IR is grateful to the Azrieli Foundation for the PhD fellowship award.

<ol>
	<li>Koski, K. J., Akhenblit, P., McKiernan, K., Yarger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+determination+of+the+complete+elastic+moduli+of+spider+silks.">Non-invasive determination of the complete elastic moduli of spider silks.</a> Nat. Mater. 12 (3), 262-267 (2013).</li><li>Palombo, F., Madami, M., Stone, N., Fioretto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+mapping+with+chemical+specificity+by+confocal+Brillouin+and+Raman+microscopy.">Mechanical mapping with chemical specificity by confocal Brillouin and Raman microscopy.</a> Analyst. 139 (4), 729-733 (2014).</li><li>Scarcelli, G., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brillouin+optical+microscopy+of+the+human+eye.">In vivo Brillouin optical microscopy of the human eye.</a> Opt. Exp. 20 (8), 9197-9202 (2012).</li><li>Scarcelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncontact+three-dimensional+mapping+of+intracellular+hydromechanical+properties+by+Brillouin+microscopy.">Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy.</a> Nat. Methods. 12 (12), 1132-1134 (2015).</li><li>Traverso, A. J., Thompson, J. V., Steelman, Z. A., Meng, Z., Scully, M. O., Yakovlev, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Raman-Brillouin+microscope+for+chemical+and+mechanical+characterization+and+imaging.">Dual Raman-Brillouin microscope for chemical and mechanical characterization and imaging.</a> Anal. Chem. 87 (15), 7519-7523 (2015).</li><li>Antonacci, G., Foreman, M. R., Paterson, C., T&#246;r&#246;k, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+broadening+in+Brillouin+imaging.">Spectral broadening in Brillouin imaging.</a> Appl. Phys. Lett. 103 (22), 221105 (2013).</li><li>Antonacci, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+plaque+stiffness+by+Brillouin+microscopy+in+experimental+thin+cap+fibroatheroma.">Quantification of plaque stiffness by Brillouin microscopy in experimental thin cap fibroatheroma.</a> J. R. Soc. Interface. 12 (112), 20150483 (2015).</li><li>Grubbs, W. T., MacPhail, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+stimulated+Brillouin+gain+spectrometer.">High resolution stimulated Brillouin gain spectrometer.</a> Rev. Sci. Instrum. 65 (1), 34-41 (1994).</li><li>Ballmann, C. W., Thompson, J. V., Traverso, A. J., Meng, Z., Scully, M. O., Yakovlev, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+Brillouin+scattering+microscopic+imaging.">Stimulated Brillouin scattering microscopic imaging.</a> Sci Rep. 5, 18139 (2015).</li><li>Remer, I., Bilenca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Background-free+Brillouin+spectroscopy+in+scattering+media+at+780+nm+via+stimulated+Brillouin+scattering.">Background-free Brillouin spectroscopy in scattering media at 780 nm via stimulated Brillouin scattering.</a> Opt. Lett. 41 (5), 926-929 (2016).</li><li>Remer, I., Bilenca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+stimulated+Brillouin+scattering+spectroscopy+at+780+nm.">High-speed stimulated Brillouin scattering spectroscopy at 780 nm.</a> APL Photonics. 1 (6), 061301 (2016).</li><li>She, C. Y., Moosm&#252;ller, H., Herring, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+light+scattering+spectroscopy+for+supersonic+flow+measurements.">Coherent light scattering spectroscopy for supersonic flow measurements.</a> Appl. Phys. B-Lasers O. 46 (4), 283-297 (1988).</li><li>Fiore, A., Zhang, j., Peng Shao, ., Yun, S. 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The authors have nothing to disclose.

The system, shown in Figure 1, was designed to be built on an 18&#39;&#39; x 24&#39;&#39; breadboard that can be mounted vertically on an optical table, facilitating placement of watery samples. As a result, it is important to strongly tighten all optical and mechanical elements and ensure that the pump and probe beams are collinear and concentric with the various elements prior to illuminating the sample in off-axis geometry.
Difficulties in observing the stimulated Brillouin...

Spontaneous Brillouin spectroscopy has been established, in recent years, as a valuable approach for the mechanical analysis of soft materials, such as liquids, real tissue, tissue phantoms and biological cells1,2,3,4,5,6,7. In this approach, a single laser illuminates the sample and light that is inelastically scattered from spontaneous thermal acoustic waves in the medium is collected by a spectrometer, providing useful information on the viscoelastic properties of the sample. The spontaneous Brillouin spectrum includes two Brillouin peaks at the acoustic Stokes and anti-Stokes resonances of the material, and a Rayleigh peak at the illuminating laser frequency (due to elastically scattered light). For a Brillouin backscattering geometry, the Brillouin frequencies are shifted by several GHz from the illuminating laser frequency and have spectral width of hundreds of MHz.
While scanning Fabry-Perot spectrometers have been the systems-of-choice for acquiring spontaneous Brillouin spectra in soft matter1,2, recent technological advances in virtually imaged phase array (VIPA) spectrometers have enabled significantly faster (sub-second) Brillouin measurements with adequate spectral-resolution (sub-GHz)3,4,5,6,7. In this protocol, we present the construction of a different, high-speed, high spectral-resolution, accurate Brillouin spectrometer based on the detection of continuous-wave-stimulated-Brillouin-scattering (CW-SBS) light from non-turbid and turbid samples in a nearly back scattering geometry.
In CW-SBS spectroscopy, continuous-wave (CW) pump and probe lasers, slightly detuned in frequency, overlap in a sample to stimulate acoustic waves. When the frequency difference between the pump and probe beams matches a specific acoustic resonance of the material, amplification or deamplification of the probe signal is provided by stimulated Brillouin gain or loss (SBG/SBL) processes, respectively; otherwise, no SBS (de)amplification occurs8,9,10,11. Thus, an SBG (SBL) spectrum can be acquired by scanning the frequency difference between the lasers across the material Brillouin resonances and detecting the increase (decrease), or gain (loss), in the probe intensity due to SBS. Unlike in spontaneous Brillouin scattering, elastic scattering background is inherently absent in SBS, enabling excellent Brillouin contrast in both turbid and non-turbid samples without any need for Rayleigh rejection filters as required in VIPA spectrometers10,11,13.
The main building blocks of a CW-SBS spectrometer are the pump and probe lasers and the stimulated Brillouin gain/loss detector. For high spectral-resolution, high speed CW-SBS spectroscopy, the lasers need to be single-frequency (&#60; 10 MHz linewidth) with sufficiently wide wavelength tunability (20 - 30 GHz) and scanning rate (&#62; 200 GHz/s), long-term frequency stability (&#60;50 MHz/h) and low intensity noise. Furthermore, linearly polarized and diffraction-limited laser beams with powers of few hundreds (tens) of mW on the sample are required for the pump (probe) beam. Finally, the stimulated Brillouin gain/loss detector should be designed to reliably detect weak backward stimulated Brillouin gain/loss (SBG/SBL) levels (10-5 - 10-6) in soft matter. To meet these needs, we selected distributed feedback (DFB) diode lasers coupled to polarization-maintaining fibers along with a stimulated Brillouin gain/loss detector combining an ultra-narrowband atomic vapor notch-filter and a high-frequency single-modulation lock-in amplifier as illustrated in Figure 1. This detection scheme doubles the intensity of the SBG signal while significantly reducing noise in the probe intensity, where the desired SBG signal is embedded11. Note that the role of the atomic vapor notch-filter used in our SBS spectrometer is to significantly reduce the detection of unwanted stray pump reflections rather than to decrease the elastic scattering background as in VIPA spectrometers that detect both spontaneous Rayleigh and Brillouin scattered light. Using the protocol detailed below, a CW-SBS spectrometer can be constructed with the capability of acquiring transmission spectra of water and tissue phantoms with SBG levels as low as 10-6 at &#60;35 MHz Brillouin-shift measurement precision and within 100 ms or less.
<img alt="Figure 1" src="/files/ftp_upload/55527/55527fig1.jpg" /> 
Figure 1: Continuous-wave Stimulated Brillouin Scattering (CW-SBS) Spectrometer. Two continuous-wave pump and probe diode lasers (DL), frequency detuned around the Brillouin shift of the sample, are coupled into polarization-maintaining single-mode fibers with collimators C1 and C2, respectively. The pump-probe frequency difference is measured by detecting the beat frequency between beams peeled from the pump and probe lasers using a set of fiber splitters (FS), a fast photodetector (FPD), and a frequency counter (FC). The S-polarized probe beam (light red), expanded using a Keplerian beam expander (L1 and L2), is right circularly polarized by a quarter-wave plate (&#955;1/4) and focused on the sample (S) by an achromatic lens (L3). For effective SBS interaction and optical isolation, the pump beam (deep red), expanded using a Keplerian beam expander (L5 and L6), is first P-polarized using a half-wave plate &#955;2/4), then transmitted through a polarizing beam splitter (PBS), and is finally left circularly polarized by a quarter-wave plate (&#955;2/4) and focused on the sample with an achromatic lens (L4; same as L3). Note that the pump and probe beams nearly counter-propagate in the sample and that an S-oriented polarizer (P) was used to prevent the P-polarized pump beam (coming out of &#955;1/4) from entering the probe laser. For lock-in detection, the pump beam is sinusoidally modulated at fm with an acousto-optical modulator (AOM). The SBG signal, manifested as intensity variations at frequency fm (see inset), is demodulated with a lock-in amplifier (LIA) following detection by a large-area photodiode (PD). For significant elimination of stray pump reflections in the photodiode, a narrowband Bragg filter (BF) and an atomic notch filter (85RB) around the pump wavelength are used alongside with a light-blocking iris (I). Data is recorded by a data acquisition card (DAQ) connected to a personal computer (PC) for further analysis of the Brillouin spectrum. All folding mirrors (M1-M6) are used to fit the spectrometer on a 18&#39;&#39;&#215;24&#39;&#39; breadboard that is vertically mounted on the optical table for facilitating placement of watery samples. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55527/55527fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Probe diode laser head and controller</td><td>Toptica Photonics</td><td>SYST DL-100-DFB</td><td>Quantity: 1</td></tr><tr><td>Pump amplified diode laser and controller</td><td>Toptica Photonics</td><td>SYST TA-pro-DFB</td><td>Quantity: 1</td></tr><tr><td>FC/APC fiber dock</td><td>Toptica Photonics</td><td>FiberDock&amp;nbsp;</td><td>Quantity: 3</td></tr><tr><td>High power single mode polarization maintaining FC/APC fiber patchcord</td><td>Toptica Photonics</td><td>OE-000796</td><td>Quantity: 1</td></tr><tr><td>FC/APC fiber collimation with adjustable collimation optics</td><td>Toptica Photonics</td><td>FiberOut</td><td>Quantity: 1</td></tr><tr><td>FC/APC fiber fixed collimator</td><td>OZ Optics</td><td>HPUCO-33A-780-P-6.1-AS</td><td>Quantity: 1</td></tr><tr><td>Single mode polarization maintaining fiber splitter 33:67</td><td>OZ Optics</td><td>FOBS-12P-111-4/125-PPP-780-67/33-40-3A3A3A-3-1</td><td>Quantity: 1</td></tr><tr><td>Single mode polarization maintaining fiber splitter 50:50</td><td>OZ Optics</td><td>FOBS-12P-111-4/125-PPP-780-50/50-40-3S3A3A-3-1</td><td>Quantity: 1</td></tr><tr><td>f=25 mm, &amp;Oslash;1/2&quot; Achromatic Doublet, SM05-Threaded Mount, ARC: 650-1050 nm</td><td>Thorlabs</td><td>AC127-025-B-ML</td><td>Quantity: 1</td></tr><tr><td>f=30 mm, &amp;Oslash;1&quot; Achromatic Doublet, SM1-Threaded Mount, ARC: 650-1050 nm</td><td>Thorlabs</td><td>AC254-30-B-ML</td><td>Quantity: 2</td></tr><tr><td>f=50 mm, &amp;Oslash;1&quot; Achromatic Doublet, SM1-Threaded Mount, ARC: 650-1050 nm</td><td>Thorlabs</td><td>AC254-50-B-ML</td><td>Quantity: 1</td></tr><tr><td>f=100 mm, &amp;Oslash;1&quot; Achromatic Doublet, SM1-Threaded Mount, ARC: 650-1050 nm</td><td>Thorlabs</td><td> AC254-100-B-ML</td><td>Quantity: 1</td></tr><tr><td>f=200 mm, &amp;Oslash;1&quot; Achromatic Doublet, SM1-Threaded Mount, ARC: 650-1050 nm</td><td>Thorlabs</td><td> AC254-200-B-ML</td><td>Quantity: 1</td></tr><tr><td>&amp;Oslash;1/2&quot; Broadband Dielectric Mirror, 750-1100 nm</td><td>Thorlabs</td><td> BB05-E03</td><td>Quantity: 4</td></tr><tr><td>&amp;Oslash;1&quot; Broadband Dielectric Mirror, 750-1100 nm</td><td>Thorlabs</td><td> BB1-E03</td><td>Quantity: 2</td></tr><tr><td>1&quot; Polarizing beamsplitter cube, 780 nm</td><td>Thorlabs</td><td> PBS25-780</td><td>Quantity: 1</td></tr><tr><td>&amp;Oslash;1&quot; Linear polarizer with N-BK7 protective windows, 600-1100 nm</td><td>Thorlabs</td><td> LPNIRE100-B</td><td>Quantity: 1</td></tr><tr><td>Shearing Interferometer with a 1-3 mm Beam Diameter Shear Plate</td><td>Thorlabs</td><td>SI035</td><td>Quantity: 1</td></tr><tr><td>6-Axis Locking kinematic optic mount</td><td>Thorlabs</td><td> K6XS</td><td>Quantity: 4</td></tr><tr><td>Compact five-axis platform</td><td>Thorlabs</td><td> PY005</td><td>Quantity: 1</td></tr><tr><td>Pedestal mounting adapter for 5-axis platform</td><td>Thorlabs</td><td> PY005A2</td><td>Quantity: 1</td></tr><tr><td>Polaris low drift &amp;Oslash;1/2&quot; kinematic mirror mount, 3 adjusters</td><td>Thorlabs</td><td> POLARIS-K05</td><td>Quantity: 4</td></tr><tr><td>Lens mount for &amp;Oslash;1&quot; optics</td><td>Thorlabs</td><td> LMR1</td><td>Quantity: 5</td></tr><tr><td>Adapter with external SM1 threads and Internal SM05 threads, 0.40&quot; thick</td><td>Thorlabs</td><td> SM1A6T</td><td>Quantity: 1</td></tr><tr><td>Rotation mount for &amp;Oslash;1&quot; optics</td><td>Thorlabs</td><td> RSP1</td><td>Quantity: 2</td></tr><tr><td>1&quot; Kinematic prism mount</td><td>Thorlabs</td><td> KM100PM</td><td>Quantity: 1</td></tr><tr><td>Graduated ring-activated SM1 iris diaphragm</td><td>Thorlabs</td><td> SM1D12C</td><td>Quantity: 1</td></tr><tr><td>Post-mounted iris diaphragm, &amp;Oslash;12.0 mm max aperture</td><td>Thorlabs</td><td>ID12</td><td>Quantity: 2</td></tr><tr><td>1/2&quot; translation stage with standard micrometer</td><td>Thorlabs</td><td>MT1</td><td>Quantity: 3</td></tr><tr><td>&amp;Oslash;1&quot; Pedestal pillar post, 8-32 taps, L = 1&quot;</td><td>Thorlabs</td><td>RS1P8E</td><td>Quantity: 1</td></tr><tr><td>&amp;Oslash;1&quot; Pedestal pillar post, 8-32 taps, L = 1.5&quot;</td><td>Thorlabs</td><td>RS1.5P8E</td><td>Quantity: 2</td></tr><tr><td>&amp;Oslash;1&quot; Pedestal pillar post, 8-32 taps, L = 2&quot;</td><td>Thorlabs</td><td>RS2P8E</td><td>Quantity: 4</td></tr><tr><td>&amp;Oslash;1&quot; Pedestal pillar post, 8-32 taps, L = 2.5&quot;</td><td>Thorlabs</td><td>RS2.5P8E</td><td>Quantity: 1</td></tr><tr><td>&amp;Oslash;1&quot; Pedestal pillar post, 8-32 taps, L = 3&quot;</td><td>Thorlabs</td><td>RS3P8E</td><td>Quantity: 4</td></tr><tr><td>Short clamping fork</td><td>Thorlabs</td><td>CF125</td><td>Quantity: 12</td></tr><tr><td>Mounting base</td><td>Thorlabs</td><td>BA1S</td><td>Quantity: 8</td></tr><tr><td>Large V-Clamp with PM4 Clamping Arm, 2.5&quot; Long, Imperial</td><td>Thorlabs</td><td>VC3C</td><td>Quantity: 1</td></tr><tr><td>&amp;Oslash;1/2&quot; Post holder, spring-loaded hex-locking thumbscrew, L = 1&quot;</td><td>Thorlabs</td><td>PH1</td><td>Quantity: 2</td></tr><tr><td>&amp;Oslash;1/2&quot; Post holder, spring-loaded hex-locking thumbscrew, L = 1.5&quot;</td><td>Thorlabs</td><td>PH1.5</td><td>Quantity: 2</td></tr><tr><td>&amp;Oslash;1/2&quot; Post holder, spring-loaded hex-locking thumbscrew, L = 2&quot;</td><td>Thorlabs</td><td>PH2</td><td>Quantity: 6</td></tr><tr><td>&amp;Oslash;1/2&quot; Optical post, SS, 8-32 setscrew, 1/4&quot;-20 tap, L = 1&quot;</td><td>Thorlabs</td><td>TR1</td><td>Quantity: 2</td></tr><tr><td>&amp;Oslash;1/2&quot; Optical post, SS, 8-32 setscrew, 1/4&quot;-20 tap, L = 1.5&quot;</td><td>Thorlabs</td><td>TR1.5</td><td>Quantity: 2</td></tr><tr><td>&amp;Oslash;1/2&quot; Optical post, SS, 8-32 setscrew, 1/4&quot;-20 tap, L = 2&quot;</td><td>Thorlabs</td><td>TR2</td><td>Quantity: 6</td></tr><tr><td>Aluminum breadboard 18&quot; x 24&quot; x 1/2&quot;, 1/4&quot;-20 taps</td><td>Thorlabs</td><td>MB1824</td><td>Quantity: 1</td></tr><tr><td>12&quot; Vertical bracket for breadboards, 1/4&quot;-20 holes, 1 piece</td><td>Thorlabs</td><td>VB01</td><td>Quantity: 2</td></tr><tr><td>Si photodiode, 40 ns Rise time, 400 - 1100 nm, 10 mm x 10 mm active area</td><td>Thorlabs</td><td> FDS1010</td><td>Quantity: 1</td></tr><tr><td>Waveplate, zero order, 1/4 wave 780nm</td><td>Tower Optics</td><td>Z-17.5-A-.250-B-780</td><td>Quantity: 2</td></tr><tr><td>Waveplate, zero order, 1/2 wave 780nm</td><td>Tower Optics</td><td>Z-17.5-A-.500-B-780</td><td>Quantity: 1</td></tr><tr><td>Fiber coupled ultra high speed photodetector</td><td>Newport</td><td>1434</td><td>Quantity: 1</td></tr><tr><td>Gimbal optical miror mount</td><td>Newport</td><td>U100-G2H ULTIMA</td><td>Quantity: 3</td></tr><tr><td>linear stage with 25 mm travel range</td><td>Newport</td><td>&amp;nbsp;M-423&amp;nbsp;</td><td>Quantity: 1</td></tr><tr><td>Lockable differential micrometer, 25 mm coarse, 0.2 mm fine,11 lb. load</td><td>Newport</td><td>&amp;nbsp;DM-25L</td><td>Quantity: 1</td></tr><tr><td>XYZ Motor linear stage</td><td>Applied Scientific Instrumentation</td><td>LS-50</td><td>Quantity: 3</td></tr><tr><td>Stage controller</td><td>Applied Scientific Instrumentation</td><td>MS-2000</td><td>Quantity: 1</td></tr><tr><td>Sample holder</td><td>Home made</td><td>Custom</td><td>Quantity: 1</td></tr><tr><td>Rubidium 85 Fused Silica spectroscopy cell with flat AR-coated windows, 150 mm length, 25mm diameter</td><td>Photonics Technologies</td><td>SC-RB85-25x150-Q-AR</td><td>Quantity: 1</td></tr><tr><td>Thermally conductive pad 300 mm x 300 mm</td><td>BERGQUIST</td><td>Q3AC 300MMX300MM SHEET</td><td>Quantity: 1</td></tr><tr><td>Heat tape 0.15 mm x 2.5&amp;nbsp; mm x 5 m, 4.29&amp;nbsp; W/m</td><td>KANTHAL</td><td>8908271</td><td>Quantity: 1</td></tr><tr><td>Polytetrafluoroethylene tape 1/2&#039;&#039; x 12 m</td><td>Teflon tape</td><td>R.G.D</td><td>Quantity: 1</td></tr><tr><td>Reflecting Bragg grating bandpass filter</td><td>OptiGrate</td><td>SPC-780</td><td>Quantity: 1</td></tr><tr><td>High frequncy aousto optic modulator</td><td>Gooch and Housego</td><td>15210</td><td>Quantity: 1</td></tr><tr><td>Aousto optic modulator RF driver, frequncy: 210 MHz&amp;nbsp;</td><td>Gooch and Housego</td><td>MHP210-1ADS2-A1</td><td>Quantity: 1</td></tr><tr><td>High frequncy lock-in amplifier&amp;nbsp;</td><td>Stanford Research Systems</td><td>SR844</td><td>Quantity: 1</td></tr><tr><td>Frequency counter</td><td>Phase Matrix</td><td>EIP 578B</td><td>Quantity: 1</td></tr><tr><td>Arbitrary function Generator</td><td>Tektronix</td><td>AFG2021</td><td>Quantity: 2</td></tr><tr><td>Data acquisition (DAQ) module</td><td>National Instruments</td><td>NI USB-6212 BNC</td><td>Quantity: 1</td></tr><tr><td>Data acquisition (DAQ) software&amp;nbsp;</td><td>National Instruments</td><td>LabVIEW 2014</td><td>Quantity: 1</td></tr><tr><td>Regulated DC power supply&amp;nbsp; dual 0-30V 5A</td><td>MEILI</td><td>MCH-305D-ii</td><td>Quantity: 1</td></tr><tr><td>Thermocouple</td><td>MRC</td><td>TP-01</td><td>Quantity: 1</td></tr><tr><td>Thermometer</td><td>MRC</td><td>TM-5007</td><td>Quantity: 1</td></tr><tr><td>Coaxial low pass filter DC-1.9 MHz</td><td>Mini Circuits</td><td>BLP-1.9+</td><td>Quantity: 1</td></tr><tr><td>20% lipid-emulsion</td><td>Sigma-Aldrich</td><td>I141-100ml</td><td>Quantity: 1</td></tr><tr><td>round 25 mm diameter cover glass thick:1 #</td><td>Menzel Glaser</td><td>150285</td><td>Quantity: 1</td></tr><tr><td>Computational software&amp;nbsp;</td><td>MathWorks</td><td>MATLAB 2015a</td><td></td></tr></tbody></table>

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.

Figures 2b and 3b display typical point SBG spectra of distilled water and lipid-emulsion tissue phantom samples (with 2.25 scattering events and an attenuation coefficient of 45 cm-1) measured within 10 ms and 100 ms, respectively. For comparison, we measured the SBG spectra in 10 s as shown in Figures 2a and 3a. In these measurements, the rubidium-85 vapor cell was heated to 90 &#176;C for attenuating stray p...

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

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 ...

high-speed-continuous-wave-stimulated-brillouin-scattering

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JoVE Journal

Engineering

10.1K Views.  Ben-Gurion University of the Negev. 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. 

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

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

Bioengineering

Fabrication And Characterization Of Photonic Crystal Slow Light Waveguides And Cavities

Slow light has been one of the hot topics in the photonics community in the past decade, generating great interest both from a fundamental point of view and for its considerable potential for practical applications. Slow light photonic crystal waveguides, in particular, have played a major part and have been successfully employed for delaying optical signals1-4 and the enhancement of both linear5-7 and nonlinear devices.8-11
Photonic crystal cavities achieve similar effects to that of slow light waveguides, but over a reduced band-width. These cavities offer high Q-factor/volume ratio, for the realization of optically12 and electrically13 pumped ultra-low threshold lasers and the enhancement of nonlinear effects.14-16 Furthermore, passive filters17 and modulators18-19 have been demonstrated, exhibiting ultra-narrow line-width, high free-spectral range and record values of low energy consumption.
To attain these exciting results, a robust repeatable fabrication protocol must be developed. In this paper we take an in-depth look at our fabrication protocol which employs electron-beam lithography for the definition of photonic crystal patterns and uses wet and dry etching techniques. Our optimised fabrication recipe results in photonic crystals that do not suffer from vertical asymmetry and exhibit very good edge-wall roughness. We discuss the results of varying the etching parameters and the detrimental effects that they can have on a device, leading to a diagnostic route that can be taken to identify and eliminate similar issues.
The key to evaluating slow light waveguides is the passive characterization of transmission and group index spectra. Various methods have been reported, most notably resolving the Fabry-Perot fringes of the transmission spectrum20-21 and interferometric techniques.22-25 Here, we describe a direct, broadband measurement technique combining spectral interferometry with Fourier transform analysis.26 Our method stands out for its simplicity and power, as we can characterise a bare photonic crystal with access waveguides, without need for on-chip interference components, and the setup only consists of a Mach-Zehnder interferometer, with no need for moving parts and delay scans.
When characterising photonic crystal cavities, techniques involving internal sources21 or external waveguides directly coupled to the cavity27 impact on the performance of the cavity itself, thereby distorting the measurement. Here, we describe a novel and non-intrusive technique that makes use of a cross-polarised probe beam and is known as resonant scattering (RS), where the probe is coupled out-of plane into the cavity through an objective. The technique was first demonstrated by McCutcheon et al.28 and further developed by Galli et al.29

Use of photonic crystal slow light waveguides and cavities has been widely adopted by the photonics community in many differing applications. Therefore fabrication and characterization of these devices are of great interest. This paper outlines our fabrication technique and two optical characterization methods, namely: interferometric (waveguides) and resonant scattering (cavities).

Slow light has been one of the hot topics in the photonics community in the past decade, generating great interest both from a fundamental point of view ...

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

Chemistry

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. ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

Optimizing Brillouin Optical Time-Domain Analyzers Based on Gain Spectrum Engineering

Demonstrated is a unique method for sensing performance enhancement in Brillouin optical time-domain analyzers (BOTDA). A Brillouin gain spectrum (BGS) is superimposed with two symmetric Brillouin loss spectra (BLS). This leads to a complex engineered spectrum shape that is more resistant to the sensing system noise. Instead of only one pump and probe interaction as in the conventional BOTDA setup, three optical probe waves are exploited, with one probe located in the BGS and the other two symmetrically in the BLS. Due to the resistance and insensitivity of the engineered spectrum shape to the noise, the sensing performance is enhanced by 60% and the measurand resolution is doubled.

A protocol for Brillouin optical time-domain analyzers based on gain spectrum engineering is presented. Enhancements in the sensing performance, including sensing range and measurand resolution are achieved and the excess Brillouin intensity noise is studied. The protocol introduces a new way to enhance distributed Brillouin sensing performance.

Demonstrated is a unique method for sensing performance enhancement in Brillouin optical time-domain analyzers (BOTDA). A Brillouin gain spectrum (BGS) is ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

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.

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 ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...