D. P. acknowledges funding from the European Union project CRIMSON under Grant Agreement No. 101016923 and the Regione Lombardia project NEWMED under Grant Agreement No. POR FESR 2014-2020. G. C. acknowledges funding from the European Union project GRAPHENE Core3 under grant agreement number 881603. G. C. also acknowledges funding from King Abdullah University of Science and Technology, Grant Award Number: OSR-2016-CRG5-3017-01.

<ol>
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G. C. declares involvement with the company, Cambridge Raman Imaging, which aims to commercialize the broadband SRS microscopy technology. The other authors declare no conflicts of interest.

Broadband SRS microscopy is a powerful imaging technique that offers authentic chemical contrast to identify and disentangle the chemical constituents of a heterogeneous sample. The potential of this analytical tool can be beneficial to several research fields, ranging from materials science to histopathology. The downside of broadband SRS microscopy is the fact that it is technically demanding; the experimentalist not only requires know-how on broadband laser sources but also needs to manipulate the laser pulses to efficiently generate SRS, a signal that, in turn, needs to be measured with sophisticated detection schemes. This paper presents a protocol that describes a workflow to produce chemical maps of mixed chemical compounds using a multiplex broadband SRS microscope. Although the work described may be trivial for some laser physicists and nonlinear microscopists, it may not be the case for readers interested in the benefits of broadband SRS microscopy whose scientific knowledge resides outside these domains. Therefore, we aimed to detail every step to guide the wide audience interested in broadband SRS microscopy.
The protocol at hand started by showing how to prepare a simple yet spectroscopically rich sample composed of several strong and well-known Raman scatterers. We discussed how to obtain the broadband pump and narrowband Stokes beams necessary to set up an SRS microscope. Figure 5C shows a scheme of the SHG and OPO setups. Note that lens f1 focuses the fundamental beam onto LBO1 to generate the SHG, while a dichroic mirror reflects the SHG radiation and transmits the residual fundamental beam. A second lens f2 collimates the SHG beam. As f2 &#62; f1, the SHG beam is expanded by a factor equal to f2/f1. A third lens f3 focuses the expanded SHG beam onto a second Type I LBO crystal (LBO2) cut at &#952; = 90&#176; and &#966; = 29.0&#176;. By pumping LBO2 with the aforementioned SGH (520 nm), radiation within the 680-910 nm range will emerge from LBO2 through difference-frequency generation (DFG), producing two beams: the signal and idler27&#160;(Figure 5D,E). The latter is discarded while the former is amplified in the OPO cavity to deliver the pump pulses employed in the SRS experiments. The pump of the OPO at 520 nm, namely, the SHG beam, should not be confused with the pump of the SRS experiments (i.e., the signal beam of the OPO).
The contrast in SRS microscopy originates from a nonlinear signal generated at the focal spot of the microscope, a signal that demands confining a large number of photons in the sample plane at a given time. This photon confinement is achieved with a high numerical aperture (NA) microscope objective, an array of lenses that also sets the spatial resolution of the system: the higher the NA, the higher the spatial resolution. However, high NA objectives are densely packed with glass, which introduces positive GDD to pulsed radiation, a frequency chirp that ultimately broadens the temporal profile of the pulses39. Thus, the GDD introduced by the microscope objective might increase the duration of the broadband pump pulses, making it even longer than the Stokes temporal envelope and reducing the effective, accessible bandwidth of the Raman signal. Furthermore, this broadening might also introduce a distortion of the spectral profile of the measured SRS spectrum.
In CARS, the spectroscopically relevant signal emerges at wavelengths that differ from those of the excitation fields. A simple photomultiplier tube or charge-coupled device (CCD) camera can be used to integrate the CARS signal in time, summing up thousands of pulses to average out the laser noise. Instead, the SRS signal appears as a faint modulation transfer embedded within a strong and fluctuating laser background. Because this modulation is weak, the laser noise can readily overwhelm it, reducing both the imaging speed and the sensitivity of the SRS microscope. Therefore, before imaging, it is imperative to measure the relative intensity noise (RIN) to determine whether the laser is suitable for high-speed SRS imaging and to select the modulation frequency with the lowest noise. The RIN is defined as the noise power spectral density [&#948;P(f), with W2/Hz units] of the laser normalized by the mean optical power (<img alt="Equation 2" src="/files/ftp_upload/63709/63709eq02.jpg" style="margin: auto;" />)40,41. In other words, the RIN describes the normalized laser fluctuations at different frequencies (Eq [4]).
<img alt="Equation 7" src="/files/ftp_upload/63709/63709eq07.jpg" style="margin: auto;" /> (4)
Thus, the RIN is a parameter of the SRS system that determines the ideal modulation frequency range for the experiments. For example, the olive bar in Figure 8 shows the ideal modulation frequency range for SRS imaging. In the case of narrowband SRS, the user should measure the RIN of both pump and Stokes to choose what beam needs to be modulated to achieve optimum performance. Note from Figure 8, for example, that the Stokes beam has a slightly higher RIN than the pump, implying that the SRG measurements would turn out noisier than their SRL counterparts. In the case of broadband SRS, the beam that should be modulated is the narrowband beam.
The angular dispersion D of the grating expresses the diffraction angle as a function of wavelength, and is defined as the derivative of the grating equation. For the Littrow configuration, the angular dispersion is given by Eq (5).
<img alt="Equation 8" src="/files/ftp_upload/63709/63709eq08.jpg" style="margin: auto;" /> (5)
To get Eq (5), we assumed&#160;&#945; = &#946;, solved Eq (2) for m/d and inserted the result into d&#946;/ d&#955;. Note that in the Littrow configuration, &#946; = sin-1(m&#955;/2d). Within the small-angle approximation, the change in position along the spectrum is fd&#946; &#8776;&#160;dl (Figure 10). Thus, by inserting d&#946; into Eq (5), we can calculate the linear dispersion, a quantity with units of nm mm-1 using Eq (6):
<img alt="Equation 9" src="/files/ftp_upload/63709/63709eq09.jpg" style="margin: auto;" /> (6)
For a diffraction grating operating in the Littrow configuration with 1,851.85 grooves/mm, d = 540 nm. If we use the first-order diffraction of light at ~789 nm, D = 0.0027 rad nm-1. With an f = 750 mm lens, we get a linear dispersion of&#160;&#8776; 0.5&#160;nm mm-1, translating into &#8776; 7.8&#160;cm-1mm-1. Thus, the focal length of the lens determines the &#34;density&#34; of nm per mm at the detector plane: The longer the focal length, the fewer nm per mm obtained, increasing the space between the spectral lines of the broadband pump. Conversely, with shorter focal lengths, there will be more nm per mm at the detector plane, reducing the space occupied by the dispersed pump.
Balanced detection improves the image quality and sensitivity of noisy setups. For example, according to the RIN spectra shown in Figure 8 and considering typical SRS with an amplitude of 1 x 10-5, the unbalanced signal-to-noise ratio (SNR) is &#8776;60. Using balanced detection (i.e., close to the shot-noise), it is possible to attain an SNR of &#8776;145. Figure 11 shows spectra and composite images under balanced and unbalanced conditions.&#160;Naturally, the effects of balanced detection impact the final results of the experiments, namely, the chemical maps. Supported by these results, we emphasize that balanced detection is a powerful technique to counter the detrimental effects of laser fluctuations on image quality. It is worth mentioning that balanced detection is best suited for noisy lasers, such as fiber oscillators. SRS microscopes operating with quiet optical light sources (e.g., solid-state lasers), may not require balanced detection.
The protocol also explains an approach based on nonlinear optics to find the spatiotemporal overlap between the pulses of these beams. We described the advantages of using the 1st instead of the 0th diffraction-order of an AOM as the modulated Stokes beam. Further, the detrimental effects of dispersion on the SRS generation efficiency were described with suggestions of ways to mitigate them via a prism compressor. In addition, the protocol explains how to align the prisms and highlights three critical aspects to consider for optimum performance. We not only discuss the relevance of the RIN for SRS microscopy but also show how to measure it with a lock-in amplifier and, with the RIN spectrum, define the best modulation frequency. With a concrete example, this paper explains how the grating equation helps in designing the detection chain. Finally, the protocol illustrates, with real SRS data, the structure of the SRS hypercube and how to analyze it with a conventionally used scientific programming language.
This protocol has three minor limitations. First, the detection scheme employed in this contribution consists of a nonconventional, multichannel lock-in detector designed and built in-house by Sciortino et al.26&#160;As demonstrated previously25, this detector can be replaced by an off-the-shelf balanced photodiode. Although this modification concerns only the detector and leaves the protocol virtually unchanged, with a single-photodiode, one needs to scan each spectral component on the detector instead of measuring them all at once. Second, this protocol employs inline balanced detection, which requires inserting several optical elements into the beam path. These optical elements increase the system complexity and lead to losses of optical power and pulse broadening.
Inline balanced detection also demands that the two pump replicas pass through the sample, a situation that may not be ideal for light-sensitive samples, such as living cells, or for strongly birefringent ones in which the two pump replicas may experience different optical properties, thus canceling the balanced detection. Third, the protocol relies on a home-built OPO, a device that may not be readily available. However, alternatives to the broadband spectra delivered by the OPO are the supercontinua from nonlinear optical fibers or bulk crystals. The latter could be employed only with low repetition rate lasers (up to 5 MHz). Thus, as with every experimental design, the protocol at hand has some limitations. However, they are minimal and do not compromise the success of this approach.
Although a reference sample is described here, this protocol can successfully disentangle chemical species within cells and animal and plant tissues, such as cellulose, lipidic species, or proteins, finding practical applications in different biochemical quests or as a diagnostics tool in histopathology. Similarly, this protocol can be a valuable tool in material sciences. For example, following this protocol, one can interrogate the molecular composition and concentration of polymeric species42. Furthermore, this methodology is compatible with other nonlinear microscopy techniques, such as broadband microscopy based on pump-probe43&#160;and heterodyne CARS44, four-wave mixing processes that, as with SRS, also require two excitation light beams and modulation-transfer measurements. Finally, some of the information contained in this paper can be applied to nonlinear imaging techniques that do not rely on modulation transfer techniques, but require aligning two or more pulsed laser beams, such as conventional CARS45&#160;and SFG microscopies46.
In summary, this protocol describes a powerful methodology based on broadband SRS microscopy to extract chemical maps and their characteristic SRS spectra from chemically heterogeneous mixtures, delivering datasets that allow straightforward quantitative data analysis. The versatility and simplicity of the method also give the interested reader the possibility to adapt it to different nonlinear techniques.

Raman microscopy is a powerful imaging technique that delivers rich chemical maps by measuring Raman scattering1, an inelastic radiative process that originates from molecules vibrating in response to incident light2,3. Each pixel of a Raman map contains a spectrum that carries direct information on the chemical composition and structure of the sample, resulting in images with intrinsic vibrational contrast. To date, Raman microscopy is the reference standpoint for microspectroscopy studies of molecular vibrations as no other imaging technique can produce images with both high chemical specificity and high spatial resolution4. Despite its outstanding chemical specificity, the generation efficiency of Raman scattering is low, calling for either extended pixel dwell times or high-power excitation, leading, respectively, to low acquisition rates and incompatibility with sensitive samples.
This single deficiency of Raman microscopy led researchers to apply coherent Raman scattering5,6,7,8,9 as a source of contrast for microscopy. This is a nonlinear optical process that enhances the vibrational response by several (up to seven) orders of magnitude, thus allowing high-speed chemical imaging10,11,12,13. In particular, the two most employed coherent Raman scattering techniques are coherent anti-Stokes Raman scattering (CARS)14 and stimulated Raman scattering (SRS)15. In contrast to CARS, SRS shows a linear dependence on the concentration of resonant molecules. It is immune to the nonresonant background, a nonlinear effect unrelated to any vibrational transition but distortive to the Lorentzian shapes characteristic of the Raman spectra of the molecular vibrations16,17. Thus, SRS microscopy yields authentic Raman information that allows direct quantitative image analysis.
SRS is a third-order, nonlinear, optical process that provides direct information on the chemical bonds of a sample. It originates from the spatiotemporal superposition of two optical fields generally in the near-infrared spectral region, namely the pump and the Stokes at frequency &#969;pu and &#969;S, respectively10,11,18. This superposition generates a beating at the pump-Stokes frequency detuning &#937; = &#969;pu-&#969;S. When &#937; matches a molecular vibration &#937;R, the molecule resonates, causing a coherent energy transfer between the light fields and the molecule. As a result, the molecule reaches a vibrationally excited state. This process can be monitored by measuring either the annihilation of pump photons (a signal known as stimulated Raman loss [SRL]) or the concomitant amplification of Stokes photons (a process known as stimulated Raman gain [SRG]). SRG and SRL are small signals (&#916;I) that sit on top of an intense and fluctuating background (I). As typical values of the SRS signal (&#916;I/I) are in the 10-6-10-4 range, the laser noise can easily obscure it. To mitigate the detrimental effects of the laser noise on the signal-to-noise ratio (SNR) and consequently on the imaging speed, SRS detection relies on modulation transfer techniques (e.g., lock-in amplifiers, resonant circuits, or box-car averagers) at high modulation frequencies (&#62;1 MHz), where the laser noise reaches its minimum values15,19,20.
Conventional SRS microscopy employs narrowband (&#8776;10 cm&#8722;1) pump and Stokes pulses to produce chemical images at a single vibrational frequency, allowing video-rate imaging with pixel dwell times as low as &#8776;100 ns21,22. However, because narrowband SRS microscopy forms chemical maps by sequentially scanning the sample at only a few vibrational frequencies, its information is limited23. SRS images with one or two vibrational contrasts may not suffice to differentiate chemical species with overlapping Raman bands, especially within heterogeneous systems. Therefore, the paradigmatic narrowband SRS microscope does not exploit the full potential of SRS, because investigating a handful of vibrational frequencies hinders its chemical specificity and neglects the wealth of information encoded within vibrational spectra. Furthermore, sequential scanning of the sample at different frequencies results in extended pixel dwell times that can trigger photodamage and prevent rigorous spatial coregistration between consecutive images, leading to motion artifacts.
Contrary to its narrowband counterpart, broadband SRS microscopy retrieves a vibrational spectrum per pixel at each sample scan10,12,24. Thus, broadband SRS provides hyperspectral imaging with strict spatial coregistration of different vibrational contrasts, allowing rigorous data analysis. This not only reveals the chemical constituents of the specimen through Raman spectra, but also helps to determine their relative concentrations. Depending on how the spectra are acquired, broadband SRS microscopy is classified either as hyperspectral SRS or multiplex SRS. In hyperspectral SRS, the SRS spectrum per scanned point of the sample is acquired sequentially (i.e., it is retrieved by sweeping the frequency detuning &#8486;), building an SRS spectrum by stacking together the SRS signals at consecutive Raman shifts. The Raman spectrum is measured simultaneously at several vibrational modes in multiplex SRS. Thus, the multiplex SRS approach combines a modulated narrowband pulse with a broadband pulse to drive the SRS signal at different frequencies, and uses a multichannel detector with a sensitivity comparable to that of narrowband SRS to detect the SRS spectra.
This paper presents a protocol to produce chemical maps of heterogeneous samples using multiplex SRS microscopy. A scheme of the SRS microscope employed in this protocol is depicted in Figure 1 and described in detail elsewhere25,26,27. Briefly, a commercial mode-locked Yb fiber laser, producing 140 fs pulses centered at 1040 nm, with 10 W average power and an 80 MHz repetition rate, drives the broadband SRS microscope. A polarizing beam splitter (PBS) separates the fundamental beam into two branches. To produce the narrowband Stokes pulses, one branch with 4 W of the fundamental beam is sent to an etalon that generates a narrowband (&#8776;15 cm-1) beam, which is then modulated at 1.6 MHz with an acoustic optic modulator (AOM). The remaining fraction with 6 W of the fundamental beam is frequency-doubled with a 2.8 mm thick lithium triborate (LBO) crystal, cut for type-I phase matching (&#952; = 90&#176;, &#966; = 13.8&#176;). The resulting second harmonic generation at 520 nm travels to an X-folded cavity to pump an optical parametric oscillator (OPO), a device that uses a 3.0 mm thick LBO crystal (type I phase matching, &#952; = 90&#176;, &#966; = 9.8&#176;) as active medium to deliver a broadband optical radiation tunable within the 680-910 nm spectral region (Figure 2). These broadband pulses serve as the pump in the SRS experiments and propagate to a prism compressor to precompensate the dispersion effects induced by the microscope objective.
After the compression stage, a &#955;/2 waveplate, combined with a YVO4 birefringent plate, produces two orthogonally polarized replicas whose electronic subtraction at the detection plane cancels the noise of the broadband pump. A dichroic mirror combines the pump and Stokes beams and sends them to an upright microscope. A water-immersion objective with a numerical aperture (NA) of 1.27 focuses the light onto the sample, while an oil-immersion objective with a NA of 1.4 collects it. Before the detection stage, a short-pass filter (SPF) removes the modulated Stokes, while a diffraction grating operating in Littrow configuration disperses the transmitted broadband pump. A second PBS2 separates the pump replicas, and a lens focuses them onto two photodiode arrays. The signals from these photodiode arrays are electronically subtracted and sent to a home-built multichannel-lock-in amplifier (M-LIA). The demodulated signal is then normalized by the direct current (DC) readings of one of the photodiode arrays, thus producing the SRL spectrum.
As an exemplary experiment, we image mixtures of several well-known Raman scatterers, each with a unique Raman spectrum. Thus, the protocol starts by describing how to prepare the reference samples. As we detect SRL, we continue to explain how to obtain narrowband Stokes pulses and set up the optical source that delivers the broadband (&#8776;250 cm-1) pump pulses, namely, the homebuilt OPO. The protocol shows the alignment and optimization of the optical beams, describing critical parameters such as the power and spectra of the narrowband Stokes and the broadband pump. The protocol describes in detail the optical path of the broadband pump because it requires special optical elements. It also explains how to find the spatiotemporal overlap between pump-Stokes pulses and shows a practical way to determine the relative intensity noise (RIN), which in turn helps define the best modulation frequency for SRS experiments. Then, we explain the working principle and calibration of the detection chain. Finally, the protocol shows the data acquisition process, chemometrics, and image processing pipeline.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collection objective</td>
			<td>Nikon</td>
			<td>CFI Apo Lambda S 60x Oil, NA=1.4, Nikon</td>
			<td>Oil immersion objective</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Fisher</td>
			<td>043211-KJ</td>
			<td>Quartz, cover slip for microscope slide, 25.4 x 25.4 x 0.15 mm</td>
		</tr>
		<tr>
			<td>Delay line</td>
			<td>Physik Instrumente (PI)</td>
			<td>M-406.6PD</td>
			<td>Precision microtranslation stage, 150 mm travel range</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Merck</td>
			<td>D8418-500ML</td>
			<td>Methylsulfinylmethane, Molecular Biology Grade DMSO, DMSO, Methyl Sulfoxide</td>
		</tr>
		<tr>
			<td>Etalon</td>
			<td>SLS Optics Ltd</td>
			<td>Custom made</td>
			<td>Anti reflective coating at 1,040 nm, Mounted in a 38 mm diameter x 35.5 mm long stainless steel cell with protective dust caps, and a 50 mm diameter &#8216;pinch-clamp&#8217; mounting ring</td>
		</tr>
		<tr>
			<td>Excitation objective</td>
			<td>Nikon</td>
			<td>CFI Plan Apo IR 60XC WI, NA=1.27, Nikon</td>
			<td>Water immersion objective</td>
		</tr>
		<tr>
			<td>Grating</td>
			<td>LightSmyth</td>
			<td>T-1850-800s Series</td>
			<td>High Efficiency Transmission Grating T-1850-800s Series</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Coherent</td>
			<td>Custom made</td>
			<td>Fidelity, HP</td>
		</tr>
		<tr>
			<td>&#955;/2</td>
			<td>Thorlabs</td>
			<td>SAHWP05M-1700</td>
			<td>Mounted superachromatic half-wave plate</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thorlabs</td>
			<td>CM5-PBS203/M</td>
			<td>16 mm Cage-Cube-Mounted Polarizing Beamsplitter Cube,</td>
		</tr>
		<tr>
			<td>PMMA beads</td>
			<td>Merck</td>
			<td>MFCD00198073</td>
			<td>Micro particles based on polymethacrylate</td>
		</tr>
		<tr>
			<td>Prisms</td>
			<td>Crisel</td>
			<td>320-8218</td>
			<td>LASER DISPERSING PRISMS in SF11</td>
		</tr>
		<tr>
			<td>PS beads</td>
			<td>Merck</td>
			<td>72986-10ML-F</td>
			<td>Micro particles based on polystyrene</td>
		</tr>
		<tr>
			<td>YVO4 crystal</td>
			<td>Dr. Sztatecsny GmbH</td>
			<td>Custom made</td>
			<td>&#160;thickness 8 mm, dia 1.00 cm, 1 689,00 689,00 suitable for 1&#34; mount, coated for 850 - 1,100 nm</td>
		</tr>
	</tbody>
</table>

multiplex chemical imaging based on broadband stimulated raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

Figure 3 shows exemplary results obtained using this protocol with PS, PMMA, and olive oil. This rotation of LBO1 will change the refractive index experienced by the SHG field, directly modifying its phase velocity. When the phase velocity of the SHG field matches that of the nonlinear polarization induced in LBO1, the nonlinearly generated field and the nonlinear polarization will be in phase, leading to intense SHG radiation. In other words, tweaking the &#966; angle of LBO1 will allow the user to attain the ideal phase matching condition for SHG. As a Type&#160;I phase matching crystal is used here, the polarization of the SHG beam will be orthogonal to that of the fundamental beam (Figure 5B).
Figure 8 shows the RIN of the optical sources used in this protocol and the shot-noise limit, which is a consequence of the quantum nature of electrons and photons that sets a fundamental limit to the laser noise. The shot-noise limited RIN is computed with <img alt="Equation 5" src="/files/ftp_upload/63709/63709eq05.jpg" style="margin: auto;" />&#160;as shown by Eq (3).
<img alt="Equation 6" src="/files/ftp_upload/63709/63709eq06.jpg" style="margin: auto;" /> (3)
Where h is Planck&#39;s constant, and &#957; is the optical frequency. Thus, the shot noise provides useful guidelines for the electronics design.
Figure 11A and Figure 11C show exemplary data of balanced and unbalanced spectra. Naturally, the effects of balanced detection impact the final results of the experiments, namely, the chemical maps. Figure 11B and Figure 11D show composite images in the unbalanced and balanced conditions, respectively. Successfully implementing the described protocol will help identify and localize the different chemical constituents of a heterogeneous sample and extract their characteristic SRS spectra. Subjecting the hyperspectral data of Figure 12 to chemometric analysis gives Figure 13. Figure 13A shows a composite of the concentration maps of the different chemical constituents of the sample, while Figure 13B shows their characteristic SRS spectra. Note that the data shown in Figure 13A not only allows the user to easily identify the different constituents of the sample but also to perform more quantitative analysis. For example, by using the concentration maps, we could calculate the mean of the fractional concentration of each chemical species: 38% DMSO, 25% PMMA, 14% PS, and 22% Olive oil.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63709/63709fig01.jpg" /> 
Figure 1: Schematic of the broadband SRS microscope employed in this protocol. Abbreviations: PBSx = polarizing beamsplitter; SHG = second harmonic generation module; OPO = optical parametric oscillator; AOM = acousto optic modulator; SPF = short-pass filter; M-LIA: Multichannel lock-in amplifier; DM = dichroic mirror. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63709/63709fig02.jpg" /> 
Figure 2: Spectra of the tunable broadband pump (blue) and the narrowband (red) Stokes beams. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63709/63709fig03.jpg" /> 
Figure 3: Brightfield image of the chemically heterogeneous sample. Note that conventional microscopy does not allow the different constituents to be distinguished. Scale bar = 100 &#181;m. Abbreviations: PS = polystyrene; PMMA = polymethyl methacrylate; DMSO = dimethyl sulfoxide. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63709/63709fig04.jpg" /> 
Figure 4: Modulation of the narrowband Stokes pulses. (A) The transparent blue trace shows the 0th diffracted beam, while the black one shows the corresponding 1st order. (B) Optical setup for optimizing the modulation efficiency of the 1st order diffracted beam and finetuning of the Stokes beam&#39;s spot size prior to arriving at the excitation objective. Abbreviations: AOM = acousto optic modulator; fx = focal length of lens X. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63709/63709fig05.jpg" /> 
Figure 5: Nonlinear optical processes required to drive the OPO. (A) Geometry of the SHG interaction. Two fundamental photons at &#969;1 bring the material system to a high-energy virtual level, from where the material system leaps down to the ground state, emitting a photon at &#969;SHG. (B) Scheme of the SHG experiment. (C) A schematic of the SHG and OPO setups. (D) Geometry of the DFG interaction. A &#969;SHG photon is split into the signal (&#969;Signal) and idler (&#969;Idler) photons. A gain of the signal beam is achieved by feeding back the signal photons and making them resonate in the cavity. (E) Scheme of the DFG experiment. Abbreviations: SMx = spherical mirror (R = 75 mm); OPO = optical parametric oscillator; SHG = second harmonic generation module; DFG = difference-frequency generation; LBO = lithium triborate; OC = oil condenser; DM = dichroic mirror; fx = focal length of lens X. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63709/63709fig06.jpg" /> 
Figure 6: Geometry of the prism compressor. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63709/63709fig07.jpg" /> 
Figure 7: Sum-frequency generation to optimize spatiotemporal overlap. (A) SFG between the pump and Stokes and their respective SHG impinging on a screen. Here, a lens focused the pump and the Stokes beams onto the crystal, while a low-pass filter removed them. (B) Intensity of the SFG between pump and Stokes as a function of time delay. Set the time zero of the SRS setup at the position that maximizes the SFG. The asymmetry of the cross-correlation in B is due to the temporal profile caused by the etalon on the Stokes beam. Abbreviations: SFG = sum-frequency generation; SHG = second harmonic generation module; SRS = stimulated Raman scattering spectroscopy. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63709/63709fig08.jpg" /> 
Figure 8: RIN spectra. The band highlighted in green shows the best spectral region for the SRS experiments. Modulating the Stokes beam at any frequency within this band guarantees that the effects of the laser noise on the SRS signal will be the lowest possible. Abbreviations: RIN = relative intensity noise; SRS = stimulated Raman scattering spectroscopy. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63709/63709fig09.jpg" /> 
Figure 9:&#160;Beam profiles. (A) Pump, (B) Stokes, and (C) pump and Stokes. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63709/63709fig10.jpg" /> 
Figure 10: Assumed geometry for a dispersion grating and a photodiode array detector. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63709/63709fig11.jpg" /> 
Figure 11. The effects of balanced detection. Effects on spectra (A, C) and chemical images (B, D). The composites shown in panels (B) and (D) are the final results of the experiments (i.e., after chemometric analysis of hyperspectral data. See protocol section 4 for details). Scale bars = 10 &#181;m. Abbreviation: SRS = stimulated Raman scattering spectroscopy. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/63709/63709fig12.jpg" /> 
Figure 12: A representative SRS hypercube acquired with broadband SRS microscopy. The x-y plane stores the coordinates of the scanned positions, while each vector along z registers an SRS spectrum. Abbreviation: SRS = stimulated Raman scattering spectroscopy. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/63709/63709fig13.jpg" /> 
Figure 13: Chemometric analysis of hyperspectral SRS data. (A) Composite of the concentration maps of the different constituents of the sample. (B) Characteristic spectra of the chemical species. In both panels, yellow: olive oil, blue: DMSO, cyan: PS, and orange: PMMA. Scale bar = 20 &#181;m (A). Abbreviations: SRS = stimulated Raman scattering spectroscopy; PS = polystyrene; PMMA = polymethyl methacrylate. <a href="https://www.jove.com/files/ftp_upload/63709/63709fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy at JoVE.com

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

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3.7K Views.  Politecnico di Milano. We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Video: Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Stimulated Stokes and Antistokes Raman Scattering in Microspherical Whispering Gallery Mode Resonators

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres can be thought as a store of energy with a huge variety of applications: compact laser sources, highly sensitive biochemical sensors and nonlinear phenomena. A protocol for the fabrication of both the microspheres and coupling system is given. The couplers described here are tapered fibers. Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS). A proof of the cavity-enhanced phenomenon is given by the lack of correlation among the pump, signal and idler: a resonant mode has to exist in order to obtain the pair of signal and idler. In the case of hyperparametric oscillations (four wave mixing and stimulated anti-stokes Raman scattering), the modes must fulfill the energy and momentum conservation and, last but not least, have a good spatial overlap.

Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS).

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Method for Recording Broadband High Resolution Emission Spectra of Laboratory Lightning Arcs

Lightning is one of the most common and destructive forces in nature and has long been studied using spectroscopic techniques, first with traditional camera film methods and then digital camera technology, from which several important characteristics have been derived. However, such work has always been limited due to the inherently random and non-repeatable nature of natural lightning events in the field. Recent developments in lightning test facilities now allow the reproducible generation of lightning arcs within controlled laboratory environments, providing a test bed for the development of new sensors and diagnostic techniques to understand lightning mechanisms better. One such technique is a spectroscopic system using digital camera technology capable of identifying the chemical elements with which the lightning arc interacts, with these data then being used to derive further characteristics. In this paper, the spectroscopic system is used to obtain the emission spectrum from a 100 kA peak, 100 &#181;s duration lightning arc generated across a pair of hemispherical tungsten electrodes separated by a small air gap. To maintain a spectral resolution of less than 1 nm, several individual spectra were recorded across discrete wavelength ranges, averaged, stitched, and corrected to produce a final composite spectrum in the 450 nm (blue light) to 890 nm (near infrared light) range. Characteristic peaks within the data were then compared to an established publicly available database to identify the chemical element interactions. This method is readily applicable to a variety of other light emitting events, such as fast electrical discharges, partial discharges, and sparking in electrical equipment, apparatus, and systems.

Emission spectroscopy techniques have traditionally been used to analyze inherently random lightning arcs occurring in nature. In this paper, a method developed to obtain the emission spectroscopy from reproducible lightning arcs generated within a laboratory environment is described.

Lightning is one of the most common and destructive forces in nature and has long been studied using spectroscopic techniques, first with traditional ...

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence signals were proven cumbersome until the advent of hyperspectral imaging, in which fluorescence sources can be separated from each other as well as from background signals and autofluorescence (given knowledge of their spectral signatures). However, traditional, emission-scanning hyperspectral imaging suffers from slow acquisition times and low signal-to-noise ratios due to the necessary filtering of both excitation and emission light. It has been previously shown that excitation-scanning hyperspectral imaging reduces the necessary acquisition time while simultaneously increasing the signal-to-noise ratio of acquired data. Using commercially available equipment, this protocol describes how to assemble, calibrate, and use an excitation-scanning hyperspectral imaging microscopy system for separation of signals from several fluorescence sources in a single sample. While highly applicable to microscopic imaging of cells and tissues, this technique may also be useful for any type of experiment utilizing fluorescence in which it is possible to vary excitation wavelengths, including but not limited to: chemical imaging, environmental applications, eye care, food science, forensic science, medical science, and mineralogy.

Spectral imaging has become a reliable solution for identification and separation of multiple fluorescence signals in a single sample and can readily distinguish signals of interest from background or autofluorescence. Excitation-scanning hyperspectral imaging improves on this technique by decreasing the necessary image acquisition time while simultaneously increasing the signal-to-noise ratio.

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different materials show variability in resistive switching properties due to the intrinsic nature of the material used, leading to discrepancies in the field because of underlying operation mechanisms. This highlights a need for a reliable technique to understand mechanisms using nanostructural observations. This protocol explains a detailed process and methodology of in&#160;situ nanostructural analysis as a result of electrical biasing using transmission electron microscopy (TEM). It provides visual and reliable evidence of underlying nanostructural changes in real time memory operations. Also included is the methodology of fabrication and electrical characterizations for asymmetric crossbar structures incorporating amorphous vanadium oxide. The protocol explained here for vanadium oxide films can be easily extended to any other materials in a metal-dielectric-metal sandwiched structure. Resistive switching crossbars are predicted to serve the programmable logic and neuromorphic circuits for next-generation memory devices, given the understanding of the operation mechanisms. This protocol reveals the switching mechanism in a reliable, timely, and cost-effective way in any type of resistive switching materials, and thereby predicts the device&#39;s applicability.

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Presented here is a protocol for analyzing nanostructural changes during in&#160;situ biasing with transmission electron microscopy (TEM) for a stacked metal-insulator-metal structure. It has significant applications in resistive switching crossbars for the next generation of programmable logic circuits and neuromimicking hardware, to reveal their underlying operation mechanisms and practical applicability.