Name: implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope
Uploaded: 2016-07-17T14:00:00
Description: 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

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...

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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">
			<td height="21" style="height:21px;width:419px;">Photomultiplier tube</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:419px;">ZEN software</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;">Bandpass filters</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td>LSM BiG 1935-176</td>
			<td>400-480 nm ; 500-550 nm ; 465-610 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dichroic mirror</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
			<td>Cutoff wavelength 760 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Silver mirrors</td>
			<td style="width:120px;">Newport</td>
			<td>10D20ER.2&#160;</td>
			<td>&#955;/10, 480-20,000 nm , Quantity 4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Single-axis translation stage with standard micrometer</td>
			<td style="width:120px;">Thorlabs</td>
			<td style="width:317px;">PT1/M</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Aluminium breadboard</td>
			<td style="width:120px;">Thorlabs</td>
			<td>MB1015/M&#160;</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Mirror mount</td>
			<td style="width:120px;">Thorlabs</td>
			<td>KMSS/M&#160;</td>
			<td>Quantity 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror holder for &#216;1&#34; Optics&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>MH25</td>
			<td>Quantity 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris diaphragms&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>ID8/M</td>
			<td>Quantity 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protective box</td>
			<td style="width:120px;">Thorlabs</td>
			<td>TB4, XE25L900/M, T205-1.0, RM1S</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</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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Research

JoVE Journal

Biology

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

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

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

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

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.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...