AF is supported by the Dutch Technology Foundation STW, which is the applied science division of NWO, and the Technology Programme of the Ministry of Economic Affairs. (STW OTP 11114).

<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51847/51847fig1highres.jpg" width="500" />
Figure 1. Schematic illustrating the CARS microscope setup with the intrinsic flow through dissolution setup. This figure has been modified from Fussell et al13.
1. System Startup
<ol>
	<li>Turn on the 20 psec pulsed 1,064 nm CARS laser and allow the laser to warm-up (approx. 1.5 hr).</li>
	<li>Turn on the deuterium lamp UV light source and allow it to warm-up (approx. 10 min).</li>
	<li>Open the shutter on the deuterium lamp UV light source by setting the shutter switch to "open".</li>
	<li>Turn on the microscope control PC and open the microscope control software.</li>
	<li>Turn on the UV spectrometer PC and open the spectrometer control software.</li>
</ol>
2. Microscope Setup
<ol>
	<li>Select the desired microscope objective. Use a 20X/0.5 NA objective to achieve the results presented in this work.</li>
	<li>Set the filters in the filter set turret to transmit excitation lasers and reflect the CARS signal. Select a 775 nm long-pass dichroic mirror and a 650 nm band-pass 40 nm filter to replicate the results shown in this work.</li>
	<li>Place appropriate filters in front of the photomultiplier tube (PMT) detector that transmit CARS signal and filter unwanted light. Filter the light with a 750 nm short-pass filter and a 650 nm band-pass 40 nm filter to reproduce the experiments conducted in this work.</li>
</ol>
3. System Testing
<ol>
	<li>Turn on the peristaltic pump and pump dissolution medium for a few minutes through the Z-shaped UV flow cell to clear previous liquid from the piping.</li>
	<li>Determine the flow rate of the pump by weighing the amount of dissolution medium pumped in 2 min. Adjust pump speed until desired flow rate is reached. Pump the dissolution medium at a flow rate of 5 ml/min to achieve the results reported in this work.</li>
</ol>
4. UV Dissolution Measurement
<ol>
	<li>In the UV spectrometer control software, click the &#8220;File&#8221; menu then click &#8220;New absorbance measurement&#8221; to open a window which lists all available spectrometers.</li>
	<li>Click on the correct UV spectrometer, and then click &#8220;Next&#8221; to open a window which displays the data acquisition parameters.</li>
	<li>Define both the integration time and the spectral averaging. Choose an integration time of 150 msec with 200 averages to replicate the results shown in this work.</li>
	<li>Click the button labelled &#8220;Next&#8221; to bring up the screen used to record the reference spectrum.</li>
	<li>Click on the button that appears as a yellow light bulb to record a reference spectrum. Pump dissolution medium continuously during this measurement.</li>
	<li>Close the shutter on the deuterium lamp UV light source by setting the switch to &#8220;closed&#8221;.</li>
	<li>Click the button labelled &#8220;Next&#8221; to bring up the screen used to record the dark spectrum.</li>
	<li>Click on the button that appears as a grey light bulb to record a dark spectrum. Pump dissolution medium continuously during this measurement.</li>
	<li>Click on the button that says &#8220;Finish&#8221; to begin the UV absorbance measurements.</li>
</ol>
5. CARS Dissolution Video
<ol>
	<li>In the CARS microscope control software click on the button that selects an &#8220;XYT&#8221; measurement.</li>
	<li>Click the drop-down box and select the image size in pixels. Select an image size of 512 x 512 pixels to reproduce the images reported in this work.</li>
	<li>Drag the imaging speed slider to either the &#8220;fast&#8221;, &#8220;medium&#8221;, or &#8220;slow&#8221; position. Use the fast scanning speed (1.12 sec per image) to achieve the results shown in this work.</li>
	<li>Click the arrows labelled &#8220;zoom&#8221; to adjust the zoom level. Select &#8220;2x&#8221; zoom to replicate the level of zoom and field of view (350 x 350 &#181;m) used for these results.</li>
	<li>Click the drop-down box and select the objective used.</li>
	<li>Click the input box and type the amount of frames required for the CARS dissolution video (depending on length of experiment). Conduct dissolution for about 15 min by recording 900 frames to reproduce the results shown in this work.</li>
</ol>
6. CARS Wavelength Tuning
<ol>
	<li>Using the optical parametric oscillator (OPO) controller adjust the settings of the OPO such as temperature, piezo position, and Lyot filter position until maximal laser output at the desired Raman frequency is reached. Tune the OPO to 2,960 cm-1 to record the same results as those presented in this article.</li>
</ol>
7. The Dissolution Experiment
<ol>
	<li>Place a tablet into sample holder of the custom built CARS flow cell, screw the sample holder closed tightly to prevent leakage.</li>
	<li>Attach the piping to the CARS flow cell connecting the CARS flow cell to the beaker containing the dissolution medium and the peristaltic pump.</li>
	<li>Place the CARS flow cell containing a tablet on the microscope stage.</li>
	<li>Check that the CARS flow cell is connected to the dissolution medium beaker, the peristaltic pump, the Z-shaped UV flow cell and the waste collection beaker.</li>
	<li>Click the &#8220;XY repeat&#8221; button to start the microscope system scanning in a continuous scan mode.</li>
	<li>Adjust the focus of the microscope by moving the objective until the surface of the tablet is in the field of view on the microscope control computer screen.</li>
	<li>Click on the slider in the microscope control software labelled &#8220;PMT&#8221;. Adjust the detector sensitivity by increasing/decreasing PMT voltage until a satisfactory image (neither too dark nor saturated) is visible on the screen. NOTE: Take care not to overload the PMT by using high voltage. For this work, we used a PMT voltage around 600 V but this can vary depending on the PMT used.</li>
	<li>Click &#8220;Stop&#8221; in the microscope control software to stop the continuous scan.</li>
	<li>Simultaneously (or as close together as possible) start pumping dissolution medium, start recording a single XYT scan, and start collecting UV absorbance spectra.</li>
	<li>During the dissolution experiment, monitor the video recording and manually adjust the microscope focus to ensure the tablet is continually in focus.</li>
</ol>
8. Post Dissolution
<ol>
	<li>Stop the peristaltic pump by turning it off.</li>
	<li>Click the &#8220;File&#8221; menu and then click &#8220;Save as video&#8221; on the microscope control software to save the XYT scan as a video.</li>
	<li>Click the &#8220;File&#8221; menu, then click &#8220;Save&#8221; and then click &#8220;Stop Export&#8221; on the spectrometer control software to stop the collection of UV absorption spectra.</li>
	<li>Remove the CARS flow cell from the microscope stage and remove the tablet from the CARS flow cell.</li>
	<li>Wash the CARS flow cell using water and ethanol, and then dry using tissue paper.</li>
</ol>

<ol>
	<li>Ku, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Biopharmaceutical+Classification+System+in+Early+Drug+Development.">Use of the Biopharmaceutical Classification System in Early Drug Development.</a> The AAPS Journal. 10, 208-212 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+United+States+Pharmacopeia.">The United States Pharmacopeia.</a> United States Pharmacopeial Convention 32nd ed. , 1-8 (2009).</li><li>Florence, A. J., Johnston, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+ATR+UV/vis+spectroscopy+in+physical+form+characterisation+of+pharmaceuticals.">Applications of ATR UV/vis spectroscopy in physical form characterisation of pharmaceuticals.</a> Spectrosc. Eur. 4, (2004).</li><li>Aaltonen, J., 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=In+situ+measurement+of+solvent-mediated+phase+transformations+during+dissolution+testing.">In situ measurement of solvent-mediated phase transformations during dissolution testing.</a> J. Pharm. Sci. 95, 2730-2737 (2006).</li><li>Savolainen, M., 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=Better+understanding+of+dissolution+behaviour+of+amorphous+drugs+by+in+situ+solid-state+analysis+using+Raman+spectroscopy.">Better understanding of dissolution behaviour of amorphous drugs by in situ solid-state analysis using Raman spectroscopy.</a> European Journal of Pharmaceutics and Biopharmaceutics. 71, 71-79 (2009).</li><li>Slipchenko, M. N., 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=Vibrational+imaging+of+tablets+by+epi-detected+stimulated+Raman+scattering+microscopy.">Vibrational imaging of tablets by epi-detected stimulated Raman scattering microscopy.</a> Analyst. 135, 2613-2619 (2010).</li><li>Parekh, S. H., Lee, Y. J., Aamer, K. A., Cicerone, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Cellular+Imaging+by+Broadband+Coherent+Anti-Stokes+Raman+Scattering+Microscopy.">Label-Free Cellular Imaging by Broadband Coherent Anti-Stokes Raman Scattering Microscopy.</a> Biophys. J. 99, 2695-2704 (2010).</li><li>Kang, E., 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=In+Situ+Visualization+of+Paclitaxel+Distribution+and+Release+by+Coherent+Anti-Stokes+Raman+Scattering+Microscopy.">In Situ Visualization of Paclitaxel Distribution and Release by Coherent Anti-Stokes Raman Scattering Microscopy.</a> Anal. Chem. 78, 8036-8043 (2006).</li><li>Kang, E., Robinson, J., Park, K., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel+distribution+in+poly(ethylene+glycol)/poly(lactide-co-glycolic+acid)+blends+and+its+release+visualized+by+coherent+anti-Stokes+Raman+scattering+microscopy.">Paclitaxel distribution in poly(ethylene glycol)/poly(lactide-co-glycolic acid) blends and its release visualized by coherent anti-Stokes Raman scattering microscopy.</a> J. Controlled Release. 122, 261-268 (2007).</li><li>Kang, E., 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=Application+of+coherent+anti-stokes+Raman+scattering+microscopy+to+image+the+changes+in+a+paclitaxel-poly(styrene-b-isobutylene-b-styrene)+matrix+pre-+and+post-drug+elution.">Application of coherent anti-stokes Raman scattering microscopy to image the changes in a paclitaxel-poly(styrene-b-isobutylene-b-styrene) matrix pre- and post-drug elution.</a> Journal of Biomedical Materials Research Part A. 87A, 913-920 (2008).</li><li>Jurna, M., 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=Coherent+anti-Stokes+Raman+scattering+microscopy+to+monitor+drug+dissolution+in+different+oral+pharmaceutical+tablets.">Coherent anti-Stokes Raman scattering microscopy to monitor drug dissolution in different oral pharmaceutical tablets.</a> Journal of Innovative Optical Health Sciences. 2, 37-43 (2009).</li><li>Windbergs, M., 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=Chemical+Imaging+of+Oral+Solid+Dosage+Forms+and+Changes+upon+Dissolution+Using+Coherent+Anti-Stokes+Raman+Scattering+Microscopy.">Chemical Imaging of Oral Solid Dosage Forms and Changes upon Dissolution Using Coherent Anti-Stokes Raman Scattering Microscopy.</a> Anal. Chem. 81, 2085-2091 (2009).</li><li>Fussell, A., Garbacik, E., Offerhaus, H., Kleinebudde, P., Strachan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+dissolution+analysis+using+coherent+anti-Stokes+Raman+scattering+(CARS)+and+hyperspectral+CARS+microscopy.">In situ dissolution analysis using coherent anti-Stokes Raman scattering (CARS) and hyperspectral CARS microscopy.</a> European Journal of Pharmaceutics and Biopharmaceutics. 85, 1141-1147 (2013).</li><li>Lua, Y. -. Y., Cao, X., Rohrs, B. R., Aldrich, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Characterizations+of+Spin-Coated+Films+of+Ethylcellulose+and+Hydroxypropyl+Methylcellulose+Blends.+.">Surface Characterizations of Spin-Coated Films of Ethylcellulose and Hydroxypropyl Methylcellulose Blends. .</a> Langmuir. 23, 4286-4292 (2007).</li><li>Skoog, F. J., Holler, S. R., Crouch, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Instrument analysis. 6 ed. , (2007).</li><li>Rodr&#237;guez-Hornedo, N., Lechuga-Ballesteros, D., Hsiu-Jean, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+transition+and+heterogeneous/epitaxial+nucleation+of+hydrated+and+anhydrous+theophylline+crystals.">Phase transition and heterogeneous/epitaxial nucleation of hydrated and anhydrous theophylline crystals.</a> Int. J. Pharm. 85, 149-162 (1992).</li></ol>

The authors declare that they have no competing financial interests.

When performing CARS microscopic dissolution experiments there are a few critical aspects that need to be monitored during the experiment. Firstly, introducing the dissolution medium to the CARS flow cell causes the focus to move. This means that the image is immediately lost and it takes a few microns of objective adjustment to find the surface again. Secondly, there is risk of liquid leakage from the CARS flow cell if the glass cover breaks during the experiment. This can potentially cause liquid damage to the optics, so it is important to listen for any cracking sound that could mean the glass has broken. Finally, there is also a small chance that the piping can become blocked due to particulate matter in the system during the experiment, this can be seen as a sudden unusual change in the UV spectra and also through periodically checking the flow during the experiment.
Particulate blockage of the piping is mainly an issue with tablets that have been designed to disintegrate during dissolution. This is one of the limitations for this technique as this system requires the surface of the tablet to remain intact throughout the dissolution to allow imaging. In addition to disintegrating tablets, it is currently not possible to image tablets that are designed to swell during dissolution as this can lead to breakage of the CARS flow cell.
Imaging tablets during dissolution provides a greater understanding of what is occurring on the surface of a dissolving tablet. Conventional pharmaceutical dissolution methods focus only on the drug content dissolved in the dissolution medium which can identify whether the tablet passes or fails the required standard. However, in the case of a failed test it is difficult to determine what caused the failure. The case of a failed dissolution test is potentially where in situ dissolution analysis using CARS microscopy can provide answers.
Future applications for in situ dissolution analysis using CARS microscopy could include investigations using more complicated tablets containing more than one drug or excipient, in particular non-swelling sustained or controlled release dosage forms during formulation development. Additionally, it could be possible to investigate samples using biorelevant dissolution media creating conditions more closely related to in vivo. 
In conclusion, this work shows that CARS microscopy is capable of rapid chemically specific imaging based on Raman vibrational frequencies allowing selective imaging of the drug in a tablet containing both drug and excipient. Additionally, CARS microscopy combined with inline UV absorption spectroscopy is a powerful tool capable of monitoring the surface of tablets undergoing dissolution and correlating surface changes seen using CARS with changes in dissolution rate.

During the development of oral pharmaceutical dosage forms such as tablets and capsules there is a strong emphasis on dissolution testing. Oral dosage forms are required to dissolve before they can be absorbed for therapeutic efficacy. Poorly soluble drugs generally have issues reaching an adequate concentration which makes dissolution testing particularly important1. Pharmacopoeial dissolution methods are most commonly used for dissolution analysis. In most cases this requires preparing the drug as a tablet or capsule which is then placed into a beaker of flowing dissolution medium. The dissolved drug concentration is then determined by analyzing samples of the dissolution medium using a standard spectroscopic technique such as UV absorption spectroscopy2. These traditional pharmaceutical dissolution methods do not provide any direct analysis of the sample or any changes that might be occurring on the dissolving surface of the dosage form. Direct analysis of the sample during dissolution can provide more information about the dissolving dosage form and potentially identify problems causing dissolution test failure.
Direct analysis of dissolving dosage forms requires the use of in situ analytical techniques which are capable of monitoring the dissolution process. To record in situ during dissolution the analytical technique must not be influenced by the presence of the dissolution medium and the technique needs a high temporal resolution to reliably measure changes to the dissolving dosage form in the order of seconds. Attenuated total reflectance UV spectroscopy has been shown to be suitable for measuring changes during dissolution but lacks spatial resolution provided by imaging techniques3. Traditional pharmaceutical imaging techniques such as scanning electron microscopy (SEM), and spontaneous Raman mapping both have limiting factors preventing their use in situ for dissolution.
SEM imaging is a high-resolution rapid imaging technique capable of imaging the surface of pharmaceutical dosage forms. However, SEM imaging is generally performed under vacuum conditions and requires sample coating making it unsuitable for in situ dissolution&#160;imaging. Fiber-coupled spontaneous Raman spectroscopy combined with a flow through cell and UV flow-through absorption spectroscopy, has been performed to monitor various drug systems in situ during dissolution, including theophylline4, carbamazepine, and indomethacin5. Raman spectroscopy was capable of identifying surface changes occurring during dissolution but it gave no spatial information about where the surface changes were occurring. Spontaneous Raman mapping uses Raman spectra and provides spatial information about the surface of the sample but imaging takes on the order of minutes to hours depending on image area, making it unsuitable for in situ dissolution imaging.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a rapid imaging technique and combined with inline UV absorption spectroscopy, it has allowed us to develop a technique capable of in situ dissolution analysis. CARS microscopy provides rapid chemically selective imaging which is not influenced by the presence of dissolution medium making it a suitable technique for in situ dissolution analysis. CARS techniques are divided roughly into two groups based on the pulse duration of the lasers; one being narrowband CARS (picosecond pulsed lasers), and the other being broadband CARS (femtosecond pulsed lasers). A typical CARS microscope system consists of two pulsed laser sources and an inverted microscope. To produce a CARS signal, one of the pulsed lasers needs to be tunable so&#160;there is a frequency difference between the two lasers which matches a Raman vibration. Additionally, the two lasers are required to overlap&#160;in space (spatial) and time (temporal), with pulses from both lasers arriving at the same area of the sample at the same time. As Raman vibrations are chemically specific and CARS signal is only generated within the focal volume of the microscope, CARS microscopy is capable of chemically selective imaging with a resolution down to the diffraction limit.
Narrowband CARS microscopy&#160;using a single Raman vibrational mode allows&#160;about 100x&#160;faster imaging compared to spontaneous Raman mapping techniques6. Broadband CARS microscopy images over a wider spectral range (600-3,200 cm-1 vs. ~4 cm-1) but has a lower spectral resolution (around 10 cm-1 vs. ~4 cm-1) and slower imaging speed (50 msec/pixel vs. ~5 &#956;sec/pixel) compared to narrowband CARS microscopy7.
Narrowband CARS microscopy has been used to image drug release from some pharmaceutical systems. In the area of pharmaceutical formulations, Kang et al.8-10 imaged drug loaded polymer films. Initially they imaged the distribution of the loaded drug, which was followed by imaging of the drug release from a static dissolution medium. Jurna et al.11 and Windbergs et al.12 went a step further and imaged firstly the theophylline distribution in lipid dosage forms followed by imaging the drug dissolution using a dynamic dissolution medium.
We have developed a new analytical method to simultaneously monitor surface changes on the tablet undergoing dissolution with narrowband CARS microscopy while recording the dissolved drug concentration with UV absorption spectroscopy. We illustrate the use of this method imaging tablets containing the model drug theophylline combined with ethyl cellulose undergoing dissolution with water as dissolution medium.

<table border="0" cellpadding="0" cellspacing="0" width="827">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of the Material/Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:99px;">Catlog number</td>
			<td style="width:189px;">Comments/Description</td>
			<td style="width:183px;">Website</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paladin 1064nm laser</td>
			<td>Coherent&#160;</td>
			<td>N/A</td>
			<td>Prototype model not for sale</td>
			<td><a href="http://www.coherent.com/">http://www.coherent.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Levante Emerald Optical parametric oscillator</td>
			<td>APE Berlin</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ape-berlin.de/en/products/levante/levante-emerald-opo#block-views-products-block-1">http://www.ape-berlin.de/en/products/levante/levante-emerald-opo#block-views-products-block-1</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IX 71 Microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.olympusamerica.com/seg_section/product.asp?product=1023">http://www.olympusamerica.com/seg_section/product.asp?product=1023</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoview 300 scanning unit</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
			<td>http://www.olympusamerica.com/seg_section/seg_product_print.asp?product=133</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photon multiplier tube R3896</td>
			<td>Hamamatsu</td>
			<td>N/A</td>
			<td></td>
			<td><a href="https://www.hamamatsu.com/jp/en/R3896.html">https://www.hamamatsu.com/jp/en/R3896.html</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Free standing optics / filters</td>
			<td>Thorlabs and Chroma</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.chroma.com/">http://www.chroma.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td><a href="http://www.thorlabs.de/index.cfm?">http://www.thorlabs.de/index.cfm?</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reglo peristaltic pump</td>
			<td>ISMATEC</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ismatec.com/int_e/pumps/t_reglo/reglo.htm">http://www.ismatec.com/int_e/pumps/t_reglo/reglo.htm</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">USB2000+ spectrometer</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/products/usb2000+.asp">http://www.oceanoptics.com/products/usb2000+.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DT-MINI-2-GS light source</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/dtmini.asp">http://www.oceanoptics.com/Products/dtmini.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FIA-Z-SMA-TEF Z shaped flow cell</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/fiazsmaflowcells.asp">http://www.oceanoptics.com/Products/fiazsmaflowcells.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QP400-2-SR-BX optical fiber</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/premgradesol.asp">http://www.oceanoptics.com/Products/premgradesol.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic piping</td>
			<td>ISMATEC</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ismatec.com/int_e/tubing/misc/tubing_home.htm">http://www.ismatec.com/int_e/tubing/misc/tubing_home.htm&#160;</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CARS dissolution tablet flow cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td colspan="2">Homebuilt at university - designed to hold 12mm diameter, 3mm thick tablets. The flowcell has a channel depth of around 0.5mm.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass beakers</td>
			<td>VWR</td>
			<td>D108980</td>
			<td></td>
			<td><a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4537423">https://us.vwr.com/store/catalog/product.jsp?product_id=4537423</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Theophylline anhydrate</td>
			<td>BASF</td>
			<td>30058079</td>
			<td></td>
			<td><a href="http://www.basf.com/group/corporate/en/brand/THEOPHYLLINE">http://www.basf.com/group/corporate/en/brand/THEOPHYLLINE</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ethylcellulose</td>
			<td>Colorcon</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.colorcon.com/products-formulation/all-products/film-coatings/sustained-release/ethocel">http://www.colorcon.com/products-formulation/all-products/film-coatings/sustained-release/ethocel&#160;</a></td>
		</tr>
	</tbody>
</table>

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.

In situ dissolution analysis using CARS microscopy was conducted on tablets (12 mm diameter, flat-faced) containing a 50:50 mixture of the model drug theophylline anhydrate and ethyl cellulose with distilled water pumped at 5 ml/min as the dissolution medium. CARS images (512 x 512 pixels) were collected every 1.12 sec at the Raman vibrational frequency 2,960 cm-1 which is selective for the theophylline content in the tablet for the duration of the dissolution experiment. Figure 2 shows selected frames from the dissolution video. At the beginning of dissolution (Figure 2, time 0 sec) there are areas of green showing the theophylline content of the tablet and there are also dark areas where there is only ethyl cellulose present on the surface of the tablet. In the dark areas on the surface of the tablet it is possible to faintly see the ethyl cellulose content. This is because it is reported that ethyl cellulose has Raman vibrational frequencies with maxima around 2,930 and 2,975 cm-1&#160;14. After about 60 sec&#160;there appears to be the beginning of theophylline monohydrate crystal growth on the surface which can be seen as narrow needle-shaped crystals growing outwards from at least one crystal nucleus at the center of the frame (Figure 2, time 60 sec). The monohydrate crystal growth can be much more clearly seen after 130 sec (Figure 2, time 130 sec). Additionally, at time point 130 sec it can be seen that the monohydrate crystal has not spread entirely across the surface of the tablet. It appears as though the presence of the ethyl cellulose regions has physically blocked the lengthening of the monohydrate needles. After 250 sec, it can be seen that the monohydrate coverage of the surface is not as prominent which suggests that the monohydrate crystals are themselves beginning to dissolve.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51847/51847fig2highres.jpg" width="500" /> 
Figure 2. Frames from CARS dissolution video. Selected CARS images (2,960 cm-1) from a dissolution video for a theophylline anhydrate with ethyl cellulose tablet. The 0 sec image is recorded at one area of the sample while the 60, 130, and 250 sec images are recorded at another area of the sample. The CARS video is available as supplementary information. Scale bar is 50 &#181;m.
Ultra-violet (UV) spectroscopy is a form of absorption spectroscopy using UV light as the excitation source. UV spectroscopy measures electron transitions from the ground state to an excited state15. Theophylline has a broad peak centered around 270 nm while ethyl cellulose is practically insoluble in the dissolution medium so is not expected to contribute to the UV spectrum recorded. Analysis of the dissolution medium using the inline z-shaped UV flow cell allows us to quantitatively determine the amount of drug dissolved during dissolution. Figure 3 shows the UV-dissolution profile for the dissolution of the theophylline anhydrate with ethyl cellulose tablet. The UV dissolution profile (Figure 3) shows that dissolution of theophylline anhydrate begins quickly reaching a maximum concentration of around 90 &#181;g/ml&#160;within 120 sec; after this time point the dissolution rate begins to decrease. The decrease in the dissolution rate could be due to the presence of the theophylline monohydrate (solubility 6 mg/ml&#160;at 25 &#176;C&#160;16) crystals on the surface which are less soluble than theophylline anhydrate (solubility 12 mg/ml&#160;at 25 &#176;C&#160;16) and clearly seen in the CARS dissolution video (Figure 2) at this time point. The gradually reducing dissolution rate could also partially be explained by a reduction in theophylline exposure to the flowing medium. This reduction occurs because ethyl cellulose is practically insoluble in water, so as the theophylline dissolves the remaining ethyl cellulose hinders theophylline exposure to the dissolution medium.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51847/51847fig3highres.jpg" width="500" /> 
Figure 3. UV dissolution profile. Concentration vs. time plot for a theophylline anhydrate combined with ethyl cellulose tablet showing the concentration of theophylline in the dissolution medium during the dissolution experiment.

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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.

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 ...

coherent-anti-stokes-raman-scattering-cars-microscopy-visualizes

Research

JoVE Journal

Engineering

18.0K 次观看.  University of Twente. 相干反斯托克斯拉曼散射（CARS）显微术是结合本征流通过溶解的设置，以允许在原位和药物片剂进行溶出度的表面的实时可视化。使用这种定制的设置，因此能够使用内联紫外吸收光谱记录药物溶出曲线相关CARS视频。 

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

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 ...

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

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 ...

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the presence of carbon nanotubes. These nanowires are extreme in the sense that they are the limit of miniaturization of nanowires and their behavior is not always a simple extrapolation of the behavior of larger nanowires as their diameter decreases. The paper then describes the methods required to obtain Raman spectra from extreme nanowires and the fact that due to the van Hove singularities that 1D systems exhibit in their optical density of states, that determining the correct choice of photon excitation energy is critical. It describes the techniques required to determine the photon energy dependence of the resonances observed in Raman spectroscopy of 1D systems and in particular how to obtain measurements of Raman cross-sections with better than 8% noise and measure the variation in the resonance as a function of sample temperature. The paper describes the importance of ensuring that the Raman scattering is linearly proportional to the intensity of the laser excitation intensity. It also describes how to use the polarization dependence of the Raman scattering to separate Raman scattering of the encapsulated 1D systems from those of other extraneous components in any sample.

The paper describes a method for producing extreme nanowires by melt infiltration into carbon nanotubes and how 1D systems may be characterized and investigated using Resonance Raman Spectroscopy to determine vibrational and optical excitation energies.

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the ...

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 ...

Generation and Coherent Control of Pulsed Quantum Frequency Combs

We present a method for the generation and coherent manipulation of pulsed quantum frequency combs. Until now, methods of preparing high-dimensional states on-chip in a practical way have remained elusive due to the increasing complexity of the quantum circuitry needed to prepare and process such states. Here, we outline how high-dimensional, frequency-bin entangled, two-photon states can be generated at a stable, high generation rate by using a nested-cavity, actively mode-locked excitation of a nonlinear micro-cavity. This technique is used to produce pulsed quantum frequency combs. Moreover, we present how the quantum states can be coherently manipulated using standard telecommunications components such as programmable filters and electro-optic modulators. In particular, we show in detail how to accomplish state characterization measurements such as density matrix reconstruction, coincidence detection, and single photon spectrum determination. The presented methods form an accessible, reconfigurable, and scalable foundation for complex high-dimensional state preparation and manipulation protocols in the frequency domain.

A protocol is presented for the practical generation and coherent manipulation of high-dimensional frequency-bin entangled photon states using integrated micro-cavities and standard telecommunications components, respectively.

We present a method for the generation and coherent manipulation of pulsed quantum frequency combs. Until now, methods of preparing high-dimensional ...

Scattering And Absorption of Light in Planetary Regoliths

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed microscopic particles. The theoretical and numerical methods constitute a framework of Radiative Transfer with Reciprocal Transactions (R2T2). The R2T2 framework entails Monte Carlo order-of-scattering tracing of interactions in the frequency space, assuming that the fundamental scatterers and absorbers are wavelength-scale volume elements composed of large numbers of randomly distributed particles. The discrete random media are fully packed with the volume elements. For spherical and nonspherical particles, the interactions within the volume elements are computed exactly using the Superposition T-Matrix Method (STMM) and the Volume Integral Equation Method (VIEM), respectively. For both particle types, the interactions between different volume elements are computed exactly using the STMM. As the tracing takes place within the discrete random media, incoherent electromagnetic fields are utilized, that is, the coherent field of the volume elements is removed from the interactions. The experimental methods are based on acoustic levitation of the samples for non-contact, non-destructive scattering measurements. The levitation entails full ultrasonic control of the sample position and orientation, that is, six degrees of freedom. The light source is a laser-driven white-light source with a monochromator and polarizer. The detector is a mini-photomultiplier tube on a rotating wheel, equipped with polarizers. The R2T2 is validated using measurements for a mm-scale spherical sample of densely-packed spherical silica particles. After validation, the methods are applied to interpret astronomical observations for asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko (Figure 1) recently visited by the NASA Dawn mission and the ESA Rosetta mission, respectively.

Numerical and experimental methods are presented for multiple scattering of light in discrete random media of densely-packed particles. The methods are utilized to interpret the observations of asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko.

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed ...

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. ...