The authors want to thank Dr. Philippe Combette (IES, UM, Montpellier, France) for the loan of the fast oscilloscope and acknowledge financial supports from Montpellier RIO Imaging (MRI). HR acknowledges ANR grants France Bio Imaging (ANR-10-INSB-04-01) and France Life Imaging (ANR-11-INSB-0006) infrastructure networks for coherent Raman imaging developments. This work was mainly supported by an European Research Council grant (FP7-IDEAS-ERC 311610) and an INSERM - AVENIR grant to NT.

<img alt="Figure 1" src="/files/ftp_upload/54262/54262fig1.jpg" /> 
Figure 1. Schematic view of the general set-up. It includes the Ti:sapphire (680 - 1,080 nm) and the OPO (1,050 - 1,300 nm) lasers, the delay line with the 4 mirrors (M1 to M4), the fast oscilloscope, the photodiode and two fixed iris diaphragms I1 and I2. Mirrors M2 and M3 are fixed on a linear translation stage enabling to change the...

<ol>
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The most challenging part of the work is the temporal synchronization of the laser beams. It requires a fast photodiode combined with a fast oscilloscope, but only a rough overlapping in time can be performed at first. Then a further adjustment of few cm is required. Finally, micrometer moves by a linear translation stage allows performing the final fine adjustment of the delay line length in order to trigger the CARS signal. This signal is maintained in a narrow range of around 20 micrometers, as observed by tuning the ...

Optical microscopy has become a major technique for nondestructive visualization of dynamic processes in living biological systems with a subcellular resolution. Fluorescence microscopy is currently the most popular imaging contrast used in live cells due to its high specificity and sensitivity1. A large palette of fluorescent probes has emerged (exogenous dyes, genetically encoded proteins, semiconductor nanoparticles). Various sample illumination fluorescent-based techniques have flourished (such as confocal or two-photon microscopy) to perform 3D imaging and to reduce a main drawback of this technique which is photobleaching2. Other limitations include the requirement of fluorophore labeling because most of molecular species are not intrinsically fluorescent and therefore these fluorophores have to be artificially introduced in the imaged sample. This artificial manipulation may be disruptive especially for small molecules or induces potential photo-toxicity. These reasons make fluorescence microscopy not well suited for in-vivo observations. Hence, the use of optical imaging techniques with high sensitivity and specific molecular contrasts without the use of fluorescent molecules is highly desirable in biomedical science.
Several nonlinear optical imaging techniques without labeling or staining have emerged, including second-harmonic generation (SHG)3,4 and third-harmonic generation (THG)5. SHG microscopy has been used to image structural arrangements at the supramolecular level such as microtubules or collagen6. THG is generated from optical heterogeneities such as interface between an aqueous medium and lipids7. THG was also demonstrated to image myelin8,9. Both techniques can be implemented on a two-photon fluorescence microscope and require only one laser beam. However they require high power laser intensity (typically 50 mW at 860 nm for SHG10, 25 - 50 mW at 1,180 nm for THG9), which is deleterious in living samples, and do not provide the chemical specificity that is required to unambiguously image specific biological structures.
Chemically selective imaging can be obtained from inherent molecular vibration signals based on Raman scattering. When a beam of light hits matter, photons can be absorbed and scattered by atoms or molecules. Most of the scattered photons will have the same energy, i.e., frequency, as the incident photons. This process is called Rayleigh scattering. However, a small number of photons will be scattered at an optical frequency different from the frequency of the incident photons, i.e., with an inelastic scattering process called Raman scattering. The difference in energy originates from excitation of vibrational modes depending on molecular structure and environment. Therefore, spontaneous Raman scattering provides chemically selective imaging as different molecules have specific vibrational frequencies. However it is limited because of its extremely weak signal. Confocal Raman microscopy has been developed and provides 3D spatial resolution, but it requires high average power and long acquisition time11. To overcome these difficulties, recent advances in laser technology have allowed the rise of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS)11,12,13.
CARS is a third-order nonlinear optical process. Three laser beams, composed of a pump beam at frequency &#969;P, a Stokes beam at frequency &#969;S and a probe beam (most often being the pump) are focused in a sample and generate an anti-Stokes beam at frequency &#969;AS= (2&#969;P - &#969;S)14. The anti-Stokes signal can be significantly enhanced when the frequency difference between the pump and the Stokes beams is tuned to a Raman molecular vibration &#937;R=(&#969;P - &#969;S). CARS signal is based on multiple photon interaction. It generates therefore a coherent signal orders of magnitude stronger than spontaneous Raman scattering.
CARS microscopy was first experimentally demonstrated by Duncan et al.15. Zumbusch et al. improved then the technique, by using two focused near-infrared femtosecond laser beams with an objective lens of high numerical aperture, allowing the phase matching condition of CARS and avoiding the two-photon non-resonant background16. CARS microscopy has therefore emerged as a powerful tool for live cell and tissues imaging, by chemically detecting molecules such as lipids (via C-H stretch vibration)17,18, water (via O-H stretch vibrations), proteins, DNA in live cells19,20 but also deuterated chemical compounds for pharmaceutical21 and cosmetic applications22.
The major limitation of nonlinear microscopy originates from the complexity and the cost of the optical sources. A CARS system requires two wavelength tunable lasers with short pulse durations and with temporally and spatially synchronized pulse trains. Early CARS microscopes were based on two synchronized picosecond Ti:sapphire lasers20. CARS imaging was also obtained from a single femtosecond Ti:sapphire laser generating a supercontinuum light source23. Recently, laser sources composed of a single femtosecond Ti:sapphire laser pumping a tunable optical parametric oscillators (OPO) have been used for CARS microscopy. This set-up allows intrinsically temporally synchronized beams with a difference of frequency between the pump and the Stokes beam covering the full molecular vibrational spectrum24. In addition, laser scanning microscopes based on a turn-key fs laser and an OPO, primarily used for two-photon fluorescence (TPF) are now available for non-physicists. The potential of such set-ups can be greatly enhanced without requiring supplementary investment by the incorporation of other nonlinear optical imaging, since each nonlinear (NLO) imaging modality is sensitive to specific structures or molecules. Multimodal NLO imaging therefore capitalizes the potential of NLO microscopy for complex biological samples25. The coupling of these techniques has allowed the investigation of many biological questions, in particular on lipid metabolism, skin or cancer development26, skeletal muscle development27, atherosclerotic lesions28. Moreover, the implementation of laser beam scanning with CARS gives the capability of high-rate imaging, i.e., an appealing tool to study dynamical processes in vivo.
The aim of this work is to show each step to implement the CARS technique on a standard multiphoton laser scanning microscope. The microscope is based on a fsec Ti:sapphire laser and an OPO (pumped by the Ti:sapphire laser) operated by a software for biologists. The integration was performed by adjusting the length of one of the laser beam path in order to synchronize in time the two beams. We describe the step-by-step implementation of this technique which requires only basic background in experimental optics. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodents, and we show this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

<table border="0" cellpadding="0" cellspacing="0" width="1157">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:419px;">Oscilloscope</td>
			<td style="width:120px;">Tektronix</td>
			<td style="width:317px;">TDS 520D&#160;</td>
			<td style="width:301px;">500 MHz&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Photodetector</td>
			<td style="width:120px;">Thorlabs</td>
			<td>DET08C/M, T4290</td>
			<td>5 GHz InGaAs, 800-1700 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Ti:Sapphire laser Chameleon Ultra Family II</td>
			<td style="width:120px;">Coherent</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Optical parametric oscillator OPO Compact Family</td>
			<td style="width:120px;">APE Berlin</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Axio Examiner microscope LSM 7 MP</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Motorized periscope</td>
			<td style="width:120px;">Newport</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Objective W Plan-Apochromat 20x/1.0</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Beam combiner</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Acousto-optic modulator</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">OPO power attenuator</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
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			<td>400-480 nm ; 500-550 nm ; 465-610 nm</td>
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			<td>&#955;/10, 480-20,000 nm , Quantity 4</td>
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			<td>Quantity 1</td>
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			<td>MB1015/M&#160;</td>
			<td>Quantity 1</td>
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			<td style="width:120px;">Thorlabs</td>
			<td>KMSS/M&#160;</td>
			<td>Quantity 4</td>
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			<td style="width:120px;">Thorlabs</td>
			<td>ID8/M</td>
			<td>Quantity 3</td>
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			<td style="width:120px;">Thorlabs</td>
			<td>TB4, XE25L900/M, T205-1.0, RM1S</td>
			<td>Quantity 1</td>
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			<td height="21" style="height:21px;">Optical posts</td>
			<td style="width:120px;">Thorlabs</td>
			<td>TR40/M, PH50/M, PH75/M, BA2/M</td>
			<td>Quantity 8 (lengths depending on the set-up)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:419px;">661-690 nm bandpass filter</td>
			<td>Semrock</td>
			<td>676/29 nm BrightLine&#174; single-band bandpass filter</td>
			<td>Quantity 1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fluorescent beads</td>
			<td style="width:120px;">ThermoFisher</td>
			<td>TetraSpeck&#8482; Fluorescent Microspheres Size Kit</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Laser viewing card</td>
			<td style="width:120px;">Thorlabs</td>
			<td style="width:317px;"></td>
			<td>IR laser viewing card</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Laser safety glass</td>
			<td style="width:120px;">Newport</td>
			<td style="width:317px;">LV-F22.P5L07&#160;</td>
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			<td height="21" style="height:21px;width:419px;">FluoroMyelin&#8482; Red Fluorescent Myelin Stain&#160;</td>
			<td style="width:120px;">ThermoFisher</td>
			<td>F34652</td>
			<td></td>
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</table>

implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

The pulse train frequency of standard Ti:sapphire laser is typically around 80 MHz. The OPO has the same frequency since it is pumped by the Ti:sapphire laser. A fast oscilloscope of at least 200 MHz is therefore required. A fast photodiode in the range 600 to 1,100 nm is also required. The maximal temporal shift occurs when the Ti:sapphire and the OPO signals are shifted of 1/(2&#215;80&#215;106) = 6.2 nanoseconds. It corresponds to a maximum beam path shift of 1.9 m. ...

Watch this Scientific Journal Video about Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope at JoVE.com

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

The overall goal of this procedure is to implement a complimentary microscopy technique called CARS, which allows label-free, chemically selective, lifestyle imaging on a standard multi-photon laser scanning microscope based on a femto-second titanium-sapphire laser and an optical parameter oscillator. This method can help answer key questions in the field of biology or neuroscience such as understanding of lipid-related diseases, skin penetration mechanisms, or skin disorders. The main advantage of this technique is that it's easy to implement on a standard laser scanning microscopy based on Ti:Sapphire and OPO lasers.Demonstrating the procedure will be Hassan Boukhaddaoui, research associate and head of the imaging platform of the laboratory and Vasyl Mytskaniuk. Turn on the titanium-sapphire and OPO lasers as presented in the text protocol. Switch on the microscope computer.Turn on the microscope component switches. Start the software by double-clicking the icon on the desktop and set it up according to the text protocol. Then, place a dichroic mirror with a cutoff wavelength at 760 nanometers in the side port slider.Locate it in the infinity space above the objective nose piece. Set the narrow band pass filter and the reflector cube in front of PMT1 to record only the CARS signal at 670 nanometers to reproduce the presented results. Then, place specific narrow band filters in front of PMT3 for fluorescence observation of the myelin, and in front of PMT4 for SHG observation.In the software, set the signal on the detector with an ad hoc band pass filter. Open the light path tool in the setup manager menu in the acquisition tab, then activate the desired PMT and select a color for the channel. For example, use green for CARS, and magenta for SHG.The two beams that originate from the same titanium sapphire laser are not in sync when they reach the microscope because the OPO beam is delayed when it is generated. Follow this procedure to resynchronize them by adjusting the length of one of the laser beam pads by incorporating a delay line. The use of a fast photo diode and a fast oscilloscope is required.First, using BNC cables, connect the input channel CH1 of the oscilloscope to the electrical BNC laser output sync out. Next, connect the input channel CH2 to the photo diode. Then, choose the CH1 channel as the trigger by pressing trigger menu, then the main menu button source and then the side menu button that corresponds to the channel selected as CH1.Now position and fix with optical mounting posts to the photo diode in the focal plane of a 10x air microscope objective. In the Channels tool, define the titanium-sapphire laser wavelength at 830 nanometers at low power, which is less than 1%of the full power. In the Acquisition Mode tool, reduce the scan area to one point in order to illuminate the photo diode with the tiniest beam.Then switch on the laser scan by clicking on the continuous button. Press Autoset on the oscilloscope and manually move the photo diode to get a pulse train on the oscilloscope screen. Press the run/stop button to freeze the display.Next, switch off the titanium-sapphire laser scan. In the software, click the Channels tool, deselect the 830 nanometer laser and set the OPO signal to 1107 nanometers and low power. Then switch on the OPO laser scan, record the pulse trains of the OPO laser on the oscilloscope and switch off the OPO laser scan.Now, compare the temporal shift between the titanium-sapphire and the OPO signals. Calculate the length of the delay line, which will be used to position the mirrors. Before proceeding, put on safety goggles and remove any chain bracelets or watches from wrists.Now, open the laser titanium-sapphire line where the delay line will be implemented by removing the protective tubes. Then select a wavelength in the visible range in order to be able to easily observe the laser beam, for example, 700 nanometers at low power, and switch on the laser scan. Now put two iris diaphragms into the open laser line using the optical mounting posts.Position one iris at the exit of the delay line and place the other iris at the entrance of the periscope. Next, decrease the iris diaphragm aperture and adjust the diaphragm positions to fit the laser beam path then fix them on the optical table. Finally, adjust the vertical position of a third mobile iris diaphragm.The size diaphragms will serve as a control for the realignment procedure to check the position of the laser beam while successively positioning the four mirrors of the delay line. Now, place the mirror M1 on a compact kinematic mirror mount at the entrance of the delay line, and adjust its position and its orientation to maintain the beam height with the use of the mobile iris diaphragm. Then place mirrors M2 and M3 at 90 degrees onto the translation stage according to the calculated delay line length and adjust their orientation using the mobile iris diaphragm.Set M4 at the exit f the delay line just before the iris and carefully adjust its position and angle to fit the laser beam path through the two fixed iris diaphragms. Now position the laser viewing card at the output of the microscope objective and check the laser beam profile. If necessary, slightly adjust the orientation of M4 to observe a uniform bright disc.Lastly, reposition the fast photo diode under the laser beam in the sample focus plane. Then observe the temporal shift between the titanium-sapphire laser beam and the OPO beam on the oscilloscope. If necessary, change the delay line length by moving the whole translation stage to synchronize the pulses.The spacial overlap of the two beams needed for a CARS signal is obtained by visualizing fluorescent polystyrene beads fixed to a microscope slide. Focus on the beads using a 20x water objective. Now, in the Channels tool of the acquisition tab, add track one, or use the existing track.Select the wavelength at 830 nanometers and low power for the titanium-sapphire laser beam. Toggle the color to green in the track one box from the Channels window, and the PMT3 or the PMT4 box from the light path window. Next, add track two for the OPO laser beam and set the wavelength at 1107 nanometers and low power.Toggle the color to red in the track two box from the Channels window and in the PMT3 or the PMT4 box from the light path window. Now, select the maximum scan area and adjust the gain of both tracks to 600. Then sequentially apply the scan of the two beams onto the sample by clicking on continuous.Observe the image in the screen area in the 2D view. Increase the power of both lasers. If necessary, slightly move the focusing drive to find the focus plane of the beads.Finally, adjust the crop and zoom the image in on a single bead or on a group of adjacent beads. Next, use the periscope controller to overlap the beams in the XY plane. In the software, open the Maintain tab.Click on the system options and display the motorized periscope tool window. Further details are discussed in the text protocol. After overlapping images from both laser beams, synchronization is achieved.To achieve the precise temporal adjustment to activate CARS, prepare a glass slide with a droplet of olive oil and cover slip. Then using a 20x water immersion objective, focus on the edge of the cover slip. Now, on track one, set the wavelength to 830 nanometrs for the titanium-sapphire laser beam and at 1107 nanometers for the OPO.Toggle both lasers in track one to get a simultaneous scan. Start with both lasers at low power. Next, on the light path window, uncheck PMT4 and select PMT1.Then choose the maximum scan area and switch on the laser scans by clicking on the continuous button. Adjust the gain to 600 and, if necessary, increase the power to both lasers. Next, adjust the display intensity in the display view option control block.Then slowly move the translation stage of the delay line until the signal becomes significantly enhanced. Next, decrease the power to both lasers. Now, check whether the signal is a CARS signal by alternatively switching off one of the two lasers.If the signal intensity becomes weaker or disappears, a CARS signal has been obtained. Since the final system is dedicated to non-physicists, enclose the light path of the delay line with an enclosure box and tubes to avoid direct access to a harmful, nonvisible, high peak powered laser beam. However, do provide access to the translation stage knob.A fluro-myelin red dye, which has selectivity for myelin, was used to observe the myelin sheath. Simultaneous illumination at 830 and at 1095 nanometers provides a CARS signal. The circles correspond to the myelin sheath surround axons in a transversal cut.The same structure is found while overlapping CARS and fluorescence images. The level of detail of CARS and fluorescent labeling is very similar. Potentially obviating the need for dye use.The system can also simultaneously generate a second harmonic signal from the collagen fibers at the outer service of the sciatic nerves. A different detector is used to record a signal at 550 nanometers since a narrow band pass filter at 670 nanometers is used for the CARS signal recording. The fibers are illustrated by a false magenta color.The CARS signal alone clearly shows the myelin sheaths. By simultaneously visualizing both signals, the myelin sheaths in green are seen surrounded by collagen fibers in magenta. A CARS signal requires less energy than SHG or THG signals.CARS signals were achieved with four milliWatts at 830 nanometers and 13 milliWatts at 1095 nanometers, while 50 milliWatts at 1095 nanometers was required to obtain an SHG signal. After watching this video, you should have a good understanding how to modify step by step one of the laser beam paths to temporally synchronize both laser beams. The implementation of the delay line can be achieved in a few weeks by biologists with basic background in experimental optics or in collaboration with physicists.Don't forget that working with high power, invisible laser beams can be extremely hazardous. And precautions, like wearing goggles while performing the procedure and final enclosure of the laser beam path should be taken.

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JoVE Journal

Biology

11.1K Views.  INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier. The overall goal of this procedure is to implement a complimentary microscopy technique called CARS, which allows label-free, chemically selective, lifestyle imaging on a standard multi-photon laser scanning microscope based on a femto-second titanium-sapphire laser and an optical parameter oscillator. This method can help answer key questions in the field of biology or neuroscience such as understanding of lipid-related diseases, skin penetration mechanisms, or skin disorders. The main advan...