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.
