Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 135–140

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Spectrochimica Acta Part A: Molecular and BiomolecularSpectroscopy

j ourna l homepage: www.e lsev ie r .com/ locate /saa

Multimodal nonlinear microscope based on a compact fiber-formatlaser source

Francesco Crisafi a,1, Vikas Kumar a,1, Antonio Perri a, Marco Marangoni a, Giulio Cerullo a, Dario Polli a,b,⁎a IFN-CNR, Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italyb Center for Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, via Giovanni Pascoli 70/3, 20133 Milano, Italy

⁎ Corresponding author at: IFN-CNR, Dipartimento dPiazza Leonardo da Vinci 32, 20133 Milano, Italy.

E-mail address: dario.polli@polimi.it (D. Polli).1 Authors contributed equally to work.

http://dx.doi.org/10.1016/j.saa.2017.06.0551386-1425/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 February 2017Received in revised form 26 May 2017Accepted 30 June 2017Available online 05 July 2017

We present a multimodal non-linear optical (NLO) laser-scanning microscope, based on a compact fiber-formatexcitation laser and integrating coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering(SRS) and two-photon-excitation fluorescence (TPEF) on a single platform.We demonstrate its capabilities in si-multaneously acquiring CARS and SRS images of a blend of 6-μm poly(methyl methacrylate) beads and 3-μmpolystyrene beads. We then apply it to visualize cell walls and chloroplast of an unprocessed fresh leaf of Elodeaaquatic plant via SRS and TPEFmodalities, respectively. The presentedNLOmicroscope, developed in house usingoff-the-shelf components, offers full accessibility to the optical path and ensures its easy re-configurability andflexibility.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Nonlinear optical microscopyCARSSRSTPEFMultimodalCoherent Raman spectroscopy

1. Introduction

Optical microscopy is an extremely powerful investigation tool in lifesciences, thanks to its ability of visualizing morphological details in cellsand tissues on the sub-micrometer spatial scale [1]. It provides a muchhigher spatial resolution compared to magnetic resonance imaging,and, at the same time, it does not require the sample to be fixed, as inelectron microscopy. Nonlinear optical (NLO) microscopy techniques, inparticular, offer additional advantages, such as inherent 3D-sectioningcapability and greater penetration depth, due to the use of infraredwave-lengths. The most widespread NLO technique is two-photon-excitationfluorescence (TPEF) [2–4]. It provides very high sensitivity, but in mostcases it requires the addition of markers to the sample to be studied, ei-ther exogenous (staining dyes or semiconductor quantum dots) or en-dogenous (fluorescent proteins). Other NLO microscopy techniqueshave the advantage of being label-free, allowing the use of pristine sam-ples. Some of these techniques, such as second-harmonic generation(SHG) [5–7], sum-frequency generation (SFG) [8,9] and third-harmonicgeneration (THG) [4,10] microscopy, do not provide chemical contrast,i.e. they are not able to selectively differentiate between specific compo-nents of a cell or a tissue. SHG observes contrast in non-centrosymmetricstructures like collagen fibers in tissue, while THG is mainly sensitive to

i Fisica, Politecnico di Milano,

local changes of nonlinear refractive index within the sample occurringat interfaces, such as lipid cell membranes or lipid droplets. Chemical se-lectivity is provided by coherent Raman scattering (CRS) techniques [11],which employ the vibrational spectrum of a molecule to provide an en-dogenous signature that can be used for its identification. CRS exploitsthe third-order nonlinear optical response of the sample to set up andde-tect a vibrational coherence within the ensemble of molecules inside thelaser focus. When the difference between pump and Stokes frequenciesmatches a characteristic vibrational frequency, then all the molecules inthe focal volume are resonantly excited and vibrate in phase; this vibra-tional coherence enhances the Raman response by many orders of mag-nitude with respect to the incoherent spontaneous Raman process.

CRSmicroscopyhas twopopular forms: coherent anti-Stokes Ramanscattering (CARS) [12,13] and stimulated Raman scattering (SRS) [14–17]. In both CARS and SRS, two synchronized narrowband pulses, thepump (at frequency ωp) and the Stokes (at frequency ωs), are focusedon a sample and their frequency difference is tuned to a Raman-activevibrational mode Ω of the targeted molecule, i.e. Ω = ωp − ωs. Whenit happens, in the case of CARS, a strong anti-Stokes signal at frequencyωas = 2ωp − ωs is generated, which is utilized to probe the moleculesunder study. Since CARS signal is at higher frequency with respect toinput pump-Stokes frequencies, it can be easily spectrally separatedand is also immune to one-photon fluorescence signals which fall atlower frequencies. This makes CARS signal naturally free from anykind of linear background. On the other hand, the CARS process hasthe serious drawback that it suffers from nonlinear non-resonant back-ground (NRB). CARS signal is given by ICARS∝ |χ(3)|2=|χR(3)(Ω)+χNR(3)|2,

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where χR(3)(Ω) is the complex resonant response of the targeted vibra-tion and χNR(3) is the real non-resonant response of the background,which does not deliver any chemically specific information. In SRS, onthe other hand, the coherent interaction with the sample induces stim-ulated emission from a virtual state of the sample to the investigated vi-brational state, resulting in a Stokes-field amplification (stimulatedRaman gain, SRG) and in a simultaneous pump-field attenuation (stim-ulated Raman loss, SRL). SRS is inherently free from NRB, however it istechnically demanding as it requires the detection of a weak signal(the SRG/SRL) on top of a large and fluctuating linear background.This typically requires sophisticated modulation-transfer techniques.

Multimodal NLOmicroscopy can capitalize the potential of differentNLO modalities by combining two or more of them in a single imagingplatform. Hence, it can provide richermicroscopic information by imag-ing different kinds of molecules or structures in a sample. MultimodalCARS/TPEF/SHG microscopes have already proven for the capability ofbiological imaging and identification of cancerous tissues in brain [18],lung [19] and kidney [20]. Cheng et al. have utilized the combinationof TPEF/SFG/CARS modalities to investigate the central nervous systemin diseased and healthy states [21], in the study of the progressionof arterial diseases [22] and in imaging and quantitative analysis of ath-erosclerosis, a major cause of cardiovascular diseases [6]. The combina-tion of THG/SHG/TPEF microscopies has been used e.g. to visualize themicrostructure of human cornea [4].

The main stumbling block which prevents widespread adoption ofmultimodal NLO microscopy techniques in the biological and medicalcommunities is the complication and cost of the experimental apparatus,both for the excitation laser and the microscope. Regarding the excita-tion source, the most critical technique is CRS. A compromise must befound between high peak laser power to enhance the nonlinear signal,which points towards shorter pulses, and the frequency resolution topreservemolecular selectivity, which points towards narrow laser band-width. In the condensed phase, Raman transitions exhibit linewidths ofthe order of tens of cm−1, so that the optimal pulse duration lies in the1–3 ps range for pump/Stokes pulses. Moreover, their frequency differ-ence should be tunable, to access a large vibrational bandwidth and thelaser repetition rate should be high (≈100 MHz) to reach the shot-noise limit in detection and avoidmulti-photon absorption sample dam-age caused by the high peak power of the pulses. An output power of≈100mWper branch is also typically required to compensate for lossesin the optical chain of the microscope. The complexity of the excitationlaser is one reason why, after its early demonstration in 1982 [23], thedevelopment of CRSmicroscopy has stopped for nearly twodecades. Fol-lowing its revival, initial CRS implementations were based on two elec-tronically synchronized picosecond Ti:sapphire oscillators [24–26],while the current “gold standard” is represented by an optical parametricoscillator synchronously pumped by a picosecond Nd:YVO4 oscillator[27–29]. Such systems are complex, expensive, and they all critically re-quire a synchronization between two independent laser sources, whichmust bemaintained over time. Drastically simplified excitation architec-tures, with lower cost and smaller footprint, are thus greatly in demand,as theywould lower the technological entrance barriers to CRSmicrosco-py. In addition, the multimodal NLO microscope platform cannot easilyrely on commercially available solutions. The detection of the differentnonlinear signals, in fact, requires full accessibility to the optical paths,in order to place different components and detectors required to imple-ment the various techniques. This is difficult in commercial microscopesystems that, being designed for the end user, are typically not accessibleand modifiable.

In this paper, we describe a multimodal NLO laser-scanning micro-scope with a highly simplified architecture, based on a compact fiber-based laser, which enables the TPEF, CARS and SRS modalities. Thefiber laser generates two synchronized beams of picosecond pulses, thepump at 780 nm and the Stokes tunable between 950 and 1050 nm,with sufficient spectral coverage to implement CARS andSRSmicroscopyin the C\\H stretching region. A single excitation beam can also be used

for other NLO microscopy modalities, such as TPEF, SHG, THG and SFG.The excitation source is coupled to a home-built multimodal scanningmicroscope, based on off-the-shelf components and allowing maximumaccessibility to the beam paths. We demonstrate CARS/SRS imaging ofpolymer beads and of leaves of the Elodea aquatic plant.

2. Materials and Methods

Fig. 1(a) shows the architecture of the compact multi-branch fiber-format laser source that generates the multi-colour pulses required forthe different NLO microscopy modalities. It is based on a mode-lockedErbium:fiber oscillator working at 40-MHz repetition rate, which feedsthree independent Er-doped fiber amplifiers (EDFAs), each producing350-mW average power at 1560-nm central wavelength. In this way,all three EDFA outputs are inherently synchronized and phase coherent[30]. Further, they are compressed to nearly transform-limited sub-100-fs duration by individual pairs of silicon prisms. Twoof the EDFAoutputs(referred to as ‘Arm 1’ and ‘Arm 2’ in Fig. 1(a)) are coupled to highlynonlinear fibers (HNLFs) that considerably broaden the laser spectrum,generating side-lobes at both longer and shorter wavelengths with re-spect to the input fundamental one. The longer wavelength peak is asoliton, produced mainly through self-frequency shift due to intra-pulse Raman scattering. On the other hand, the shorter wavelengthpeak is propagating in the normal dispersion regime, also phase-matched to the soliton. The spectral positions of the two peaks sensi-tively depend on the overall dispersion of the pulse coupled to theHNLF. Acting on the silicon prism-pair, the position of soliton peak canbe continuously tuned and precisely controlled between 1700 and2100 nm.

The third EDFAoutput [referred to as ‘Arm0’ in Fig. 1(a)] at 1560nm issent to a non-linear crystal to generate the pump pulses for the CRS pro-cesses at a fixed wavelength of 778 nm. We employed a 10-mm-longMgO-doped periodically poled lithium niobate (PPLN) crystal with a pol-ing period of 19.3 μm. It generates a narrow-bandwidth (15-cm−1

linewidth) second harmonic, according to the spectral compression tech-nique [31], with 120-mW average power [see green spectrum inFig. 1(b)]. In the same manner, the redshifted soliton output of one oftheHNLFs (in ‘Arm1’) is frequencydoubled in a 10-mm-longPPLN crystalhaving a fan-out grating design, spanning the poling period range of 26–33 μm. It generates tunable Stokes pulses for the CRSmodalities from 950to 1050 nm [see red spectra in Fig. 1(b)] with power up to 10 mW andlinewidth ranging between 18 and 30 cm−1. Tuning of the Stokes wave-length is achieved simply by transversely translating the fan-out crystalusing amotorized stage, calibrated to guarantee a rapid (within a fractionof a second) and reproducible selection of the vibrational Raman shift. Thepump-Stokes frequency detuning in the range 2330–3330 cm−1 fullycovers the CH-vibrational region, which is the most commonly used inCRS microscopy. Before entering the microscope, the pump and Stokespulses are temporally matched by a mechanical delay line and thencollinearly combined by a dichroic beam splitter (Semrock, LP02-785RS-25). For the SRS modality, the pump beam is modulated at 1 MHz by anacousto-optic modulator placed in the beam before the combiner. Thenarrowband pump or Stokes pulses can individually be utilized as an ex-citation pulse for TPEF or SHG/THG experiments as per demand of thesample. Furthermore, a part of the compressed sub-100 fs output of theEDFA at 1560 nm just before the PPLN (in Arm 0) can be split and usedas an excitation pulse for THG microscopy.

The third branch [referred to as ‘Arm 2’ in Fig. 1(b)] is used, by prop-erly adjusting the silicon prism pair before the HNLF, to generate abroadband spectrum in the 840- to 1100-nm wavelength region. Aftercompression in a SF-10 prism pair, we obtain sub-20-fs pulses, whichcan be used either as Stokes pulses to implement broadband CARS/SRS configurations [32,33] or as excitation pulses for TPEF or SHG exper-iments performed on the microscope. In the following, we will presentexperimental results obtained coupling only ‘Arm 0’ and ‘Arm 1’beams to the multimodal NLO microscope.

Fig. 1. (a) Schematic of amulti branched Er: fiber laser source formultimodal nonlinear microscopy. AOM, acousto-opticmodulator; DM, dichroicmirror; PPLN, periodically poled lithiumniobate; EDFA, erbium-doped fiber amplifier; HNLF, highly nonlinear fiber; BS, beam splitter; Si, silicon. (b) Spectra of narrowband pump (Arm0) and tunable narrowband Stokes (Arm1)beams; The curve in blue indicates thewavelength dependent averagepower of the Stokes beamarriving on the sample. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

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We have developed a simple and low-cost multimodal NLO laser-scanning microscope, following the design in Fig. 2. A pair of galvano-metric mirrors (Cambridge Technology: ProSeries II Scan Head-10 mm) raster scans the input beams over the sample area at thefocus of the microscope objective. For this purpose, two telecentriclenses, the scan and tube lenses (Thorlabs: LSM05 BB, EFL 110 mm),are arranged in a standard 4-f configuration to form an image relay sys-tem between the scan head and the back focal plane of the focusing ob-jective. This objective (Zeiss 100×, NA = 0.75, WD = 4 mm, fieldnumber 25) provides a flat field-of-view (FOV) of 250 × 250 μm2 areaand a lateral spatial resolution of approximately 1 μm. The samples aremounted on a computer-controlled X-Y-Z stage, composed of a motor-ized X-Y-scanning stage (Standa: model 8MTF-102LS05) and a motor-ized Z-translation stage (Mad City Labs Inc.: model MMP1). This stageenables us to bring the desired sample area under the FOV of the focus-ing objective and to acquire images of large sample areas by tiling

Fig. 2. Sketch of the multimodal NLO laser-scanning microscope, depicting detectionschemes for SRS and CARS/TPEF modalities in forward direction; DM, dichroic mirror;PMT, photomultiplier tube; AOM, acousto-optic modulator.

consecutivemeasurements. Alternatively, it can also be utilized as a ras-ter scanning stage for acquiring images of the sample without scanningthe beams. This method is particularly important for microscopic tech-niques that are sensitive either to polarization, such as balanced-detec-tion Raman induced Kerr-effect microscopy [34], or to beam pointing,such as broadband Fourier-transform SRS using a birefringent interfer-ometer [33] or broadband SRS spectroscopy using a photonic timestretcher [35].

After the sample, the generated forward CARS, SRS and TPEF signalsare collected by a second identical microscope objective. A dichroicbeam splitter transmits the Stokes beam, bearing the SRG signal, and re-flects the shorter wavelengths, containing CARS/TPEF signals and thepump beam itself. A photomultiplier tube (Hamamatsu: R3896) readsCARS or TPEF signals after spectral filtering by applying appropriatebandpass interferential filters. A balanced photodiode (Thorlabs:PDB210A/M) and a high-speed lock-in amplifier (Zurich Instruments:HF2LI) detect the transmitted SRG signal by a modulation-transferscheme. A reference beam for the balanced photodiode is obtained bysplitting a small portion of the Stokes beam just before the combiner(shown in Fig. 1(a) as ‘Reference arm’). A set of lenses in 4-f configura-tion are placed after the sample to conjugate the back aperture of thecollimating objective with the detectors, thus guaranteeing a propercollection of light within the active areas of the detectors. Our NLO mi-croscope can also be readily reconfigured to work in epi (back-scatter-ing) detection, just by placing a dichroic beam splitter between thetube lens and the focusing objective to reflect epi- CARS, TPEF, SHG orTHG signals, to be collected by their respective PMTs/detectors.

3. Results and Discussion

We first tested our NLO microscope on a sample made of a mixtureof two different polymer beads, namely poly-methyl methacrylate(PMMA)with 6-μmdiameter and polystyrene (PS)with 3-μmdiameter,dispersed on a glass substrate.We simultaneously acquire CARS and SRSimages (with 200 × 200 pixels) in galvanometric-mirrors scanningmode of an 80 × 80 μm2 area of the sample with 3-ms pixel dwelltime. The average pump and Stokes powers on the sample are 12 mW(modulated at 1 MHz) and 5 mW, respectively. Fig. 3(a) and (b) reportthe CARS and SRS images at pump-Stokes frequency detuning of2953 cm−1, in resonance with PMMA vibrational response. Fig. 3(c)and (d) show the CARS and SRS images, at pump-Stokes frequencydetuning of 3060 cm−1, mainly in resonance with PS response. Fig.3(e) and (f) are the overlay images of CARS signals (a, c) and of SRG sig-nals (b, d), respectively. One can clearly see in false colour mapping

Fig. 3. Images (200× 200 pixels) of amixture of 6-μmPMMAbeads and 3-μmPS beads for CARS (a, c) and SRS (b, d)modalities, at 2953 cm−1 (a, b) and 3060 cm−1 (c, d)with 3-ms pixeldwell time. (e, f): overlay images of CARS (a, c) and SRS (b, d) images. CARS (g) and SRS (h) spectra taken at points ‘A’ (in red) and ‘B’ (in green). (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article.)

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(PMMA in red and PS in green) the chemical selectivity of CRS micros-copy. We have also acquired the CARS/SRS spectra of PMMA and PS[Fig. 3(g) and (h)] by parking the microscope at points ‘A’ and ‘B’ (asshown in the panels a–d), respectively. Theywere recorded by scanningthe Stokes wavelength with the help of the motorized translation stageof the fan-out PPLN in ‘Arm 1’. SRS spectra [Fig. 3(h)], being inherentlyfree from NRB, display the main PMMA resonance at 2953 cm−1 andthe two peaks of PS at 3060 cm−1 and 2910 cm−1 with symmetricallineshape and with central positions in good agreement with literature.As expected, CARS spectra [Fig. 3(g)], suffering from interference withNRB, show characteristic dispersive lineshapes and blue-shifted centralpositions of the peaks.

Being NLO techniques, both CARS and SRS have three-dimensional(3D) sectioning capabilities. We performed 3D-CARS chemical imagingfor our composite sample of polymer beads. To this purpose, we ac-quired a set of CARS images (over 50 × 50 μm2 area with 125×125pixels) at different z positionswithin the sample at two frequencydetunings: 2953 cm−1, in resonance with PMMA, and 3060 cm−1, inresonance with PS. In particular, we varied the axial (z) position of thesample throughout the laser focus over 30-μm travel range with a stepsize of 0.5 μm, for a total of 61 steps for each of the two vibrational fre-quencies. Fig. 4(a) shows the overlay of two CARS images collected at aparticular z position for the two frequency detunings (red, PMMA and

Fig. 4. (a) Overlay of two CARS images collected at a fixed z position (acquired at 2953 cm−1 indwell time. (b) 3D reconstruction of the sample volume by rendering the two sets of 61 imagesthe reader is referred to the web version of this article.)

green, PS). Fig. 4(b) shows the resulting 3D reconstruction of the samplevolume, employing the entire acquired dataset.

We further acquired CARS images of the same polymer beads sam-ple at several pump-Stokes frequency detunings in the 2600 to3200 cm−1 range, at 10 cm−1 intervals. We mapped a sample area of50 × 50 μm2 with 125 × 125 pixels, at a fixed z axial position. Wethen performed multivariate curve resolution (MCR) analysis [36,37]on the acquired 125 × 125 × 61 data matrix, obtaining two dominantspectral components related to PMMA and PS [shown in Fig. 5(c)]. Theconcentration maps associated with these two components are plottedin Fig. 5(a) and (b), respectively.

To demonstrate the multimodal imaging capability of the micro-scope on a biological sample, we imaged an unprocessed fresh leaf ofElodea aquatic plant. A small part of the leaf with a drop of water issandwiched between two thin microscope glass slides. Fig. 6(a) showsan SRS image (200 × 200 pixels, 100 × 100 μm2 area) of the sample ob-tained at a Raman shift of 2890 cm−1 (pump, 778 nm and Stokes1004 nm), mainly targeting the cellulose, a constituent molecule forplant cell walls. The (thicker) cell walls mostly made of cellulose canclearly be seen.We also performed TPEFmicroscopy of the same samplearea targeting mainly the intracellular Chlorophyll a, excited by two-photon absorption of pump pulses at 778 nm. This wavelength is wellsuited for the purpose, because Chlorophylls have negligible linear

resonancewith PMMA, red, and 3060 cm−1, in resonancewith PS, green)with 200 μs pixelfor the two resonances. (For interpretation of the references to colour in this figure legend,

Fig. 5.MCR analysis of the recorded hyperspectral CARS dataset (acquired with 200-μs pixel dwell time) of the polymer beads sample. (a, b) Concentration maps of the first and secondcomponents, associated to PMMA and PS, respectively. (c) Retrieved spectral shapes of the two components (PMMA, blue, and PS, red). (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

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absorption at the 778-nm pump wavelength [38,39]. Even if the Stokeswavelength at 1004 nmdoes not contribute to the TPEF process, in com-bination with the pump it can generate CARS or four-wave-mixing sig-nals that can spectrally overlap with TPEF. To avoid these processes, we

Fig. 6. 200 × 200 pixels images of a 100 × 100 μm2 area of an unprocessed fresh leaf of ‘Elodeamainly targeting cellulose, a major constituent of plant cell walls; (b) TPEF image acquired witof the two images.

merely go out of time overlap between the pump and Stokes pulses.Under this situation, the TPEF image acquired in forward direction onthe PMT in the spectral window 600-700 nm is shown in Fig. 6(b).Round shaped Chlorophyll-rich areas can be seen within the

’ aquatic plant; (a) SRS image acquired at pump-Stokes frequency detuning of 2890 cm−1,h pump pulse excitation, depicting round shaped intracellular chloroplasts, (c) an overlay

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intracellular volume. Fig. 6(c) is the overlay of the two, SRS (in red) andTPEF (in green) images.

4. Conclusions

We have presented a multimodal NLO laser-scanning microscope,integrating CARS, SRS and TPEF imagingmodalities on a single platform.The system combines a compact, turnkey and potentially low-cost fiber-format excitation laserwith amicroscope, developed in house using off-the-shelf components, which offers full accessibility to the optical pathand ensures its easy re-configurability and flexibility. This system,while performing very closely to state-of-the-art setups, provides acost-effective alternative to the commercially available expensive mul-timodal nonlinear optical imaging systems. Experimental results pre-sented here on CARS/SRS and TPEF microscopy in forward direction ofpolymer and plant samples, are quite promising in view of applicationsof this microscope to mainstream biomedical problems.

Conflict of Interest

The authors declare no competing financial interest.

Acknowledgements

This work is supported by European Research Council: ConsolidatorGrant VIBRA (ERC-2014-CoG No. 648615), and Advanced Grant STRA-TUS (ERC-2011-AdG No. 291198).

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