Name: Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy (Video) | JoVE Journal
Uploaded: 2011-05-18T12:00:00.000+00:00
Duration: 15 min 4 s
Description: Learn to construct an all-optical gate that isolates Raman signals from fluorescence background using ultrafast switching. This method enables high signal-to-noise biochemical quantification for biophysical research.

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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 cemented to the opening by means of a silicone epoxy. The bottle is then inverted for measurement. </li>
 <li>Because our system depends on precise timing of the pump and signal pulses, we are constrained not to use the conventional focus adjustment of our microscope, which translates the objective up and down. This adds and subtracts optical path from our system. Instead, we place our samples on a secondary stage mounted on top of the microscope stage that has its own independent focus control.</li>
</ol>


2.	In order to take time-gated Raman spectra, the excitation beam must be properly prepared. 
<ol> 
 
 <li>We start with light emerging from a 2.4 W tunable pulsed Ti:Sapph laser (Chameleon, Coherent Systems, Santa Clara, CA). Each pulse in the 80 MHz pulse train has 30 nJ of energy, has a temporal width of 140 fs, and has a spectrum centered at 808 nm with a spectral bandwidth of ~6 nm. </li>
 <li>To prevent back-reflections from re-entering the laser cavity, the light is passed through a Faraday isolator. A half-wave plate placed before the Faraday isolator allows continuous tuning of the total power sent into the system. </li>
 <li>Because 6 nm is too broad a bandwidth to resolve most Raman modes, the beam is sent through a very narrow (0.8 nm FWHM, Andover Corporation, Salem, NH) bandpass filter centered at 808 nm. </li>
 <li>The light is then focused by an achromatic doublet (f = 100 mm) onto a 5 mm β-Barium Borate (BBO) crystal (CASIX, Fuzhou, Fujian, P. R. China) to halve the wavelength from 808 nm to 404 nm. The BBO crystal is placed in a mount with tip and tilt controls, and also mounted on a translation stage. To maximize the efficiency of the wavelength conversion, the crystal must be placed precisely symmetric about the focus of the doublet, and with its crystal axis aligned to the polarization of the incoming beam. </li>
 <li>Because efficiency of the wavelength conversion is polarization-dependent, control over the amount of light sent to the sample can be obtained by placing a second half-wave plate after the Faraday isolator. By rotating this waveplate, the intensity of light sent to the sample can be adjusted independently of the intensity sent in the pump beam (to be discussed below).</li>
 <li>The wavelength-converted light is recollimated by a second achromatic doublet (f = 500 mm) chosen such that the exiting beam is large enough to fill the back aperture of the microscope objective, and directed into an inverted microscope (IX-71, Olympus, Center Valley, PA) by means of two steering mirrors. </li>
 <li>The microscope objective, being the optical element housed within the most monolithic component of the system, defines the optic axis. To align the excitation beam to this axis, a mirror is placed in the sample plane of the microscope. The two steering mirrors are then iteratively tuned while observing the back-reflected laser beam on a CCD cam-era (μEye, IDS, Obersulm, Germany) attached to the imaging port of the microscope. Assuming that the image on the camera is centered on the microscope's field-of-view, the beam is on-axis when the focal spot is centered on the microscope chip and translation of the objective along the z-axis does not change the center point of the defocused beam. </li>
 <li>When a sample is placed in the sample plane and irradiated with laser light Raman scattering occurs. A dichroic filter placed below the microscope objective separates the wavelength-shifted Raman scattered light (and fluorescence) from the excitation beam, directing the Raman scattered light to the side port of the microscope. The microscope was modified to remove any lenses within this path, such that the signal light emerges from the microscope collimated. </li>
 <li>Because the signal beam emerging from the microscope is larger than the clear aperture of the Glan-thompson polarizers, a 0.47x telescope constructed of two achromatic doublets (f1 = 75 mm, f2 = 35 mm) is used to shrink the beam. </li>
 <li>The signal light is then polarized by a Glan-Thompson polarizer oriented at 0° with respect to vertical in the lab frame, and directed to a dichroic mirror where it is recombined with the pump beam. </li>
</ol>
3.	In order for the optical gate to operate at peak efficiency, care must be taken in the preparation of the pump beam (Kerr beam) as well. 

<ol>
	<li>The pump pulse is first magnified by 0.35x with a telescope constructed with two achromatic doublets (f1 = 35 mm, f2 = 100 mm) to match the final size of the signal beam. </li>
 <li>The pump beam is then sent into a delay line composed of a right angle prism placed on a linear translation stage that can be tuned to ensure temporal overlap of the pump and signal pulses (tuning to be discussed below).</li>
 <li>After the delay line, the beam is sent through a half-wave plate and polarizer oriented at 45° with respect to vertical in the lab frame. This ensures the proper polarization state of the pump beam when it reaches the nonlinear medium. </li>
 <li>The light is then reflected off of two steering mirrors, one with piezo-electric controls, that are used to finely adjust the position of the pump beam such that it overlaps spatially with the signal beam. The overlap is obtained by observing the pump and signal beams at two locations, one close and one far from the dichroic mirror where the beams are combined. By using the first steering mirror to overlap the two beams at the near point, and the piezo mirror to overlap the beams at the far point, the pump beam can be made exactly collinear with the signal beam. </li>
 <li>With the two beams combined, the Kerr gate and collection system are set up to maximize the collected time-gated signal. </li>
</ol>
4.	With the two beams combined, the Kerr gate and collection system are set up to maximize the collected time-gated signal. 
<ol> 
 <li>The pump and signal beams are first passed through a dichroic filter that has an OD of 6 at 404 nm to prevent any residual excitation light from exciting Raman scattering within the nonlinear medium. </li>
 <li>The pump and signal beams are then both focused by an achromatic doublet (f = 35 mm) into a 1 cm pathlength quartz cuvette containing the nonlinear material. Any nonlinear material having a suitably high nonlinear index (higher than CS2), and suitably short temporal response (&lt;2 ps), can be utilized here. For these experiments, we use carbon disulfide, CS2 , that has a nonlinear index n2 = 3.1 x 10−18 m2/W. The light is then recollimated by a second doublet with a focal length identical to the first.</li>
 <li>The beams are then passed through and Glan-Thompson analyzer on a rotating mount, and then through a set of absorption and interference filters that combined have an OD of 10 at 808 nm. </li>
 <li>Finally, the signal light is focused by an achromatic doublet (f = 35 mm) into a 50 μm multimode optical fiber, where the fiber is mounted in a stage that allows translation in x, y, and z. The fiber is then coupled to a commercial imaging spectrograph with attached CCD camera (SP2300i and Pixis 100B, respectively, both manufactured by Princeton Instruments, Trenton, NJ). </li>
 <li>To align the collection system to maximize collected signal, the analyzer is set to 0° and a test sample of toluene is placed in the sample plane. By adjusting the x, y, and z controls of the fiber mount, the collected Raman signal is optimized. </li>
 <li>To ensure proper spatial and temporal overlap of the pump and signal beams, a mirror is placed in the sample plane of the microscope. The 404 nm filter is removed from the system. With the analyzer rotated to 90° the retroreflected 404 nm beam is sent into the spectrograph with the intensity adjusted such that it does not saturate the camera. With the pump beam off, the analyzer is rotated to minimize the transmitted 404 nm signal. With the pump beam turned back on, the delay stage is slowly tuned until the transmission of the 404 nm light begins to increase. Then, by iteratively tuning the delay stage, the piezo-electric mirror, and the x, y, and z controls of the fiber, one maximizes the signal.</li>
 <li>Because the retro-reflected 404 nm beam and the Raman scattered light may take slightly different paths through the system, final adjustments are made by placing a strong Raman scatterer (such as toluene) on the sample stage and the slightly tweaking the alignment by changing the delay stage, piezeo-electric mirror, and x, y, and z controls of the fiber to optimize the Raman signal. </li>
</ol>

5.	Collection of a single time-gated Raman spectrum requires acquisition of several spectra to correct for system artifacts.

<ol>
	<li>With the analyzer set to 0°, the excitation beam on and the pump beam off, an "ungated" spectrum is taken, representing the spectrum that would be acquired without the benefit of the time-gating system. </li>
 <li>With the analyzer set to 90°, the excitation beam and the pump beam still off, a back-ground spectrum is taken, representing the amount of stray light leaking through the polarizers and other elements in the system. </li>
 <li>With the analyzer remaining at 90° and all beams on, the "gated" spectrum is taken, representing the Raman scattered light allowed through the crossed polarizers by the presence of the pump beam. </li>
 <li>Finally, with the analyzer still at 90°, the excitation beam off and the pump beam on, a second background spectrum is taken, representing the contribution to the "gated" spectrum of residual amounts of 808 nm light entering the spectrograph. </li>
 <li>Additionally, a spectrum is taken with all lasers off, characterizing the baseline "dark" signal level of the camera and electronics. </li>
 <li>Because the spectra often require lengthy integration times, it is imperative to break this integration time into several pieces (frames) to allow for proper correction of cosmic rays - a common problem when acquiring data over spans of several minutes. </li>
</ol>
6.	Once acquired, several processing steps are helpful to improve the quality and appearance of the data.
<ol>
	<li>First, all spectra are dark corrected by subtracting off the "dark" spectrum. </li>
 <li>To correct for cosmic rays, the several frames constituting one acquisition are compared to each other on a pixel-by-pixel basis. For any pixel of any frame that falls outside of 2 median deviations from the median value for that pixel across all frames, that pixel's value is replaced by the median value across all frames.</li>
 <li>The cosmic ray corrected frames are then averaged and smoothed by a Savitzky-Golay filter 1. </li>
 <li>To extract the "true" gated spectrum, we subtract the "excitation-only" and "pump-only" spectra from the measured gated spectrum. </li>
 <li>Finally, the gated and ungated spectra are then background corrected by subtracting a 5th-order polynomial, determined using the method of Lieber and Mahadevan-Jansen 2.
</li>
</ol>
7. Representative Results: 
<img src="/files/ftp_upload/2592/2592fig1.jpg" alt="Ultrafast laser spectroscopy diagram; optical path, SHG, Raman process, piezo-actuated mirror setup." data-altupdatedat="1788235205000" data-alt="Figure 1">
 Figure 1. Schematic diagram of the Kerr gating system. The pump beam path is shown in red, while the SHG path is shown in blue. The path where Raman and Fluorescence are overlapped is shown in green, while the path where the Fluorescence has been temporally filtered out is shown in yellow. Abbreviations as follows: BPF, band pass filter; CCD, charge-coupled device; DCM, dichroic mirror; FI, Faraday isolator; λ/2, half wave plate; LPF, long-pass filter; NLM, nonlinear medium; P, polarizer; SHG, second harmonic generation crystal.
<img src="/files/ftp_upload/2592/2592fig2.jpg" alt="Raman spectroscopy results; intensity vs. relative wavenumbers; gated spectrum analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Top: Raw spectra of coumarin dissolved in immersion oil. Red curve shows the spectrum taken with the gate held open (analyzer set for maximum transmission). Black curve shows the spectrum taken with the analyzer aligned for minimum transmission and a pump beam applied (the gated spectrum). Blue curve shows the spectum taken with the analyzer aligned for minimum transmission and no pump beam applied (gate held closed). Green curve shows the spectrum taken with only the pump beam applied. Dashed magenta lines indicate spectral region shown in panel below. All spectra have been smoothed with a 11 point, 3rd order Savitzky-Golay filter. Bottom: Spectra of coumarin dissolved in immersion oil after fluorescence background subtraction. Red curve is the spectrum with the gate held open, and the blue curve is the gated spectrum. The gated spectrum clearly shows the convoluted high-wavenumber peak characteristic of oils.

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No conflicts of interest declared.

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><tbody><tr><td>Lenses</td><td>Thorlabs Inc.</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 Inc.</td><td>Chameleon</td><td>30 nJ, 200 fs, 80 MHz</td></tr><tr><td>40X oil immersion objective</td><td>Olympus Corporation</td><td>UApo/340</td><td>NA = 1.35</td></tr><tr><td>Inverted microscope</td><td>Olympus Corporation</td><td>IX-71</td><td>Modified to remove all lenses in side port</td></tr><tr><td>Half wave plate</td><td>Thorlabs Inc.</td><td>AHWP05M-600</td><td></td></tr><tr><td>Glan-Thompson polarizer</td><td>Thorlabs Inc.</td><td>GTH10M</td><td>&amp;#8764;10% transmission loss</td></tr><tr><td>Spectrometer</td><td>Princeton Instruments/Acton</td><td>SP2300i</td><td></td></tr><tr><td>CCD</td><td>Princeton Instruments/Acton</td><td>Pixis 100B</td><td></td></tr><tr><td>Mathmatical software</td><td>Mathworks</td><td>MATLAB</td><td>version 2008a</td></tr><tr><td>Faraday isolator</td><td>EOT</td><td>BB8-5I</td><td></td></tr><tr><td>Piezo-electric mirror</td><td>Newport Corp.</td><td>AG-M100</td><td></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 &amp;#177; 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>CS&lt;sub&gt;2&lt;/sub&gt;</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>Cargill Labs</td><td>16242</td><td>Type DF</td></tr></tbody></table>

rejection of fluorescence background in resonance and spontaneous raman microspectroscopy

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

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

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

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

The aim of this procedure is to construct an all optical gait that passes Raman scattered light while rejecting fluorescent signals. This is accomplished by first exciting and polarizing the Raman scattering. The second step of the procedure is to prepare the pump beam to operate the gate.Thirdly, the pump and ram and pulses must be adjusted so that they overlap. The final step of the procedure is to acquire time gated spectra. Ultimately, results can be obtained that show biochemical quantification and classification through analysis of ramen signals with high signal to noise ratios.The main advantage of this technique over existing methods, such as software removal of the fluorescence background, is that the shot noise produced by the fluorescence is significantly reduced. This method can help answer key questions in the biological and biomedical fields, such as non-invasive characterization of chemical composition of endogenous fluor in bacteria cells and tissues, understanding cellular processes and diseases such as cancer, vascular, or neurodegenerative diseases by using intrinsic markers. This method may also help with the development of new probes that could be used both as fluorescence and RAM labels, as well as for non-invasive medical sensors for blood analysis.Though this method can provide insight into biological systems for biomedical engineering. It can also be applied to other fields such as biofuel, research, or telecom industry. Biological samples are typically placed on a number one thickness cover slip mounted in an ATO Fluor cell chamber.Liquid samples, particularly those toxic to humans, are placed in a small glass bottle with a cover slip cemented to the opening by means of silicone epoxy, which is then inverted for measurement place samples on the secondary stage mounted on top of the microscope stage that has its own independent focus control in order to take time gated ramen spectra, first, the excitation beam must be properly prepared. Start with light emerging from a 2.4 watt tunable pulsed GI sapphire laser. Each pulse in the 80 megahertz pulse train should have 30 nano joules of energy, a temporal width of 140 femtosecond and a spectrum centered at 808 nanometers with a spectral bandwidth of around six nanometers to prevent back reflections from reentering the laser cavity, the light should be passed through a Faraday isolator place, a half wave plate before the Faraday isolator to allow for continuous tuning of the total power sent into the system.Because six nanometers is too broad a bandwidth to resolve most ramen modes, the beam is sent through a very narrow band pass filter. Send it at 808 nanometers. Next, use an aromatic doublet to focus the light onto a five millimeter beta barium bore.Eight crystal to half the wavelength from 808 nanometers to 404 nanometers. Place the beta barium bate crystal in a mount with tip and tilt controls mounted on a translation stage. To maximize the efficiency of the wavelength conversion, the crystal must be placed precisely symmetric about the focus of the doublet and with its crystal axis aligned to the polarization of the incoming beam.Because the efficiency of the wavelength conversion is polarization dependent control over the amount of light sent to the sample can be obtained by placing a second half wave plate after the faraday isolator. By rotating this wave plate, the intensity of light sent to the sample can be adjusted independently of the intensity sent in the pump beam. The wavelength converted light is then re collimated by a second aromatic doublet chosen such that the exciting beam is large enough to fill the back aperture of the microscope objective and directed into an inverted microscope by means of two steering mirrors.The microscope objective defines the optic axis to align the excitation beam to this axis. Place a mirror in the sample plane of the microscope. The two steering mirrors are then iteratively tuned while observing the back reflected laser beam on a CCD camera attached to the imaging port of the microscope.Assuming that the image on the camera is centered on the microscope's field of view, the beam is on axis. When the focal spot is centered on the microscope chip and translation of the objective along the Z axis does not change. The center point of the defocused beam ramen scattering occurs when the sample is placed in the sample plane and is irradiated with laser light.A dichroic filter placed below the microscope objective separates the wave shifted. Ramen scattered light from the excitation beam directing the ramen scattered light to the side port of the microscope. The microscope has been modified to remove any lenses within this path, such that the signal light emerges from the microscope coated because the signal beam emerging from the microscope is larger than the clear aperture of the gland.Thompson polarizers, a 0.47 times telescope constructed of two aromatic doublets is used to shrink the beam. The signal light is then polarized by a gland Thompson polarizer oriented at zero degrees with respect to vertical in the lab frame and directed to a dichroic mirror where it is recombined with the pump beam. The pump beam is sent into a delay line composed of two mirrors at right angles to each other, both placed on a linear translation stage that can be tuned to ensure temporal overlap of the pump and signal pulses.After the delay line, the beam is sent through a halfway plate and polarizer oriented at 45 degrees with respect to vertical in the lab frame. This ensures the proper polarization state of the pump beam when it reaches the non-linear medium. The light is then reflected off of two steering mirrors, one with piezoelectric controls, which should be used to finally adjust the position of the pump beam such that it overlaps spatially with the signal beam.To obtain this overlap, observe the pump and signal beams at two locations, one close and one far from the dichroic mirror where the beams are combined by using the first steering mirror to overlap the two beams at the near point and the pizo mirror to overlap the beams at the far point, the pump beam can be made exactly coline with the signal beams. Next, the corrugate and collection system should be set up to maximize the collected time gated signal. To do this, the pump and signal beams are first passed through a diic filter that has an OD of six at 404 nanometers to prevent any residual excitation light from exciting and scattering within the non-linear medium.The pump and signal beams are then focused by an aromatic doublet into a one centimeter path length quartz s vete containing the non-linear material, any non-linear material, having a suitably high non-linear index and suitably short temporal response can be utilized.Here. For these experiments, we use carbon di sulfide. The light is then re collimated by a second doublet with a focal length identical to the first.The beams are then passed through a gland Thompson analyzer on a rotating mount, and then through a set of absorption and interference filters that combined have an OD of 10 at 808 nanometers. Finally, the signal light is focused by an aromatic doublet into a 50 micron multimode optical fiber, where the fiber is mounted in a stage that allows translation in X, Y, and Z.The fiber is then coupled to a commercial imaging spectrograph with attached CCD camera to align the collection system to maximize collected signal, set the analyzer to zero degrees and place a test sample of toluene in the sample plane, optimize the collected Raman signal by adjusting the X, Y, and Z controls of the fiber mount to ensure proper spatial and temporal overlap of the pump and signal beams. Place a mirror in the sample plane of the microscope.Then remove the 404 nanometer filter from the system. Rotate the analyzer to 90 degrees so that the retro reflected 404 nanometer beam is sent to the spectrograph with the intensity adjusted such that it does not saturate the camera. Now with the pump beam off, rotate the analyzer to minimize the transmitted 404 nanometer signal.Then turn the pump beam back on and slowly adjust the delay stage until the transmission of the 404 nanometer light begins to increase. Next iteratively turn, the delay stage, the piezoelectric mirror and the X, y, and Z controls of the fiber to maximize the signal because the retro reflected 404 nanometer beam and the Raman scattered light may take slightly different paths through the system. Make final adjustments by placing a strong Raman Scatterer such as toluene on the sample stage, replacing the Raman filter and slightly tweaking the alignment by changing the delay stage pizo electric mirror, and X, Y, and Z controls of the fiber to optimize the Raman signal.Now the system is ready to collect spectra. This first requires the acquisition of several background curves to correct for system artifacts. First, with the analyzer set to zero degrees, the excitation beam on and the pump beam off.Obtain an ungated spectrum, then set the analyzer to 90 degrees and collect a background spectrum representing stray light leaking through the polarizers. Next, with the analyzer remaining at 90 degrees, turn the ramen beam off and pump beam on. Collect a second background spectrum representing the amount of pump light leaking through the dichroic filters.Finally, with all lasers off, collect a baseline dark spectrum representing the dark current level of the camera and electronics. Finally turn all beams on and collect a gated spectrum. To get the true spectrum of just the light through the gate, the two background spectrum and the dark spectrum must be subtracted from this gated spectrum.Here we see a schematic diagram of the corrugating system. The pump beam path is shown as a solid red line while the SHG path is shown as a solid navy line. The path where ramen and fluorescence are overlapped is shown in green.While the path where the fluorescence has been temporarily filtered out is shown in yellow. Here we see the raw spectra of coumarin dissolved in immersion oil. The red curve shows the spectrum taken with the gate held open with the black curve shows the spectrum taken with the analyzer aligned for minimum transmission and a pump beam applied.The blue curve shows the spectrum taken with the analyzer aligned for minimum transmission and no pump beam applied, and the green curve shows the spectrum taken with only the pump beam applied. All spectra have been smooth with an 11 point third order KY gole filter. The dashed magenta lines indicate the spectral region shown in the following graph.Here we see spectra of coumarin dissolved in immersion oil after fluorescence background subtraction. The red curve is the spectrum with the gate held open, and the blue curve is the gated spectrum. The gated spectrum clearly shows the convoluted high wave number, peak characteristic of oils.While attempting this procedure, it is important to remember that the laser must first have enough pulse energy to drive the ga, and that the system requires exquisite spatial and temporal overlap of the two pulses. After watching this video and reading the attached protocol, you should have a good understanding of how to separate the laser beam into an excitation and curve beam. How to overlap those two beams, both spatially and temporally.How to record a ramen signal via spectrograph and C, c, D, and also how to visualize and analyze the ramen signal. Don't forget that working with lasers can be extremely dangerous, and precautions such as wearing laser goggles should always be taken while attempting this procedure. Additional safety rules apply for the use of the nonlinear material and for the use of your specific sample.

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Research

JoVE Journal

Bioengineering

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

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.

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

We present a technique for Raman spectroscopic analysis of highly radioactive samples compatible with any standard micro-Raman spectrometer, without any radioactive contamination of the instrument. We also show some applications using actinide compounds and irradiated fuel materials.

A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

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

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

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

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

A wide-field Fourier-transform microscope, based on a compact and ultra-stable birefringent interferometer, allows the parallel acquisition of spectra for all pixels of a 2D detector. The time-domain approach enables the disentanglement of photoluminescence and Raman signals, and allows rapid Raman mapping (~5 ms/pixel) with ~1-µm spatial and 23-cm-1 spectral resolution.

A Multimodal Wide-Field Fourier-Transform Raman Microscope

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

Learn to construct an all-optical gate that isolates Raman signals from fluorescence background using ultrafast switching. This method enables high signal-to-noise biochemical quantification for biophysical research.

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

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