The multiplex CARS spectra shown originate from a 70 mM solution of cholesterol in CCI4 (solid line) and the nonresonant background of coverglass (dashed line) at a Raman shift centered at 2900 cm-1. B Energy level diagram for a multiplex CARS process. C Schematic of the multiplex CARS microscope (P polarizer HWP/QWP half/quarter-wave plate BC dichroic beam combiner Obj objective lens F filter A analyzer FM flip mirror L lens D detector S sample). D Measured normalized CARS spectrum of the cholesterol solution. E Maximum entropy method (MEM) phase spectrum (solid line) retrieved from (D) and the error background phase (dashed line) determined by a polynomial fit to those spectral regions without vibrational resonances. F Retrieved Raman response (solid line) calculated from the spectra shown in (E), directly reproducing the independently measured spontaneous Raman response (dashed line) of the same cholesterol sample...

Multiplex CARS microspectroscopy, in conjunction with appropriate spectral analysis tools, was successfully applied to the study of phospholipid bilayer model systems [120, 121, 142, 70, 143], lipids within cells [144, 127, 145-147, 141], a single pollen grain [148], a single bacterial endophore [140], a molecular J-aggregate microcrystal [149], silicon components on a wafer [130], separated phases in polymer blends [123, 135], and concentration profiles in a microreactor [150]. 

Fig. 6.9. A Spontaneous Raman spectrum of d62-DPPC lipids and its decomposition into Lorentzian line profiles. B Normalized multiplex CARS spectra (dots) of a planar-supported bilayer and monolayer formed by d62-DPPC on a glass-water interface for parallel-polarized input beams, together with the fit using the center frequency and line width parameters extracted from the decomposition analysis in (A) (solid line). The spectrum exposure time was 0.64 s. Error bars indicate the shot-noise standard deviation (Copyright American Chemical Society [70])...

Fig. 6.10. In vivo multiplex CARS microspectroscopy of a NIH 3T3-L1 fibroblast cell in the high-wavenumber region where C-H stretch vibrations reside. A CARS image revealing the intracellular distribution of constituents with high densities of lipids, such as the membrane envelope of the nucleus and intracellular lipid droplet (LD) organelles. Typical MEM-reconstructed Raman spectra taken for (B) a single LD organelle that is indicated by the arrow in A, (C) the nucleus, and (D) the cytoplasm. The spectrum exposure time was 0.3 s...

While in the frequency domain all the spectroscopic information regarding vibrational frequencies and relaxation processes is obtained from the positions and widths of the Raman resonances, in the time domain this information is obtained from coherent oscillations and the decay of the time-dependent CARS signal, respectively. In principle, time- and frequency-domain experiments are related to each other by Fourier transform and carry the same information. However, in contrast to the driven motion of molecular vibrations in frequency-multiplexed CARS detection, time-resolved CARS allows recording the Raman free induction decay (RFID) with the decay time T2, i.e., the free evolution of the molecular system is observed. While the non-resonant contribution dephases instantaneously, the resonant contribution of RFID decays within hundreds of femtoseconds in the condensed phase. Time-resolved CARS with femtosecond excitation, therefore, allows the separation of nonresonant and vibrationally resonant signals [151]. 

Analogous to the principal concept of multiplex CARS microspectroscopy (cf. Sect. 6.3.5), in multiplex SRS detection a pair of a broad-bandwidth pulse, eg., white-light femtosecond pulse, and a narrow-bandwidth picosecond pulse that determine the spectral width of the SRS spectrum and its inherent spectral resolution, respectively, is used to simultaneously excite multiple Raman resonances in the sample. Due to SRS, modulations appear in the spectrum of the transmitted broad-bandwidth pulse, which are read out using a photodiode array detector. Unlike SRS imaging, it is difficult to integrate phase-sensitive lock-in detection with a multiplex detector in order to directly retrieve the Raman spectrum from these modulations. Instead, two consecutive spectra, i.e., one with the narrow-bandwidth picosecond beam present and one with that beam blocked, are recorded. Their ratio allows the computation of the linear Raman spectrum that can readily be interpreted in a quantitative manner [49]. Unlike the spectral analysis of a multiplex CARS spectrum, no retrieval of hidden phase information is required to obtain the spontaneous Raman response in multiplex SRS microspectroscopy. 

Measurement accuracy. Our r.m.s. shot to shot fluctuations are 5%, thanks to the use of reference. This accuracy does not depend on the mode of operation (spatial resolution and choice of field polarizations). It is not as good, however, in multiplex CARS, or in dilute samples, or in the vicinity of narrow lines, because other sources of noise such as photoelectron statistics and laser frequency instabilities (however small) then play a major role. 

It should be noted that the multiplex CARS signal obtained from a single laser shot is comparatively small. However, averaging over many shots gives CARS spectra of similar quality as scanning CARS spectra if the lasers have comparable output powers. 

Furusawa K, Hayazawa N, Kawata S (2010) Two-beam multiplexed CARS based on a broadband oscillator. J Raman Spectrosc 41 840... 

In the above analysis, the pump frequency (cOp) and the Stokes frequency (coj have been assumed to be ideally monochromatic, which is applicable when each vibration-rotation line is scanned. In a broadband CARS system as described here the Stokes (dye) laser has a broad spectral profile so that the multiplexed CARS spectral profile of the probed species, is generated with each laser pulse. 

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M. Pealat, P. Bouchardy, M. Lefebvre and J. P. Taran, Precision of Multiplex CARS Temperature Measurements, Appl. Opt. 24 1012 (1985). 

Wurpel, G.W.H., Schins, J.M., Muller, M. Direct measurement of chain order in single phosopholipid mono- and bilayers with multiplex CARS. J. Phys. Chem. B 108, 3400-3403... 

Wurpel, G.W.H., Rinia, H.A., Muller, M. Imaging orientational order and lipid density in multilamellar vesicles with multiplex CARS microscopy. J. Microscopy (Oxford) 218,37-45... 

H. Rinia, K. N. Burger, M. Bonn and M. Muller, Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy, Biophys. J., 2008, 95, 4908-4914. 

Bonn, M., Muller, M, Rinia, H.A., and Burger, K.N.J. (2009) Imaging of chemical and physical state of individual cellular Upid droplets using multiplex CARS microscopy. /. Raman Spectrosc., 40, 763-769. 

A., and Motzkus, M. (2011) Chemose-lective imaging of mouse brain tissue via multiplex CARS microscopy. Biomed. Opt Express, 2, 2110—2116. doi 10.1364/BOE.2.002110 [doi] 147302... 

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