Coherent Raman Spectroscopy

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Email Address. Sign In. Access provided by: anon Sign Out. Coherent Raman gain spectroscopy using CW laser sources Abstract: The recent demonstration of stimulated Raman gain loss spectroscopy SRS using CW laser sources introduces a powerful tool to coherent Raman spectroscopy. In this paper we undertake an experimental and analytical evaluation of several variations of SRS using CW laser sources including 1 direct stimulated Raman gain loss measurements, 2 optically heterodyned polarization interferometry, and 3 two-beam nonlinear interferometry.

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Home Research Explore. Menu Explore Research topics Find a research unit Academic publications Research Institutes Research facilities Innovation and impact Research impact Honours and accolades Strategic alliances Quality and performance Our research environment Supporting researchers Working collaboratively Integrity and ethics Funding and awards News and features. Coherent Antistokes Raman Scattering CARS micro-spectroscopy: Technology development and life-science applications Optical microscopy is an indispensable tool that is driving progress in cell biology. Figure 1.

Learn more. Coherent Raman scattering CRS techniques, by coherently exciting vibrational oscillators in the focal volume, increase signal levels by several orders of magnitude under appropriate conditions. Different technical solutions for broadband CARS and SRS, working both in the frequency and in the time domains, are compared and their merits and drawbacks assessed.

Fluorescence microscopy using exogenous such as dyes or semiconductor quantum dots 2 or endogenous such as fluorescent proteins 3 markers offers a superb sensitivity, down to the single molecule limit. However, the addition of fluorescent markers can hardly be implemented within certain cells or tissues, and in many cases, it gives a strong perturbation to the investigated system. This is particularly true for small molecules, for which the size of the marker is comparable to or even bigger than that of the molecule itself, so that it heavily interferes with its biological function. Every component of a biological specimen cell or tissue is characterized by a vibrational spectrum that reflects its molecular structure and provides an endogenous and chemically specific signature that can be exploited for its identification.

SR is able to identify selectively many types of biomolecules found in human tissues and cells. In a typical Raman spectrum there are two vibrational frequency intervals of interest, where specific molecular signatures are clearly identified. Here the Raman spectrum contains multiple contributions from proteins and nucleic acids. The main drawback of SR microscopy is its very low scattering cross section, about 10—12 orders of magnitude lower than the absorption cross section of molecules, resulting in a weak incoherent signal which is emitted isotropically from the irradiated volume.

As a result, many practical difficulties arise.

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This vibrational coherence enhances the Raman response by many orders of magnitude with respect to the incoherent SR process. CRS microscopy thus provides the following advantages. In comparison with fluorescence microscopy it is label free, because it does not require fluorophores or staining, allowing the study of unaltered cells and tissues;. It typically works out of resonance, that is, without population transfer into electronic excited states of the molecule, thus minimizing photobleaching and damage to biological samples;. Since CRS exploits a coherent superposition of the vibrational responses from the excited oscillators, it provides a considerably stronger signal than SR microscopy, allowing for much higher imaging speeds;.

As will be discussed in detail in the following, CARS and SRS present advantages and drawbacks, so they are both actively investigated in view of applications. SR, on the other hand, provides the full vibrational spectrum and thus contains the maximum amount of chemical information, allowing subtle differentiation between species; however, it suffers from exceedingly long integration times.

In the CARS regime Figure 4a , the discrimination between the levels necessarily requires that the vibrational coherence is read out by a narrowband beam, which in the simplest case consists of the pump beam itself. The latter configuration is also known as inverse Raman scattering IRS. Note that, while can be approximated to be real and frequency independent, is the superposition of several complex Lorentzian responses related to the different vibrational transitions of the molecules 3 where is the concentration of Raman active scatterers, is the cross section, the resonance frequency, and the linewidth of the vibrational transition.

Let us first consider the CARS process. In a microscopy configuration, due to the short interaction length, one can neglect the phase mismatch between the interacting fields. Since NRB may derive from any molecule in the focal volume, its contribution easily becomes relevant for biological samples. On the other hand, for the case of broadband detection, the CARS signal in the limit becomes 6.

This expression shows that the resonant signal is multiplied by the NRB, which is phase coherent with the CARS signal and may then act as a local oscillator LO providing its heterodyne amplification.

Broadband Coherent Raman Scattering Microscopy

In this regime, the retrieved signal scales linearly with and thus with the concentration of the probed species. On the other hand, in the regime when the NRB is negligible , as, for example. As a drawback, the CARS signal here scales quadratically with the concentration of oscillators N , thus complicating a quantitative determination of the species concentration and preventing the measurement of highly diluted species.

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It is worth observing that in a broadband configuration where the Stokes pulses typically extend over hundreds of wavenumbers with a given spectral shape, the CARS and SRS signals must be properly normalized against the Stokes power density for a quantitative evaluation. In SRS, on the other hand, we have and the process is intrinsically phase matched.

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Such an advantage does not automatically translate into a better SNR for a detailed discussion see, e. The SRG signal can then be calculated as On the other hand, it suffers from the NRB, generated both by the molecular species under study and by the surrounding medium, which does not carry any chemically specific information and, when the concentrations of the target molecules are low, can distort and even overwhelm the resonant signal of interest. In addition, even in the case where the NRB is negligible, due to the homodyne detection the CARS signal scales as the square of the number of oscillators in the focal volume, so that its sensitivity rapidly degrades with decreasing concentration, making it difficult to detect the less abundant biomolecules.

Furthermore, SRS scales linearly with the concentration N , thus allowing the quantitative detection of weakly concentrated species. For these reasons, both techniques will be treated on equal footing in the remainder of this Review. Most of the early demonstrations of CARS microscopy employed two independent electronically synchronized picosecond Ti:sapphire oscillators, resulting in a very bulky and complex system. However, an extra requisite on the laser sources of the SRS technique is the low intensity noise at high frequencies, which is required to detect small differential signals.

Pump and Stokes pulses are synchronized by a delay line, collinearly combined by a dichroic mirror DM , focused on the sample by a microscope objective after going through a scanning unit, and the transmitted light is collimated by a similar objective. In principle, given the similarity of the configurations, CARS and SRS signals can be detected on the same experimental setup or even simultaneously.

Many efforts have been devoted to the suppression of NRB, which however significantly increase the experimental complexity. Another configuration that allow the suppression of NRB is the dual CARS, 69 in which a double OPO system and a laser are used to simultaneously probe two different vibrational resonances.

By properly mixing the two signals it is possible to retrieve only the resonant contribution of the nonlinear susceptivity. Excess laser intensity noise can be cancelled using balanced detection schemes, 52 which split off a fraction of the probe beam before the sample and send it to a reference detector. Such noise suppression, however, turns out to be quite difficult in real microscopy applications, since the spatially dependent transmission and scattering of the sample strongly vary the intensity of the probe beam, bringing it out of balance with the reference beam.

Lipid metabolism was studied also in human liver cells by monitoring the size and distribution of lipid droplets, and allowed to assess the effect of therapeutic drugs on specific pathologies with metabolic pathways leading to lipid accumulation.


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In the simplest configuration of hyperspectral CARS, a fixed narrowband pump and a tunable narrowband Stokes simultaneously excite a sample and the generated CARS signal is acquired sequentially by changing pump—Stokes frequency detuning, to construct the full CARS spectra for every pixel of the image. While a TL pulse has an instantaneous frequency which is equal to the carrier frequency across the whole pulse temporal profile, in a chirped pulse the phase term has a quadratic dependence on time, such that, within the temporal envelope of the pulse, the instantaneous frequency deviates from the carrier frequency in a linear fashion.

This configuration can be exploited in a CRS experiment to excite a vibrational resonance whose energy matches the IFD. Another typically adopted solution is the use of blocks of highly dispersive glass such as SF57 or SF6 whose length is selected according to the desired chirp parameter at the pump and Stokes wavelengths. Calculations can be done through Sellmeier equations, which express the refractive index as a function of the wavelength. The delay between pulses is adjusted through mechanical delay lines after recording a calibration curve using a known Raman spectra as a reference.

Pump and Stokes pulses were chirped for SF by propagation in appropriate lengths of SF57 glass blocks. A dichroic mirror split the laser output into the pump and the Stokes pulses. To impart an equal linear chirp to the two pulses, an extra GVD was added to the Stokes pulse through an SF57 glass block before it was recombined on a second dichroic mirror with the pump with an adjustable delay.

Coherent Anti-Stokes Raman Scattering Microspectrometer

After that both pulses propagated through another SF57 glass block before entering the microscope. This configuration was later adopted in many experiments. In this way, the on—off state of the Pockels cell determined the path length of the pump pulses and the corresponding frequency detuning of the SF. It starts from a femtosecond Ti:sapphire oscillator split into two beams.

Coherent anti-Stokes Raman spectroscopy of single and multi-layer graphene

Multiplex CARS imaging was achieved, with typical pixel dwell times ranging from tens to hundreds of milliseconds, thus too slow for biomedical applications. This distortion is particularly relevant in the fingerprint region, where the resonant contribution is weak. Under these conditions, the NRB exceeds the resonant signal and distorts its shape, leading to dispersive features due to the real part of. On the other hand, the availability of the complete CARS spectrum enables one to apply analytic techniques to retrieve the spectral phase or equivalently, the real and imaginary part of the nonlinear susceptibility.

The idea of the MEM method is to express the complex nonlinear response as 14 where. The MEM method extracts from the experimental data an estimated value of the phase, , by nonlinear polynomial fitting, and uses it to retrieve the imaginary part of the estimated resonant nonlinear susceptibility which, considering the real nature of the NRB, can be expressed as In a parallel development, multiplex CARS has also been pursued starting from a nanosecond laser system.

The laser output is divided by a beam splitter: one beam directly constitutes the pump for the CARS process, while the other one is launched in a 6 m long air—silica PCF for supercontinuum SC generation. Pump and Stokes pulse are collinearly recombined by a notch filter and focused on the sample; the transmitted CARS signal, after filtering, is detected by a spectrometer with a CCD camera. Since both pump and Stokes pulses have approximately nanosecond duration, their synchronization is not critical and is not affected by the chirp acquired by the Stokes pulse in the PCF.

It has been used, in combination with the MEM method, to extract the fingerprint Raman spectra of living yeast cells and extract dynamical information. The combination of these factors leads to rather long pixel dwell times, of the order of tens to hundreds of milliseconds.

The ISRS process creates a vibrational coherence in all modes with frequencies falling within the excitation laser bandwidth, provided that the pulse has a temporal duration close to the TL value, so that all frequencies interact nearly simultaneously with the sample. As a rule of thumb, one can say that ISRS is able to create a vibrational coherence at frequencies up to the reciprocal of the pulse duration, so that the 16 fs SC pulse can efficiently excite frequencies in the fingerprint region.

This vibrational coherence is then read out by a further interaction with the narrowband nm probe pulse, which generates the CARS signal. This combination of efficient impulsive excitation and heterodyne amplification by the NRB enables to greatly enhance the weak fingerprint signal. In this way the resonant nonlinear susceptibility , which corresponds to the vibrational coherence generated by the pump and Stokes pulses and which persists on the picosecond timescale that characterizes vibrational dephasing in molecules, is sampled by the probe pulse. By delaying the narrowband with respect to the broadband pulse, one suppresses the NRB and recovers a clean vibrational spectrum, as demonstrated by measuring solvents and by imaging the lipid distribution in C.

On the other hand, it suffers from the complications inherent with pulse shaping. Note that this phase pattern corresponds to excitation of the sample with a sequence of pulses with period where T is the period of the vibration; coherent population of a vibrational level can thus only occur if the period of the pulse sequence is an integer multiple of the corresponding vibrational period. In this case the relative displacement of the minima of the phase parabolas can be scanned to address different vibrational frequencies. These approaches exploit the capability of pulse shapers to provide independent and simultaneous control of phase, polarization, and amplitude of the pulse frequencies.

This splits the probe pulse into two spectrally distinct longer duration probe pulses with opposite phases. Due to the instantaneous and equal nonresonant response, the NRBs generated from the two probes destructively interfere to cancel each other. However, a small NRB leakage from the polarizer can still be observed. By taking the difference of the acquired spectra and after a suitable normalization, implementing the double quadrature spectral interferometry DQSI method, it was possible to retrieve the pure real and imaginary resonant CARS signals upon changing the phase of the narrowband probe pulse.

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By recording two spectra at slightly different notch positions, spectrally separated by the notch width, and taking their difference normalized to the nonresonant spectrum, multiplex Raman spectra were retrieved. Additionally, polarization shaping was applied to make the two pulses orthogonally polarized. The two shaped pulses, the main pulse and the reference pulse, are sent to the sample and the reflected pulses are separated by a dichroic beam splitter into blue and red parts and sent to fast photodetectors. As a consequence of the Raman process within the main pulse, the blue part experiences SRL and the red part SRG, while the reference pulse is unaltered.

The pulses are sent to a Michelson interferometer, producing two collinear replicas with variable delay which are focused on the sample by a microscope objective. The CARS signal is detected in the forward direction, collected by a second objective and focused on a photomultiplier followed by a data acquisition card. This enables to amplify the CARS signal by almost two orders of magnitude.



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