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ADVANCES IN RAMAN TECHNIQUES

Laser requirements and advances for Raman techniques

Andreas Isemann Quantum GmbH, 78467 Konstanz, Germany

INTRODUCTION 473 nm and 1064 nm, a narrow Raman as a probe of bandwidth output of few tens of GHz vibrational transitions has made or below 1 MHz if needed within the leaps and bounds since its discovery, linewidth of vibrational transitions and various schemes based on this for high resolution, low noise (less phenomenon have been developed than 0.02%) and excellent beam with great success. quality (fundamental transversal Applications range from basic electromagnetic mode TEM00) scientific research, to medical and provides optimised performance industrial instrumentation. Some for the resolution of the Raman schemes utilise linear Raman measurement needed. scattering, whilst others take advantage The is chosen based of high peak-power to probe on the sample under investigation, nonlinear Raman responses. with 532 nm being commonly used This article intends to provide a for the necessary virtual electronic brief overview of the differences and transition. In the following section, benefits, together with the laser source four examples from different areas of requirements and the advancements Raman applications show the diverse in techniques enabled by recent applications of linear Raman and what developments in . advances have been achieved. An example of studying a real-world LINEAR RAMAN application, the successful control of Figure 1 An example of the RR microfluidic device counting of The advent of the laser in providing a food quality using Raman photosynthetic microorganisms. As the cells of the model strain high-intensity coherent light source and multivariate analysis, is described Synechocystis sp. PCC 6803 flow through the Raman detection area 1 of the microfluidic device, RR spectra were acquired continuously has helped to make by Jernshøj et al. . Examining the about 27 times every second. The v1 RR band was used to a useful method for spectroscopy quality of meat and vegetables is differentiate 12 C- and 13 C-cells. The intensity of this band was by increasing signal levels of the discussed, together with the source plotted against the temporal axis and displayed in green and for spontaneous event to enable use of specification, when constructing a 12 C- and 13 C-cells, respectively. A part of the figure near 23.3 s was readily available detectors. system for conditions outside of a enlarged to show a 13 C- and 12 C- passed through the Raman detection area sequentially only about 0.1 s apart from each other; The most widely used method to laboratory environment. the untreated RR spectra of those two cells show a distinctive red shift date is linear Raman, which takes For real-world applications, the of all of the RR bands. advantage of commercial continuous- laser source needs not only to work Reprinted (adapted) with permission from Li et al2. Copyright (2012) American Chemical Society wave lasers. The choice of excitation at the right output parameters, but wavelength depends on the sample also it necessitates a robust design used, where in general, a shorter making it portable and highly efficient wavelength would yield a higher for integration into instruments and efficiency yet suffers from higher being able to interface with operating scattering and may induce damage software. in biological samples within the UV The two next experiments benefit region. from the high resolution that a narrow A diode pumped solid state (DPSS) linewidth laser source offers as well as laser with a wavelength between its high beam quality.

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Application of a single longitudinal enhanced Raman scattering (SERS) or mode (SLM) laser at 532 nm with as a subvariant, tip enhanced Raman a linewidth of 1 MHz enables scattering (TERS) have been developed. high resolution resonance Raman In SERS, structured surfaces are used (RR) spectra from the excitation to increase the Raman signal level of carotenoids to differentiate with the electrical field enhancements 13 12 between CO2 and CO2, used for due to those structures. Recently, high throughput profiling of cell also hybrid structures making use of populations2, see fig. 1. The cells nano-sized semiconductors, including analysed are from photosynthetic magnetic materials, have attracted microorganisms that are of interest due to their excellent SERS extraordinary value in the area of performance, as described by Prakash renewable energy and biotechnology. and colleagues5. In this research, a high throughput In TERS, the excitation light is Raman-activated cell sorting (RACS) brought onto the sample via an system has been developed, to perform atomic force microscope (AFM) tip Raman-activated cell counting. using plasmonic effects. This helps Figure 2 Single- TERS of a porphyrin derivate examined Being able to tightly focus the laser to overcome the diffraction limit of with UHV-STM at low . (A) The Raman spectrum from beam provides for necessary spatial the laser spot, increasing the spatial powder represents many orientations, whereas (B) TERS was able to resolution in the order of 0.5 – 1 µm to resolution, as well as giving a field resolve a molecule tilt, and (C) also shows single-molecule resolution look at individual cells. enhancement due to the tip geometry. on a flatly oriented molecule. Especially the differences between TERS spectra (B) and (C) are remarkable. Adapted with permission from A novel approach for precise In some cases, depending on the Xhang, et al.8 quantitative measurements of optical sample, the increase of the signal due Copyright Macmillan Publishers Ltd. 2013 mobility in atomically thin films based to the field enhancement overcomes on high resolution Raman is presented the decrease due to a lower number by Ji et al.3. Using a large change in of oscillators () inside the the intensity of the Raman mode with excitation volume. An excellent laser an applied strong magnetic field that beam quality with a high degree of occurs in one plane, but not the out enables it to focus down of plane Raman mode. It stems from to a micrometre onto the AFM needle a magnetic field induced symmetry as well as providing efficient excitation breaking. of the polarization dependent surface A quantitative analysis of the field , the mechanism responsible dependent Raman intensity allows the for electric field enhancement at determination of the optical mobility. the tip of the needle. The articles by For the quantitative measurements, the Kumar6 and Langelüddecke7 provide low noise and high power stability are an overview of the method and variety important. The excellent beam quality of applications. of the SLM laser used (Laser Quantum As always with input coupling into torus), enables confocal micro- setups, a superb beam in the measurement setup. pointing stability with fast warmup a The more commonly used linewidth times on the order of a few minutes in (CW) Raman is help with reliable day to day operation a few 10s of GHz, where most robust to concentrate on the experiment. lasers are readily available. In cancer In the following, two examples of research, screening cells for potentially achievements using TERS are given, cancerous behaviour calls for an before turning to non-linear Raman. automated analysis system. Such a The high spatial resolution of TERS system based on laser tweezers Raman has enabled the visualisation of the b spectroscopy (LTRS) has been presented tilt of a single molecule by means of by Casabella et al.4. An excellent beam measuring a spectrum, see figure 2 c quality of TEM00 ensuring a tight from the paper by Zhang et al.8 Also, focus as well as a long-term stable the authors were able to resolve the beam pointing are needed for trapping ring system of a single porphyrin the individual cells in the microfluidic derivative with a spatial resolution of channels and for the Raman 0.5 nm, see fig. 2 (right side). measurement. In another fascinating experiment, Two lasers of different wavelength TERS has been able to reveal spin were used (1064 nm for trapping, waves in iron oxide nanoparticle, as 532 nm for Raman, both from the described by Rodriguez et al.9 and Figure 3 a) Tip-enhanced Raman spectra in a line scan along the Laser Quantum ventus series), which depicted in fig. 3 using micro-Raman nano-particles/glass interface. The stacking representation shows integrated well into the overall system experiments. several first order modes below 700 cm−1 , the 2LO mode around 1323 cm−1 and the spin-wave mode around 1587 cm−1 . The step size control by use of their remote control used to obtain this set of spectra was 1 nm. In the inset is shown the capability. NON-LINEAR RAMAN AFM image acquired during the TERS line scan (b) intensity profiles Some samples allow increased To drastically increase the efficiency of of three modes along the line scan showing the sharp increase in the efficiency of the scattering when the the Raman process, various coherent spin-wave intensity when the TERS tip is on the nanoparticle. This is Raman transition happens to occur (nonlinear) Raman processes have been followed by a decrease in the intensity of all modes when the tip was retracted (tip off). (c) A sketch of the tip–nanoparticle configuration close to a real transition. However, studied. The Nonlinear Raman process shows the anisotropy between the two sides of the particle with not all samples of interest would offer uses at least two different , respect to the tip. Crystallographic facets and edges are set according this benefit. To increase the sensitivity but uses the same Raman active to Rodriguez et al.27 and spatial resolution of linear vibrational mode as the linear Raman Reprinted (adapted) with permission from Rodriguez et al.9. Copyright (2007) American Chemical Society] Copyright Macmillan Publishers Ltd. 2013. Raman, variations such as surface technique.

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Typically, laser setups using two parallel detection may require laser beams of different colour and in dedicated sophisticated detectors and pulsed mode enable a more elaborate, electronics18. but coherent process, that generates With extremely broadband laser a signal which is generally orders oscillators offering typical bandwidth of magnitude higher. In practise, on the order of 400 nm in the NIR the advantage of coherent Raman and also high repetition rate amplified scattering over linear Raman is this systems like an OPCPA offering overall higher signal. a bandwidth of greater 300 nm In Coherent Antistokes Raman being readily available, broadband Scattering (CARS), the difference single beam Raman techniques between the two wavelengths is used to have become possible. The available coherently drive a Raman transition. bandwidth allows a -1 Typically, two narrow bandwidth range of up to 4000 cm . It also Figure 4 TPF (green), CARS from subcutaneous lipid deposits (red) picosecond electronically synchronized allows for an efficient combination of and SHG from collagen () of tissue from a mouse tail Reprinted (adapted) with permission from by Pope et al.9 laser pulse trains have been used, one multiple modalities, or for very fast Copyright (2013) Optical Society of America of which is scanned in the wavelength spectroscopic CARS modality using to do CARS spectroscopy. The shape a quite simple detector in form of a of the CARS signal is different photo diode. from that of linear Raman, also the One implementation of CARS using overall sensitivity is limited by the broad bandwidth uses the principle distinction between the resonant and of spectral focussing19. A simple non-resonant part of the signal. An implementation relying on glass In this method, the CARS spectrum overview of the method is given in a stretchers to measure two vibrational is obtained through the Fourier book by Xie and colleagues10. frequencies with two (or more) pairs of transform of the CARS interferogram A variant of Coherent Raman is pump/stokes pulses simultaneously is – i.e. the CARS emission as a function Stimulated Raman Scattering (SRS), called differential CARS (or D-CARS). of time delay between two identical which again uses lasers of two different One advantage of this method is the collinear excitation pulse trains colours with synchronized output fast acquisition speed of a few seconds with a generated time delay between pulses to coherently drive a nonlinear for complete images and the effectively successive pulses. process. The advantage is greatly rejection of non-resonant background The non-resonant background can reduced non-resonant background, using the D-CARS signal. The simple be removed by simply removing the and that the output trace is the same analysis scheme allows for rapid contribution around zero time delay. as conventional Raman allowing distinguishing between saturated and The detection of the CARS signal existing databases of compound results unsaturated lipids20. can be accomplished with a simple to be used, as well as quantitative The laser source providing a Si-diode. interpretation of the measured data11. sufficiently large bandwidth not Using a 5 fs oscillator with a SRS makes use of modulation only enables single beam CARS, but spectral bandwidth of 4000 cm-1 the of one of the laser beams, a double also has been used in a multimodal, applicability of FT CARS to biological modulation technique Stimulated or hyperspectral scheme combining samples covering the bandwidth Raman and Opposite Loss D-CARS covering a wavenumber range in a single measurement has been Detection (SRGOLD) proposes of 2600 cm-1 with a resolution of demonstrated23. An image of unstained background free detection and is the 10 cm-1 together with second harmonic HeLa cells has been obtained by FT latest variant using a three colour laser generation (SHG) as well as two CARS (fig. 6) shows the differentiating beam excitation scheme12. (TPF) imaging – all from of mitochondria or endoplasmic Both CARS and SRS can in a single beam at the same time, using a reticulum, the nucleus and . principal be done in versions using pulse shaper21, 22. An example image is The speed of the acquisition of a femtosecond or picosecond lasers (and given in fig. 4 providing much richer complete CARS spectrum using FT even nanosecond lasers), each with information than from one imaging CARS depends on how fast the two different advantages and drawbacks. modality alone. pulses can pass through the needed Historically, two electronically An early implementation of spectral delay times that contribute to the synchronized tuneable laser sources focusing using an octave spanning CARS interferogram. have been used13, or more typically oscillator (Laser Quantum venteon) The refresh time then depends on today, optical parametric oscillators, and a spatial light modulator to do how fast this cycle can be repeated. providing optically synchronised single-pulse (single beam) CARS The time delays which contribute pulses. is described by Isobe to the interferogram depend on However, todays available et al23. In fig. 5, CARS images of the dephasing time of the coherent commercial implementations are often unstained HeLa cells are shown from Raman excitation, which is typically hampered with limited tuning speed, this experiment demonstrating the on the picosecond (ps) timescale. and hence slow in acquisition of a applicability to biological samples. The interferometric, non-resonant CARS/SRS spectrum14. The use of an octave spanning background being present at short One elegant way for considerably oscillator allowed the researchers to delay times only and hence can be increasing the acquisition speed or focus into narrow spectral regions completely suppressed by suitably even enabling single beam capability ranging from approx. 1000 cm-1 to positioning the numerical apodization is the use of multiplex CARS15, 16 4800 cm-1. window26. or multiplex SRS17, 18. With this Aimed at fast measurement of In the implementation of the work technique, a narrow band pump and spectroscopic CARS, the method of published by Isobe and colleagues a a broadband Stokes pulse, or even one Fourier Transform (FT) CARS shows mechanical delay line is used23. The ultra-broadband spectrum with phase great potential. The principle has first step to speed up the measurement shaping supply all wavelengths needed been shown by Ogilve et al24 using a is to use an ASynchronous OPtical to simultaneously record a large 100 MHz oscillator and is based on Sampling (ASOPS) dual comb type number of Raman shifts. However, a pump probe type dual pulse setup. setup where two repetition rate

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locked laser oscillators with a slight a b c offset in their repetition rate are used to generate the pulse pair24. In this scenario, the refresh time is given by the inverse of the laser repetition rate. The measurement time of one scan depends on how fast the delay of the two pulses covers the timeframe of the interferogram. The benefit of using oscillators with 1 GHz repetition rate over a 100 MHz repetition rate in the implementation from Ideguchi25 is a 10 times higher refresh rate, i.e. 10 times higher duty cycle – number of measurements for a b c the same integration time – as well as much higher density of sampling steps at the same difference in repetition rates. In other words, using GHz repetition rates, at the same sampling step size, one can operate at much higher refresh times, i.e. more measurements per unit time. The fast acquisition time with a high refresh rate enables the measurement of as well as enabling multimodality Instrumentation D, 2(1) (2015). Figure 5, top, CARS transients or short lived species. efficiently. Finally, Fourier Transform 7 L. Langelüddecke, P. Singh, V. images of an unstained HeLa cell by selective Recently, a first implementation CARS in combination with dual Deckert, Exploring the Nanoscale: excitation at 2930 cm−1 of such a dual comb CARS system comb technology enables fast Fifteen Years of Tip-Enhanced (a), 3200 cm−1 (b) and using two GHz oscillators has been spectroscopic CARS measurements , Applied 3400 cm−1 (c). Scale demonstrated26. The bandwidth of the within microseconds. The use of GHz Spectroscopy 69(12) (2015) 1357 – bar is 6 µm. Reprinted (adapted) with emitted pulses is 60 nm which allows repetition rate oscillators provides 1371. permission from Isobe et al23. Copyright (2009) for sub-20 fs compressed pulses. They for a factor of 10 higher refresh rates 8 R. Zhang, Y. Zhang, Z.C. Dong, S. Optical Society of America enabled measurements with a span of the spectral acquisition compared Jiang, C. Zhang, L.G. Chen, L. Zhang, from 400 – 1400 cm-1 and a resolution to commonly available 100 MHz Y. Liao, J. Aizpurua, Y. Luo, J.L. Figure 6, above, (a, of 6 cm-1 given by the time window of oscillators. Yang, J.G. Hou, Chemical Mapping b) CARS images of an unstained HeLa the Fourier transformation, at a kHz of a Single Molecule by Plasmon- cell constructed from refresh rate with the actual acquisition REFERENCES Enhanced Raman scattering, Nature integration in the time of one scan as short as 5 µs. 1 K. Jernshøj, S. Hassing, L. 498(7452) (2013) 82-86. spectral bands of 2800- To demonstrate that this method Christensen, A Non-Destructive 9 R. Rodriguez, E. Sheremet, T. 2900 cm−1 (a), and can measure at sufficient resolution and On-site Tool for Control of Deckert-Gaudig, C. Chaneac, M. 2900-3000 cm−1 (b). to discriminate different substances, Food Quality ?, Food Quality, Kostas Hietschold, V. Deckert, D. Zahn, (c) Combined image of three CARS images a spectrum of a mixture of toluene, Kapiris. InTech - Open Access Surface- and tip-enhanced Raman of an unstained HeLa chloroform and cyclohexane has been Publisher, (2012) 47-72. spectroscopy reveals spin-waves in cell reconstructed after taken, see fig. 7. The two neighbouring 2 M. Li, P. Ashok, K. Dholakia, Wei iron oxide nanoparticles Nanoscale 7 the NSC process using peaks of toluene and cyclohexane are E. Huang, Raman-Activated Cell (2015) 9545-9551. the CARS spectrum only 13 cm-1 apart, yet clearly resolved. Counting for Profiling Carbon 10 X. Xie, J.-X. Cheng, E. Potma, from mitochondria or endoplasmic reticulum The measurement revealed a linear Dioxide Fixing Microorganisms, Coherent Anti-Stokes Raman (blue), the nucleus dependency of the line maximum The Journal of Physical A, Scattering Microscopy, Handbook of (blue) and water (red). with concentration as well as pulse 116 (25) (2012) 6560-6563. Biological , Third Scale bar is 8 µm. Reprinted (adapted) with energy, which will be helpful looking 3 J. Ji, A. Zhang, J. Fan, Y. Li, X. Wang, Edition, edited by James B. Pawley, permission from Isobe towards quantitative analysis. The use J. Zhang, E. Plummer, Q. Zhang, Springer Science+Business Media, et al23. Copyright (2009) of oscillators with sub-20 fs allows to Giant magneto-optical Raman LLC, New York, 33 (2006) 595-606. Optical Society of America extend the setup with multimodal or effect in a layered transition metal 11 P. Nandakumar, A. Kovalev, A. hyperspectral capabilities. compound, PNAS, 113(9) (2016) Volkmer, Vibrational imaging 2349-2353. based on stimulated Raman CONCLUSIONS 4 S. Casabella, P. Scully, N. Goddard, scattering microscopy, New Journal In conclusion, a selection of linear P. Gardner, Automated analysis of of Physics 11 (2009) 033026. Raman applications has been presented single cells using Laser Tweezers 12 P. Berto, E. R. Andresen, H. that make use of the excellent beam Raman Spectroscopy, Analyst, 141 Rigneault, Background free quality and beam pointing and power (2016) 689. stimulated Raman spectroscopy and stability with low noise of modern 5 O. Prakash, S. Kumar, P. Singh, V. microscopy, Physical Review Letters laser sources as well as to the narrow Deckert, S. Chatterjee, A. Ghosh, 112 (2014) 053905. linewidth properties where applicable. R. Singh, Surface-enhanced 13 E. Potma, D. Jones, J.-X. Cheng, A high efficiency, robustness and Raman scattering characteristics X. Xie, J. Ye, High-sensitivity remote operation capability benefits all of CuO : Mn/Ag heterojunction coherent anti-Stokes Raman applications, these being a prerequisite probed by methyl orange: effect scattering microscopy with two for integration into industrial or of Mn2+ , Journal of Raman tightly synchronized picosecond automated systems. Spectroscopy, 47 (2016) 813–818. lasers, Letters 27 (2002) The availability for broadband 6 N. Kumar, S. Mignuzzi, W. Su, 1168–1170. sources covering spectra D. Roy, Tip-enhanced Raman 14 S. Brustlein, P. Ferrand, N. Walther, of several hundred nm have enabled spectroscopy: principles and S. Brasselet, C. Billaudeau, D. fast coherent Raman measurements applications, J EPJ Techniques and Marguet, H. Rigneault, Optical

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biography Dr. Andreas Isemann is Area Sales Manager as well as Business Development Manager with Laser Quantum GmbH. He has studied physics with his initial background in ultrafast lasers from his research in directly diode pumped femtosecond oscillators and amplifiers at the Laser Center Hannover, Germany. Figure 7 Dual-comb CARS spectrum of toluene, chloroform and cyclohexane with With his transition into industry, he a mixing ratio of 1:1:1. The difference in repetition frequencies isδ f rep. 600 Hz. The has held various positions in sales apodized resolution is 6 cm−1 and 120 spectra are averaged. The measurement time is 2 and marketing as well as product ms and the total experimental time is 200 ms. Reprinted (adapted) with permission from Mohler et al26. Copyright (2013) Optical Society of America management at Femtolasers GmbH and APE GmbH, before joining Laser Quantum GmbH. One focus has been in application management parametric oscillator-based 22 J. Rehbinder, L. Brückner, A. understanding the requirements light source for coherent Raman Wipfler, T. Buckup, M. Motzkus, on the laser source as well as scattering microscopy: practical Multimodal nonlinear optical investigating new applications, which overview, Journal of Biomedical Optics microscopy with shaped 10 fs include few cycle pulses, biomedical, 16(2) (2011) 021106. pulses, Optics Express 22(23) (2014) as well as non-linear microscopy. 15 J.-X. Cheng, A. Volkmer, L. D. 28790-28797. Book, X. Xie, Multiplex Coherent 23 K. Isobe, A. Suda, M. Tanaka, H. abstract Anti-Stokes Raman Scattering Hashimoto, F. Kannari, H. Kawano, Raman spectroscopy found its Microspectroscopy and Study of H. Mizuno, A. Miyawaki, K. place in many areas applying the Lipid Vesicles, The Journal of Physical Midorikawa, Single-pulse coherent linear as well as the non-linear Chemistry B 106 (2002) 8493-8498. anti-Stokes Raman scattering implementations of the technique. 16 A. Wipfler, T. Buckup, and M. microscopy employing an octave A summary of the various methods Motzkus, Multiplexing Single-Beam spanning pulse, Optics Express cites selected publications illustrating CARS with Heterodyne Detection, 17(14) (2009) 11259-11266. advances. In the linear case, Applied Physics Letters 100 (2012) 24 J. P. Ogilvie, E. Beaurepaire, A. application profit from industrial 071102. Alexandrou, and M. Joffre, Fourier- grade low noise stable laser sources. 17 D. Fu, F.K. Lu, X. Zhang, C. transform coherent anti-Stokes In non-linear Raman, availability Freudiger, D. R. Pernik, G. Holtom, Raman scattering microscopy, of ultra-broadband femtosecond X. Xie, Quantitative Chemical Optics Letters 31(4) (2006) 480–482. laser sources and novel pulse Imaging with Multiplex Stimulated 25 T. Ideguchi, S. Holzner, B. shaping or sampling techniques Raman Scattering Microscopy, Bernhardt, G. Guelachvili, N. have enabled new schemes e.g. Journal American Chemical Society, Picque, T. W. Hänsch, Coherent multiplexing non-linear Raman with 134 (2012) 3623−3626. Raman spectro-imaging with laser second and 18 C.-S. Liao , M. N. Slipchenko, P. frequency combs, Nature 502 (2013) third harmonic generation giving Wang, J. Li, S.-Y. Lee, R. A. Oglesbee, 355-539. rich information content, as well as J-X Cheng, Microsecond scale 26 K. J. Mohler, B. J. Bohn, M. Yan, G. enabling extremely fast acquisition vibrational spectroscopic imaging Mélen, T. W. Hänsch, N. Picque, of Raman spectroscopic data within by multiplex stimulated Raman Dual-comb coherent Raman microseconds for investigation of scattering microscopy, Light: Science spectroscopy with lasers of 1-GHz dynamic or transient systems. & Applications, 4 (2015) e265. pulse repetition frequency, Optics 19 T. Hellerer, A.M.K. Enejder, A. Letters 42(2) (2017) 318-321. acknowledgements Zumbusch, Spectral focusing: High 27 R. D. Rodriguez, D. Demaille, E. The author would like to thank Andy spectral resolution spectroscopy Lacaze, J. Jupille, C. Chaneac and Wells, Jennifer Szczepaniak Sloane with broad-bandwidth laser pulses, J.-P. Jolivet, Rhombohedral Shape of and Danielle Hardie from Laser Applied Physics Letters 85(1) (2004) Hematite Nanocrystals Synthesized Quantum Ltd, Dave Stockwell, PhD 25-27. via Thermolysis of an Additive-free from Laser Quantum Inc and Dr. 20 C, Di Napoli, F. Masia, I. Ferric Chloride Solution, The Journal Gregor Klatt from Laser Quantum Pope, C. Otto, W. Langbein, of C, 111(45) 2007 GmbH for their various contributions. P. Borri, Chemically-specific 16866–16870. dual-differential CARS micro- Corresponding author spectroscopy of saturated and ©John Wiley & Sons Ltd. 2017 details unsaturated lipid droplets, Journal of Andreas Isemann: aisemann@ 7(1–2) (2014) 68-76. laserquantum.com 21 I. Pope, W. Langbein, P. Watson, P. Borri, Simultaneous hyperspectral Microscopy and Analysis 31(4): differential-CARS, TPF and SHG 19-23 (AM), August 2017 microscopy with a single 5 fs Ti:Sa laser, Optics Express 21(6) (2013) 7096-7106.

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