HHS Public Access Author manuscript Author Manuscript
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
High Speed sub-GHz Spectrometer for Brillouin Scattering Analysis Kim Berghaus1, Seok H. Yun2,3, and Giuliano Scarcelli1,* 1Fischell
Department of Bioengineering, JH Kim Engineering Building, University of Maryland, College Park, Maryland 20742, USA
Author Manuscript
2Harvard
Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 65 Landsdowne St., Cambridge, Massachusetts 02139, USA
3The
Harvard-MIT Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
Abstract
Author Manuscript
Brillouin spectroscopy allows non-contact, direct readout of viscoelastic properties of a material and has been a useful tool in material characterization1, structural monitoring2 and environmental sensing3,4. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis which require high illumination power and long acquisition times, which prevents using this technique in biomedical applications. Our newly developed spectrometer overcomes this challenge by employing two virtually imaged phased arrays (VIPAs) in a cross-axis configuration, which enables us to do sub-GHz resolution spectral analysis with sub-seconds acquisition time and illumination power within the safety limits of biological tissue application5. This improvement allows for multiple applications of Brillouin spectroscopy, which are now being broadly explored in biological research and clinical application6.
Introduction
Author Manuscript
Brillouin scattering, first described by Brillouin8 in 1922, is the inelastic scattering of light from the thermal acoustic modes in a solid and from the random thermal density fluctuations in a liquid or gas. The spectral shift of the scattered light, usually in the sub GHz-range, gives information about the interaction between the incident light and the acoustic phonons in a sample. As a result, in can provide useful information regarding the viscoelastic properties of the material under examination. In its spontaneous version, Brillouin scattering generally has cross-sections in the order of Raman scattering, so it is a very weak signal; and, Brillouin frequency shifts are orders of magnitude smaller than Raman shifts; as a result, elastically scattered light (from Rayleigh or Mie scattering) or stray light, or backreflections off of the sample easily overshadow Brillouin spectral signature. Therefore, in order to accurately measure the Brillouin spectrum, a spectrometer needs to not only achieve sub-GHz spectral resolution but also high spectral contrast.
*
[email protected]. A complete version of this article that includes the video component is available at http://dx.doi.org/10.3791/53468.
Berghaus et al.
Page 2
Author Manuscript
In common Brillouin spectrometers, these requirements are met by scanning-grating monochromators, optical beating methods, and, most popularly, by multiple-pass scanning Fabry-Perot interferometers5. All these methods measure each spectral component sequentially which results in acquisition time of a single Brillouin spectrum of a few minutes to several hours, depending on the instrument. Here, we show that the two-stage VIPA spectrometer, has the ability of collecting all spectral components simultaneously, within less than a second with sufficient extinction (>60 dB) to effectively suppress other spurious signals5.
Instrument Overview
Author Manuscript
The integration of the VIPA etalons is the key element of the spectrometer. A VIPA is a solid etalon with three different coating areas; a highly reflective (HR) coating at the front, a partially reflective coating at the back, and a narrow anti-reflection coating strip at the front which allows the light to enter the VIPA. When the light beam is focused onto the narrow entrance of the slightly tilted VIPA the beam gets reflected into sub-components with fixed phase difference within the VIPA5. Due to the interference of the sub components, high spectral dispersion is achieved. Aligning two VIPAs sequentially in cross-axis configuration introduces spectral dispersion in orthogonal directions4. The spectral dispersion in orthogonal directions spatially separates the Brillouin peaks from crosstalk which allows us to block out the crosstalk with masks. Figure 1 displays a schematic of the two stage VIPA spectrometer. The arrows below the optical elements indicate the degree of freedom in which the translational stages should be oriented in.
Author Manuscript
The following protocol describes how to build and use a two-stage VIPA spectrometer. The spectrometer can be used in combination with a variety of standard optical probes (e.g. confocal microscope, endoscope, slit-lamp ophthalmoscope), as it has been shown recently (REF). The description of these optical setups is however outside the scope of this protocol.
Protocol A single-longitudinal mode laser is required for Brillouin spectral analysis. To align the spectrometer, a strongly attenuated portion of this laser beam is utilized. 1. Initial setup of fiber and CCD camera
Author Manuscript
1.
Find about 2000 mm free space to align and mount the camera at the end. Turn the camera on and disable the gain. Set a low integration time (0.1s). Adjust laser power using optical density filters to avoid camera saturation.
2.
Mount the fiber collimator about 1600 mm in front of the camera. Check if the beam is collimated. Place a pinhole in front of the fiber collimator. Adjust the height of the pinhole to the beam. Move the pinhole along the beam path. Use the adjustment screws of the fiber collimator mount until the beam cleanly passes through the pinhole along the entire beam path.
3.
Mount a matched achromatic pair (f =30 mm) in front of the camera.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 3
4.
Author Manuscript
Mount the horizontal mask on top of a translation stage which can slide the filter in and out of the beam, as indicated in figure 1. Check if horizontal mask is in focus by imaging the edge of the filter onto the camera. If the edges do not appear sharp, slightly adjust the spatial filter along the beam path to obtain a sharp edge image on the CCD.
2. Horizontal Stage of Spectrometer
Author Manuscript Author Manuscript
1.
Mount the spherical lens (f=200mm) S1 600 mm in front of the horizontal spatial filter. Prior to mounting the lens, place two pinholes, aligned with the optical beam, before and after the spherical lens position. When you mount the lens, make sure the beam and the back reflection off of the lens go through the two pinholes.
2.
Mount VIPA2 in the focal plane of the spherical lens. Make sure to mount the VIPA such that the translation stage can move the VIPA in and out of the beam, as indicated in figure 1. At this stage, slide the VIPA out of the beam path.
3.
Mount and align the spherical lens S2 (f=200mm) 200 mm after the VIPA and 200 mm in front of the horizontal spatial filter. Make sure to mount the spherical lens on a translation stage with the degree of freedom oriented along the optical axis.
4.
Use the translation stage to slide the VIPA2 entrance into the beam path. Observe vertical lines on the camera image. Use the translation stage of the S2 to put the lines in focus.
5.
To adjust the spectrum you have two degrees of freedom: horizontal translation to tune the entrance position of the beam into the etalon, and horizontal tilt to tune the input angle of the beam into the etalon.
6.
Measure Finesse by dividing the distance between two vertical lines by their full width at half maximum. Aim for >40. You will find that there is a tradeoff between Finesse and throughput. Measure throughput by measuring power directly before and after VIPA. Aim for >50%.
7.
If Finesse and throughput is not satisfactory go back and fine adjust the alignment. Make sure the VIPA is in the focal plane of the first lens. The beam waist needs to be within the VIPA. Repeat step 4–6.
3. Vertical Stage of Spectrometer
Author Manuscript
1.
Slide the VIPA (VIPA2) aligned in the first part out of the beam using the translation stage. The horizontal stage of the spectrometer now behaves as a 1:1 imaging system.
2.
Mount the vertical spatial filter on top of a vertical translation stage 200 mm in front of S1. Check if the vertical filter is in focus by looking at the sharpness of its imaged edges. Adjust the position of the filter along the beam path until edges are sharp.
3.
Mount and align height, tilt and lateral position of the cylindrical lens (f=200mm) C1 600 mm in front of the horizontal spatial filter.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 4
Author Manuscript Author Manuscript
4.
Mount VIPA1 on top of a vertical translation stage in the focal plane of the cylindrical lens. Mount the VIPA such that the translation stage can move the VIPA in and out of the beam path.
5.
Mount and align the cylindrical lens C2 (f=200mm) 200 mm after VIPA1 and 200 mm in front of the vertical spatial filter. Make sure to mount the cylindrical lens on a translation stage with the degree of freedom oriented along the optical axis as indicated in figure 1.
6.
Use the vertical translation stage to slide the VIPA1 entrance into the beam. Observe vertical lines on the camera image. Use the translation stage of the second cylindrical lens to put the lines in focus.
7.
To adjust the spectrum you have two degrees of freedom: vertical translation to tune the entrance position of the beam into the etalon, and vertical tilt to tune the input angle of the beam into the etalon
8.
Measure Finesse by dividing the distance between two horizontal lines by their full width at half maximum. Aim for >50. You will find that there is a tradeoff between Finesse and throughput. Measure throughput by measuring power directly before and after VIPA. Aim for >50%.
9.
If Finesse and throughput is not satisfactory go back and fine adjust the VIPA mount. Make sure the VIPA is in the focal plane of the first lens. The beam waist needs to be within the VIPA. Repeat step 6–8.
4. Combination of the Two Stages and Final Alignment
Author Manuscript
1.
Slide in VIPA2. Instead of lines you will see horizontally and vertically spaced dots. These dots are from the laser. Adjust the translational stages of the VIPAs until you can see the laser modes clearly. Measure the Finesse of the two stage spectrometer by dividing the diagonal distance between the laser modes by its full width at half maximum.
2.
Build a black box around your spectrometer using construction rails and Blackout Fabric. Allow access (while in the dark) to both VIPAS and both masks.
3.
Connect the fiber to an optical probe carrying Brillouin scattering signal such as a confocal microscope.
5. Measuring the Brillouin shift
Author Manuscript
1.
Close both, the vertical and the horizontal spatial filter, until the laser signature disappear. In order to do so, move the translation stages accordingly.
2.
Enable the gain and increase the integration time of your camera as much as possible without saturating the camera.
3.
Observe the Brillouin shift of your sample.
4.
To optimize the spectrometer throughput, open one mask at a time and scan through the spectrometer orders tilting the VIPA and adjusting its translation stage.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 5
Author Manuscript
Find the order in which your signal appears the strongest. Close mask again until laser signal disappear. Repeat with the other mask and VIPA. 5.
Take measurement of your sample by taking an image of the spectrum using the Andor software provided with the camera. Save the image in TIF format.
6.
Since you have removed the laser signatures, you will need a calibration to convert the pixel separation in Brillouin shifts. Take calibration measurement of two samples with known Brillouin shift.
6. Calibration and Analysis of Brillouin Spectrum
Author Manuscript
Export the data into Matlab. Use the calibration measurements to determine the free spectral range (FSR) and the CCD-pixel to optical frequency conversion ratio (PR) in the Brillouin spectrometer. To determine these parameters, fit the known Brillouin shifts of for example water (7.46 GHz) and glass (29.3 GHz; The shift of glass is aliased into the FSR of 20 GHz and can be treated as a shift of 9.3 GHz for calibration purposes). Overlap the CCD-frame of both calibration samples (figure 1a). Take a line scan across the frame and interpolate your data. Fit Lorentzian curves through each of the peaks to determine the peak position in terms of pixels. Figure 1b displays four distinct peaks; the two inner peaks corresponding to glass and the two outer peaks corresponding to water. Determine FSR and PR by using the following equations, where PGlass-S, PGlass-AS and PWater-S denote the measured value of Brillouin peaks in pixel units and ΩGlass-S and ΩWater-S represent the actual value of Brillouin peaks in GHz units.
Author Manuscript
Determine the Brillouin peak of the sample in pixel units PX by Lorentzian curve fitting. Use the FSR and PR to calculate the Brillouin shift of your sample using the following equations.
Author Manuscript
7. Representative Results Figure 2 shows representative Brillouin spectra and their fits for different materials. Our VIPAs both have a thickness of 5 mm which results in a FSR of about 20 GHz. The integration time for the following measurements were 0.1s. 100 measurements were taken and averaged. One calibration measurement was taken prior to acquiring the following spectra.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 6
8. Discussion
Author Manuscript
The obtained Brillouin shifts agree with previously published data4,8,9. Due to the blocking of the Rayleigh scattering it is impossible to use the laser modes as reference for the spectrum. Any drift of the laser frequency due to temperature fluctuations or other reasons therefore remains undetected and affects the accuracy of the measurements. In addition, the spectral dispersion along the dispersion axis is not linear. The lower the interference order, the higher is the nonlinearity. These sources of error can be minimized by taking calibration measurements frequently.
Author Manuscript
Given the low scattering cross section, for applications in biological materials where integration time and laser power must be kept as low as possible, it is important to maximize throughput. On the other hand, finesse must be maximized as well to achieve high spectral contrast. There is a trade-off between the two of them, as both are affected by the tilt of the VIPA. The larger the tilt, the more light can enter the VIPA, thus the higher is the throughput. However, as the tilt increases, the number of bounces within the VIPA decreases, which reduces the interference between the components and decreases the finesse. Due to its low acquisition time and illumination power such a spectrometer can be used for measuring biomedical materials noninvasively, enabling Brillouin spectroscopy to become a useful tool through high resolution in vivo-mechanical imaging4,6,10,11. Several groups using this type of spectrometer have demonstrated a variety of applications such as measuring the rheological properties of the eye lens12, detecting bacterial meningitis in spinal fluid13, and analyzing the corneal elastic modulus14.
Author Manuscript
Since the spectrometer can be used in combination with a variety of standard optical probes for example confocal microscopes, endoscopes and slit-lamp ophthalmoscopes we expect many more new biomedical applications making use of the two-stage VIPA spectrometer configuration in the future.
References
Author Manuscript
1. Dil JG. Brillouin-scattering in condensed matter. Rep Prog Phys. 1982; 45:285–334. 2. Culshaw B, Michie C, Gardiner P, McGown A. Smart structures and applications in civil engineering. Proc IEEE. 1996; 84:78–86. 3. Eloranta, EW. High spectral resolution lidar. In: Weitkamp, C., editor. Lidar: Range-Resolved Optical Remote Sensing of the Atmosphere. Springer-Verlag; New York: 2005. p. 143-164. 4. Scarcelli G. Confocal Brillouin microscopy for three-dimensional mechanical imaging. Nat Phot. 2008; 2:39–43. 5. Scarcelli G, Yun SH. Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy. Opt Exp. 2011; 19(11):10913–10922. 6. Scarcelli G, Yun SH. In vivo Brillouin optical microscopy of the human eye. Opt Exp. 2012; 20(8): 9197–9202. 7. Brillouin L. Diffusion de la lumiere et des rayonnes X par un corps transparent homogene; influence del’agitation thermique. Ann Phys (French). 1922; 17:88–122. 8. Koski KJ, Yarger JL. Brillouin imaging. Appl Phys Lett. 2005; 87:061903. 9. Faris GW, Jusinski LE, Hickman AP. High-resolution stimulated Brillouin gain spectroscopy in glasses and crystals. J Opt Soc Am B. 1993; 10:587–599.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 7
Author Manuscript
10. Antonacci G. Spectral broadening in Brillouin imaging. Appl Phys Lett. 2013; 103:221105. 11. Meng Z, Yakovlev V. Background clean-up in Brillouin microspectroscopy of scattering medium. Opt Exp. 2014; 22(5):5410–5415. 12. Reiss S, Stolz H. Spatially resolved Brillouin spectroscopy to determine the rheological properties of the eye lens. Biomed Opt Express. 2011; 2(8):2144–2159. [PubMed: 21833354] 13. Steelman Z, Meng Z, Traverso AJ, Yakovlev VV. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis. J Biophotonics. 2014; 9999:1–7. 14. Scarcelli, G.; Yun, SH. Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus. 15. Scarcelli G, Kling S, Quijano E, Pineda R, Marcos S, Yun SH. Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus. Invest Ophthalmol Vis Sci. 2013; 54:1418–1425.10.1167/iovs.12-11387 [PubMed: 23361513]
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 8
Author Manuscript
Summary The goal of this protocol is to build a parallel high-extinction and high-resolution optical spectrometer to enable rapid Brillouin scattering analysis. Using VIPA etalons we can achieve a measurement speed that is of more than 1000 times faster than traditional scanning Fabry-Perot spectrometers, which enables Brillouin analysis of biomaterials and biological tissue at low power levels in vivo.
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 9
Author Manuscript Author Manuscript
Fig. 1.
An optical fiber delivers the Brillouin scattering into the spectrometer. A cylindrical lens (f=200mm) C1 focuses the light into the entrance of the first VIPA (VIPA1). Another cylindrical lens (f=200mm) C2maps the spectral angular dispersion into a spatial separation in the focal plane of C2. In this plane, a vertical mask is used to select the desired portion of the spectrum. An analogous configuration, but tilted at 90 degrees, then follows. The beam passes through a spherical lens (f=200mm) S1 and is focused into the entrance slit of the second VIPA (VIPA2). A spherical lens (f=200mm) S2 finally creates the 2D spectrally separated pattern in its focal plane, where another, horizontal, mask is placed. The horizontal mask is imaged onto the camera using an achromatic pair lens pair..
Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 10
Author Manuscript Author Manuscript
Figure 1.
Spectrometer calibration. (a) CCD frame obtained from calibration sample. (b) Lorentzian curve fit (red) to the measured data (blue).
Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 11
Author Manuscript Figure 2.
Brillouin Spectra of different materials. Lorentzian curve fit (red) to the measured data (blue). (a) Brillouin spectrum of Methanol. The measured Brillouin shift is 5.56 GHz. (b) Brillouin spectrum of Ethanol. The measured Brillouin shift is 5.85 GHz. (c) Brillouin spectrum of Polystyrene. The measured Brillouin shift is 14.12 GHz..
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
High Speed sub-GHz Spectrometer for Brillouin Scattering Analysis Kim Berghaus1, Seok H. Yun2,3, and Giuliano Scarcelli1,* 1Fischell
Department of Bioengineering, JH Kim Engineering Building, University of Maryland, College Park, Maryland 20742, USA
Author Manuscript
2Harvard
Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 65 Landsdowne St., Cambridge, Massachusetts 02139, USA
3The
Harvard-MIT Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
Abstract
Author Manuscript
Brillouin spectroscopy allows non-contact, direct readout of viscoelastic properties of a material and has been a useful tool in material characterization1, structural monitoring2 and environmental sensing3,4. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis which require high illumination power and long acquisition times, which prevents using this technique in biomedical applications. Our newly developed spectrometer overcomes this challenge by employing two virtually imaged phased arrays (VIPAs) in a cross-axis configuration, which enables us to do sub-GHz resolution spectral analysis with sub-seconds acquisition time and illumination power within the safety limits of biological tissue application5. This improvement allows for multiple applications of Brillouin spectroscopy, which are now being broadly explored in biological research and clinical application6.
Introduction
Author Manuscript
Brillouin scattering, first described by Brillouin8 in 1922, is the inelastic scattering of light from the thermal acoustic modes in a solid and from the random thermal density fluctuations in a liquid or gas. The spectral shift of the scattered light, usually in the sub GHz-range, gives information about the interaction between the incident light and the acoustic phonons in a sample. As a result, in can provide useful information regarding the viscoelastic properties of the material under examination. In its spontaneous version, Brillouin scattering generally has cross-sections in the order of Raman scattering, so it is a very weak signal; and, Brillouin frequency shifts are orders of magnitude smaller than Raman shifts; as a result, elastically scattered light (from Rayleigh or Mie scattering) or stray light, or backreflections off of the sample easily overshadow Brillouin spectral signature. Therefore, in order to accurately measure the Brillouin spectrum, a spectrometer needs to not only achieve sub-GHz spectral resolution but also high spectral contrast.
*
[email protected]. A complete version of this article that includes the video component is available at http://dx.doi.org/10.3791/53468.
Berghaus et al.
Page 2
Author Manuscript
In common Brillouin spectrometers, these requirements are met by scanning-grating monochromators, optical beating methods, and, most popularly, by multiple-pass scanning Fabry-Perot interferometers5. All these methods measure each spectral component sequentially which results in acquisition time of a single Brillouin spectrum of a few minutes to several hours, depending on the instrument. Here, we show that the two-stage VIPA spectrometer, has the ability of collecting all spectral components simultaneously, within less than a second with sufficient extinction (>60 dB) to effectively suppress other spurious signals5.
Instrument Overview
Author Manuscript
The integration of the VIPA etalons is the key element of the spectrometer. A VIPA is a solid etalon with three different coating areas; a highly reflective (HR) coating at the front, a partially reflective coating at the back, and a narrow anti-reflection coating strip at the front which allows the light to enter the VIPA. When the light beam is focused onto the narrow entrance of the slightly tilted VIPA the beam gets reflected into sub-components with fixed phase difference within the VIPA5. Due to the interference of the sub components, high spectral dispersion is achieved. Aligning two VIPAs sequentially in cross-axis configuration introduces spectral dispersion in orthogonal directions4. The spectral dispersion in orthogonal directions spatially separates the Brillouin peaks from crosstalk which allows us to block out the crosstalk with masks. Figure 1 displays a schematic of the two stage VIPA spectrometer. The arrows below the optical elements indicate the degree of freedom in which the translational stages should be oriented in.
Author Manuscript
The following protocol describes how to build and use a two-stage VIPA spectrometer. The spectrometer can be used in combination with a variety of standard optical probes (e.g. confocal microscope, endoscope, slit-lamp ophthalmoscope), as it has been shown recently (REF). The description of these optical setups is however outside the scope of this protocol.
Protocol A single-longitudinal mode laser is required for Brillouin spectral analysis. To align the spectrometer, a strongly attenuated portion of this laser beam is utilized. 1. Initial setup of fiber and CCD camera
Author Manuscript
1.
Find about 2000 mm free space to align and mount the camera at the end. Turn the camera on and disable the gain. Set a low integration time (0.1s). Adjust laser power using optical density filters to avoid camera saturation.
2.
Mount the fiber collimator about 1600 mm in front of the camera. Check if the beam is collimated. Place a pinhole in front of the fiber collimator. Adjust the height of the pinhole to the beam. Move the pinhole along the beam path. Use the adjustment screws of the fiber collimator mount until the beam cleanly passes through the pinhole along the entire beam path.
3.
Mount a matched achromatic pair (f =30 mm) in front of the camera.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 3
4.
Author Manuscript
Mount the horizontal mask on top of a translation stage which can slide the filter in and out of the beam, as indicated in figure 1. Check if horizontal mask is in focus by imaging the edge of the filter onto the camera. If the edges do not appear sharp, slightly adjust the spatial filter along the beam path to obtain a sharp edge image on the CCD.
2. Horizontal Stage of Spectrometer
Author Manuscript Author Manuscript
1.
Mount the spherical lens (f=200mm) S1 600 mm in front of the horizontal spatial filter. Prior to mounting the lens, place two pinholes, aligned with the optical beam, before and after the spherical lens position. When you mount the lens, make sure the beam and the back reflection off of the lens go through the two pinholes.
2.
Mount VIPA2 in the focal plane of the spherical lens. Make sure to mount the VIPA such that the translation stage can move the VIPA in and out of the beam, as indicated in figure 1. At this stage, slide the VIPA out of the beam path.
3.
Mount and align the spherical lens S2 (f=200mm) 200 mm after the VIPA and 200 mm in front of the horizontal spatial filter. Make sure to mount the spherical lens on a translation stage with the degree of freedom oriented along the optical axis.
4.
Use the translation stage to slide the VIPA2 entrance into the beam path. Observe vertical lines on the camera image. Use the translation stage of the S2 to put the lines in focus.
5.
To adjust the spectrum you have two degrees of freedom: horizontal translation to tune the entrance position of the beam into the etalon, and horizontal tilt to tune the input angle of the beam into the etalon.
6.
Measure Finesse by dividing the distance between two vertical lines by their full width at half maximum. Aim for >40. You will find that there is a tradeoff between Finesse and throughput. Measure throughput by measuring power directly before and after VIPA. Aim for >50%.
7.
If Finesse and throughput is not satisfactory go back and fine adjust the alignment. Make sure the VIPA is in the focal plane of the first lens. The beam waist needs to be within the VIPA. Repeat step 4–6.
3. Vertical Stage of Spectrometer
Author Manuscript
1.
Slide the VIPA (VIPA2) aligned in the first part out of the beam using the translation stage. The horizontal stage of the spectrometer now behaves as a 1:1 imaging system.
2.
Mount the vertical spatial filter on top of a vertical translation stage 200 mm in front of S1. Check if the vertical filter is in focus by looking at the sharpness of its imaged edges. Adjust the position of the filter along the beam path until edges are sharp.
3.
Mount and align height, tilt and lateral position of the cylindrical lens (f=200mm) C1 600 mm in front of the horizontal spatial filter.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 4
Author Manuscript Author Manuscript
4.
Mount VIPA1 on top of a vertical translation stage in the focal plane of the cylindrical lens. Mount the VIPA such that the translation stage can move the VIPA in and out of the beam path.
5.
Mount and align the cylindrical lens C2 (f=200mm) 200 mm after VIPA1 and 200 mm in front of the vertical spatial filter. Make sure to mount the cylindrical lens on a translation stage with the degree of freedom oriented along the optical axis as indicated in figure 1.
6.
Use the vertical translation stage to slide the VIPA1 entrance into the beam. Observe vertical lines on the camera image. Use the translation stage of the second cylindrical lens to put the lines in focus.
7.
To adjust the spectrum you have two degrees of freedom: vertical translation to tune the entrance position of the beam into the etalon, and vertical tilt to tune the input angle of the beam into the etalon
8.
Measure Finesse by dividing the distance between two horizontal lines by their full width at half maximum. Aim for >50. You will find that there is a tradeoff between Finesse and throughput. Measure throughput by measuring power directly before and after VIPA. Aim for >50%.
9.
If Finesse and throughput is not satisfactory go back and fine adjust the VIPA mount. Make sure the VIPA is in the focal plane of the first lens. The beam waist needs to be within the VIPA. Repeat step 6–8.
4. Combination of the Two Stages and Final Alignment
Author Manuscript
1.
Slide in VIPA2. Instead of lines you will see horizontally and vertically spaced dots. These dots are from the laser. Adjust the translational stages of the VIPAs until you can see the laser modes clearly. Measure the Finesse of the two stage spectrometer by dividing the diagonal distance between the laser modes by its full width at half maximum.
2.
Build a black box around your spectrometer using construction rails and Blackout Fabric. Allow access (while in the dark) to both VIPAS and both masks.
3.
Connect the fiber to an optical probe carrying Brillouin scattering signal such as a confocal microscope.
5. Measuring the Brillouin shift
Author Manuscript
1.
Close both, the vertical and the horizontal spatial filter, until the laser signature disappear. In order to do so, move the translation stages accordingly.
2.
Enable the gain and increase the integration time of your camera as much as possible without saturating the camera.
3.
Observe the Brillouin shift of your sample.
4.
To optimize the spectrometer throughput, open one mask at a time and scan through the spectrometer orders tilting the VIPA and adjusting its translation stage.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 5
Author Manuscript
Find the order in which your signal appears the strongest. Close mask again until laser signal disappear. Repeat with the other mask and VIPA. 5.
Take measurement of your sample by taking an image of the spectrum using the Andor software provided with the camera. Save the image in TIF format.
6.
Since you have removed the laser signatures, you will need a calibration to convert the pixel separation in Brillouin shifts. Take calibration measurement of two samples with known Brillouin shift.
6. Calibration and Analysis of Brillouin Spectrum
Author Manuscript
Export the data into Matlab. Use the calibration measurements to determine the free spectral range (FSR) and the CCD-pixel to optical frequency conversion ratio (PR) in the Brillouin spectrometer. To determine these parameters, fit the known Brillouin shifts of for example water (7.46 GHz) and glass (29.3 GHz; The shift of glass is aliased into the FSR of 20 GHz and can be treated as a shift of 9.3 GHz for calibration purposes). Overlap the CCD-frame of both calibration samples (figure 1a). Take a line scan across the frame and interpolate your data. Fit Lorentzian curves through each of the peaks to determine the peak position in terms of pixels. Figure 1b displays four distinct peaks; the two inner peaks corresponding to glass and the two outer peaks corresponding to water. Determine FSR and PR by using the following equations, where PGlass-S, PGlass-AS and PWater-S denote the measured value of Brillouin peaks in pixel units and ΩGlass-S and ΩWater-S represent the actual value of Brillouin peaks in GHz units.
Author Manuscript
Determine the Brillouin peak of the sample in pixel units PX by Lorentzian curve fitting. Use the FSR and PR to calculate the Brillouin shift of your sample using the following equations.
Author Manuscript
7. Representative Results Figure 2 shows representative Brillouin spectra and their fits for different materials. Our VIPAs both have a thickness of 5 mm which results in a FSR of about 20 GHz. The integration time for the following measurements were 0.1s. 100 measurements were taken and averaged. One calibration measurement was taken prior to acquiring the following spectra.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 6
8. Discussion
Author Manuscript
The obtained Brillouin shifts agree with previously published data4,8,9. Due to the blocking of the Rayleigh scattering it is impossible to use the laser modes as reference for the spectrum. Any drift of the laser frequency due to temperature fluctuations or other reasons therefore remains undetected and affects the accuracy of the measurements. In addition, the spectral dispersion along the dispersion axis is not linear. The lower the interference order, the higher is the nonlinearity. These sources of error can be minimized by taking calibration measurements frequently.
Author Manuscript
Given the low scattering cross section, for applications in biological materials where integration time and laser power must be kept as low as possible, it is important to maximize throughput. On the other hand, finesse must be maximized as well to achieve high spectral contrast. There is a trade-off between the two of them, as both are affected by the tilt of the VIPA. The larger the tilt, the more light can enter the VIPA, thus the higher is the throughput. However, as the tilt increases, the number of bounces within the VIPA decreases, which reduces the interference between the components and decreases the finesse. Due to its low acquisition time and illumination power such a spectrometer can be used for measuring biomedical materials noninvasively, enabling Brillouin spectroscopy to become a useful tool through high resolution in vivo-mechanical imaging4,6,10,11. Several groups using this type of spectrometer have demonstrated a variety of applications such as measuring the rheological properties of the eye lens12, detecting bacterial meningitis in spinal fluid13, and analyzing the corneal elastic modulus14.
Author Manuscript
Since the spectrometer can be used in combination with a variety of standard optical probes for example confocal microscopes, endoscopes and slit-lamp ophthalmoscopes we expect many more new biomedical applications making use of the two-stage VIPA spectrometer configuration in the future.
References
Author Manuscript
1. Dil JG. Brillouin-scattering in condensed matter. Rep Prog Phys. 1982; 45:285–334. 2. Culshaw B, Michie C, Gardiner P, McGown A. Smart structures and applications in civil engineering. Proc IEEE. 1996; 84:78–86. 3. Eloranta, EW. High spectral resolution lidar. In: Weitkamp, C., editor. Lidar: Range-Resolved Optical Remote Sensing of the Atmosphere. Springer-Verlag; New York: 2005. p. 143-164. 4. Scarcelli G. Confocal Brillouin microscopy for three-dimensional mechanical imaging. Nat Phot. 2008; 2:39–43. 5. Scarcelli G, Yun SH. Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy. Opt Exp. 2011; 19(11):10913–10922. 6. Scarcelli G, Yun SH. In vivo Brillouin optical microscopy of the human eye. Opt Exp. 2012; 20(8): 9197–9202. 7. Brillouin L. Diffusion de la lumiere et des rayonnes X par un corps transparent homogene; influence del’agitation thermique. Ann Phys (French). 1922; 17:88–122. 8. Koski KJ, Yarger JL. Brillouin imaging. Appl Phys Lett. 2005; 87:061903. 9. Faris GW, Jusinski LE, Hickman AP. High-resolution stimulated Brillouin gain spectroscopy in glasses and crystals. J Opt Soc Am B. 1993; 10:587–599.
J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 7
Author Manuscript
10. Antonacci G. Spectral broadening in Brillouin imaging. Appl Phys Lett. 2013; 103:221105. 11. Meng Z, Yakovlev V. Background clean-up in Brillouin microspectroscopy of scattering medium. Opt Exp. 2014; 22(5):5410–5415. 12. Reiss S, Stolz H. Spatially resolved Brillouin spectroscopy to determine the rheological properties of the eye lens. Biomed Opt Express. 2011; 2(8):2144–2159. [PubMed: 21833354] 13. Steelman Z, Meng Z, Traverso AJ, Yakovlev VV. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis. J Biophotonics. 2014; 9999:1–7. 14. Scarcelli, G.; Yun, SH. Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus. 15. Scarcelli G, Kling S, Quijano E, Pineda R, Marcos S, Yun SH. Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus. Invest Ophthalmol Vis Sci. 2013; 54:1418–1425.10.1167/iovs.12-11387 [PubMed: 23361513]
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 8
Author Manuscript
Summary The goal of this protocol is to build a parallel high-extinction and high-resolution optical spectrometer to enable rapid Brillouin scattering analysis. Using VIPA etalons we can achieve a measurement speed that is of more than 1000 times faster than traditional scanning Fabry-Perot spectrometers, which enables Brillouin analysis of biomaterials and biological tissue at low power levels in vivo.
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 9
Author Manuscript Author Manuscript
Fig. 1.
An optical fiber delivers the Brillouin scattering into the spectrometer. A cylindrical lens (f=200mm) C1 focuses the light into the entrance of the first VIPA (VIPA1). Another cylindrical lens (f=200mm) C2maps the spectral angular dispersion into a spatial separation in the focal plane of C2. In this plane, a vertical mask is used to select the desired portion of the spectrum. An analogous configuration, but tilted at 90 degrees, then follows. The beam passes through a spherical lens (f=200mm) S1 and is focused into the entrance slit of the second VIPA (VIPA2). A spherical lens (f=200mm) S2 finally creates the 2D spectrally separated pattern in its focal plane, where another, horizontal, mask is placed. The horizontal mask is imaged onto the camera using an achromatic pair lens pair..
Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 10
Author Manuscript Author Manuscript
Figure 1.
Spectrometer calibration. (a) CCD frame obtained from calibration sample. (b) Lorentzian curve fit (red) to the measured data (blue).
Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
Berghaus et al.
Page 11
Author Manuscript Figure 2.
Brillouin Spectra of different materials. Lorentzian curve fit (red) to the measured data (blue). (a) Brillouin spectrum of Methanol. The measured Brillouin shift is 5.56 GHz. (b) Brillouin spectrum of Ethanol. The measured Brillouin shift is 5.85 GHz. (c) Brillouin spectrum of Polystyrene. The measured Brillouin shift is 14.12 GHz..
Author Manuscript Author Manuscript Author Manuscript J Vis Exp. Author manuscript; available in PMC 2016 February 29.
