Name: rejection of fluorescence background in resonance and spontaneous raman microspectroscopy
Uploaded: 2011-05-18T12:00:00
Description: Watch this Scientific Journal Video about Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy at JoVE.com

This work was funded by NSF award DBI 0852891. Part of this work was also funded by the Center for Biophotonics Science and Technology, a designated NSF Science and Technology Center managed by the University of California, Davis, under Cooperative Agreement No. PHY0120999.

1.	Some care must be taken in preparing and placing a Raman sample within this system.
<ol>
	<li>Because the system typically makes use of very high numerical aperture objectives with very short working distances, the samples are placed on a coverslip. Biological samples are typically placed on a No. 1 thickness coverslip mounted in an Attofluor cell chamber (Invitrogen, Carlsbad, CA). </li>
 <li>Liquid samples, particularly those toxic to humans, are placed in a small glass bottle with a coverslip cem...

<ol>
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The field of biomedical Raman spectroscopy has seen increasing interest over the past several years as a result of its demonstrated potential for solving several difficult challenges in biological diagnostics. For example, Raman spectra have been shown to have diagnostic value in cancer detection 3, 4, 5, 6. Raman spectroscopy has also been used in bacterial quantitation 7, 8 and bacterial drug response 9. It has also found application in a broad range of other biomedical applications ran...

<table border="1">
	<tbody>
 <tr>
 	<th>Name</th>
 <th>Company</th>
 <th>Catalog Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Lenses</td>
 <td>ThorLabs</td>
 <td>Various</td>
 <td>All lenses coated to have maximum transmission losses of 1% each</td>
 </tr>
 <tr>
 	<td>Tunable Ti:Sapph laser</td>
 <td>Coherent</td>
 <td>Chameleon</td>
 <td>30 nJ, 200 fs, 80 MHz</td>
 </tr>
 <tr>
 	<td>40X oil immersion objective</td>
 <td>Olympus</td>
 <td>UApo/340</td>
 <td>NA = 1.35</td>
 </tr>
 <tr>
 	<td>Inverted microscope</td>
 <td>Olympus</td>
 <td>IX-71</td>
 <td>Modified to remove all lenses in side port</td>
 </tr>
 <tr>
 	<td>Half wave plate</td>
 <td>Thorlabs</td>
 <td>AHWP05M-600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Glan-Thompson polarizer</td>
 <td>Thorlabs</td>
 <td>GTH10M</td>
 <td>&tilde;10% transmission loss</td>
 </tr>
 <tr>
 	<td>Spectrometer</td>
 <td>PI Acton</td>
 <td>SP2300i</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CCD</td>
 <td>PI Acton</td>
 <td>Pixis 100B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Mathmatical software</td>
 <td>The MathWorks</td>
 <td>MATLAB</td>
 <td>version 2008a</td>
 </tr>
 <tr>
 	<td>Faraday isolator</td>
 <td>EOT</td>
 <td>BB8-5I</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Piezo-electric mirror</td>
 <td>Newport</td>
 <td>AG-M100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>BBO crystal</td>
 <td>CASIX</td>
 <td>custom</td>
 <td>1 mm thickness</td>
 </tr>
 <tr>
 	<td>Bandpass filter 1</td>
 <td>Andover</td>
 <td>008FC14</td>
 <td>808 &plusmn; 0.4 nm</td>
 </tr>
 <tr>
 	<td>Dichroic mirror</td>
 <td>Semrock</td>
 <td>FF662-FDI01</td>
 <td>band edge at 662 nm</td>
 </tr>
 <tr>
 	<td>Long-pass filter</td>
 <td>Semrock</td>
 <td>BLP01-405R</td>
 <td>band edge at 417 nm</td>
 </tr>
 <tr>
 	<td>Bandpass filter 2</td>
 <td>Semrock</td>
 <td>FF02-447/60</td>
 <td>417-447 nm</td>
 </tr>
 <tr>
 	<td>CS2</td>
 <td>Sigma-Aldrich</td>
 <td>335266</td>
 <td>99% purity</td>
 </tr>
 <tr>
 	<td>Coumarin 30</td>
 <td>Sigma-Aldrich</td>
 <td>546127</td>
 <td>99% purity</td>
 </tr>
 <tr>
 	<td>Immersion oil</td>
 <td>Cargille</td>
 <td>16242</td>
 <td>Type DF</td>
 </tr>
 
 </tbody>
</table>

rejection of fluorescence background in resonance and spontaneous raman microspectroscopy

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

Watch this Scientific Journal Video about Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy at JoVE.com

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

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

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

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