This protocol can help scientist interested in nonlinear microscopy to understand the key components, the setting up, and the alignment procedure of a microscope based on stimulated Raman scattering. The main advantages of SRS microscopy are its abilities to perform label-free imaging based on vibrational contrast and to acquire an image in a few seconds. SRS microscopy has taken label-free imaging to new heights especially studies of complex biological structures such as lipids, which are fundamental to cells and cellular architecture.
SRS signal is detected as a small change in the intensity of the probe beam and it can be corrupted by noise. Therefore an accurate sign is crucial. To begin, align the OPO and the Titanium-sapphire laser beams so that they both reach the microscope.
Next, place the laser beam position sensor's detectors in two positions in between dichroic mirror one and mirror six. The first position is located close to dichroic mirror one and the second one is close to mirror six. For each position, use the sensors to detect the X and Y coordinates of the OPO beam.
Importantly, verify that the X and Y coordinates of the Titanium-sapphire laser beam are of the same OPO in both positions of the sensor's detectors. If, in some positions, the coordinates to not coincide, tune the tilt of the adjacent mirror to compensate. Follow this same procedure to align the Titanium-sapphire beam positions with respect to OPO for the path in-between mirror's six and seven.
Install an additional mirror on a flip-flop mount in-between mirror six and seven and flip the mount of the mirror to direct the beam into the autocorrelator. Power on the autocorrelator controller, start the software application on the computer controlling it, and set the beam distance adjustment screw of the autocorrelator to its normal position at 8.35 millimeters. Then, stop the Titanium-sapphire beam and release and project the OPO beam from the additional mirror to the input mirror of the autocorrelator.
Try to adjust the input mirror to maximize the laser pulse signal as shown here. Next, stop the OPO beam and release and project the Titanium-sapphire beam from the flip-flop mounted mirror to the input mirror and into the autocorrelator. Repeat the optimal beam adjustment until the autocorrelator signal, shown here, is obtained.
Now, set the beam distance adjustment screw to the cross position at 7.30 millimeters. Release both beams and scan the delayed line to overlap the two beams. Obtain resulting cross-correlator signal, as shown here.
Then, flip the flip-flop mounted mirror so that the beams can reach mirror seven and the scan head of the microscope. Remove the condenser and use the escape button to temporarily retract the 60x subjective lens. Then rotate the nose-piece to move the 60x subjective lens off the optical path.
Next, mount the detector to the upper part of the microscope using the external mechanical mount. Connect the detector's output through a 50 Ohm Low Pass Filter to an oscilloscope. Now, turn on the processor that controls the scanner head and project the OPO beam into the scanner head of the microscope.
Monitor the OPO signal and maximize the power measured by the detector using an XY translator. Then, switch the beam from the OPO laser to the Titanium-sapphire laser and verify that a signal is also obtained for the Titanium-sapphire laser. This indicates that both beams are well aligned.
Finalize the beam alignment by rotating back the nose-piece to introduce the 60x subjective. Then, use the refocus button on the microscope to regain the finalized focus to the 60x microscope objective lens. Finally, place the objective, with a magnification of 40x, in place of the condenser without touching or disturbing the sample.
Set the power of the Titanium-sapphire and OPO lasers measured before the microscope to 30 milliwatts for both beams. Then, set the wavelength of the OPO laser to a different value, with respect to the previous one, so that the pump and probe are not in resonance with the vibrational frequency of the beads. Next, release both beams, so that they enter the microscope.
Run the scanning delay line computerized translator and record the measured intensity using the lock-in amplifier for each position of the delay line. Wait until the delay line scanning is complete. Now, set the wavelength of the OPO back to 1076 nanometers, so that the pump and probe are in resonance with the vibrational frequency of the beads.
Run the scanning delay line computerized translator and wait until the delay line scanning is complete. Finally, set the obtained overlap beam position and the delay line for next acquisition of stimulated Raman scattering images. To optimize the spatial synchronization of the beams, begin by stopping the Titanium-sapphire beam, and reducing the OPO power to eight milliwatts.
Next, connect the detector readout to the data acquisition card. Run the data acquisition program along with the microscope scanning console. When finished, save the file and process the data to get an image like the one shown here.
Next, stop the OPO beam and reduce the Titanium-sapphire power to 2.5 to 4.5 milliwatts. Connect the detector with the lock-in amplifier and its readouts with the data acquisition card. Then, again run the data acquisition program along with the microscope scanning console.
When finished, save the file and process the data to get an image like the one shown here. Introduce a stack of filters in-between the 40x objective and the photodiode to remove the pump pulses and acquire only the stoke signal. Then, set the pump signal to 810 nanometers with a focused power of eight milliwatts.
Set the probe signal to 1076 nanometers with the same focused power of eight milliwatts to investigate a typical Carbon-Hydrogen bond of polystyrene with a Raman shift of 3054 inverse centimeters. Connect the detector with the lock-in amplifier and the lock-in amplifier's readout to the data acquisition card. Finally, set the pixel format and acquisition time in the microscope program and run it and the data acquisition system, saving the matrix file once it is complete.
An example measurement from a single point of the sample is displayed here. When the beam is not overlapped in time or space, the obtained result is off resonance, where the amplitude of signal, as measured by a lock-in amplifier, is zero. The phase of this signal, however, jumps between negative and positive values.
If the beams are overlapped in space, the signal increases and reaches its maximum when the beams are perfectly overlapped in time, while the phase starts to achieve a fixed value during the time at which the beams are overlapped. In a transmission image, a single beam is used and the transmitted beam intensity from the sample is measured by a photodiode. On the left, the transmission image was obtained using OPO, while on the right, the transmission image was obtained using Titanium-sapphire.
A typical example of an SRS image is shown here, in which label-free images of polystyrene beads with diameters of three micrometers are reported. In order to obtain a high-quality image, based on SRS microscopy, the alignment of a microscope is critical. Therefore, all indicated step in the protocol have to be carried out carefully.
