We appreciate V. Tufano from IMM CNR for his valuable technical assistance and Giacomo Cozzi, product specialist from Nikon Instruments, for useful discussions and continuous support. This work was partially supported by Italian National Operative Programs PONa3 00025 (BIOforIU) and by Euro-Bioimaging large-scale panEuropean research infrastructure project.

1. Starting up the laser system
<ol>
	<li>Check if the temperature of chillers is maintained at or below 20 &#176;C.</li>
	<li>Check if the humidity control unit is working properly and humidity is maintained at a value around 40%.</li>
	<li>Turn on the Ti:Sa laser, strictly following the instructions in the manual.</li>
	<li>Set the wavelength to 810 nm.</li>
	<li>Turn on the OPO and the connected mini-computer. Run the application that controls the OPO laser.</li>
	<li>Select bypass if 100% of the Ti:Sa laser output is required at the exit of the OPO box.</li>
	<li>Deselect bypass if 20% of the Ti:Sa laser output and the OPO laser output are required at the exit of the OPO box.</li>
	<li>Open the shutter of Ti:Sa and release the Ti:Sa beam to the OPO input.</li>
	<li>Release the two laser beams at the OPO exit by clicking signal-out and pump-out.</li>
	<li>Wait until both lasers Ti:Sa and OPO are stabilized (around 45&#8211;60 min).</li>
	<li>Verify the beam spot at the exit of the OPO box for both Ti:Sa and OPO using a paper detector card and check the power using a power meter.</li>
	<li>Tune the OPO laser wavelength to 1076 nm.</li>
	<li>Reduce the power to ~10 mW for each laser beam to perform alignment.</li>
</ol>
2. Setting up the microscope
<ol>
	<li>Implementation of high frequency modulation transfer method
	<ol>
		<li>Carry out the optical alignment procedure of the modulator such that the Ti:Sa beam enters and exits without any distortion.</li>
		<li>Turn on the function generator and generate a TTL signal (square wave with amplitude = 5 V, offset = 2.5 V, frequency = 5 MHz).</li>
		<li>Divide the TTL signal into two parts using a T junction; one for EOM and the other for the lock-in amplifier (LIA) (see Figure 2).</li>
		<li>Check all signal levels with an oscilloscope.</li>
		<li>Turn on the LIA and connect the generator output channel to the reference channel of the LIA.</li>
		<li>Connect the generator output channel to the high voltage power amplifier of EOM.</li>
		<li>Turn on the amplifier and set the voltage to almost the maximum level. Monitor the beam power at the exit of EOM.</li>
	</ol>
	</li>
	<li>Integration of mechanical mounting to fix PD and assign x and y relative motion 
	NOTE: With the microscope, an external mount is introduced, equipped with a micrometer that has motion control in x and y directions.
	<ol>
		<li>Mount two travel translation stages to allow movements along the x and y directions (see Figure 3).</li>
		<li>Fix the stage on a &#216;1.5&#34; post of appropriate height.</li>
		<li>Mount the PD to an external mount.</li>
		<li>Maximize the beam power&#160;at PD, adjusting the PD positions (x and y coordinates) using the micrometers attached to the mount (see Figure 3).</li>
	</ol>
	</li>
</ol>
3. Alignment of microscope
<ol>
	<li>Spatial overlap of the beams 
	NOTE: Considering the OPO beam as a reference and taking advantage of a position sensitive detector, the Ti:Sa should be overlapped to OPO according to the following procedure:
	<ol>
		<li>Align the OPO and the Ti:Sa laser beams so that they both reach the microscope.</li>
		<li>Place the laser beam position sensors detectors in two positions in between dichroic mirror 1 and mirror 6, the first position is located close to dichroic mirror 1 and the second one is close to mirror 6. For each position, use the sensors to detect the x and y coordinates of the OPO beam (follow Figure 1).</li>
		<li>Verify that the x and y coordinates of the Ti:Sa laser beam are the same OPO in both positions of the sensors detectors. If in some positions the coordinates of Ti:Sa and OPO do not coincide, tune the tilt of the adjacent mirror to compensate the difference (follow Figure 1).</li>
		<li>Follow the same procedure to align the Ti:Sa beam positions with respect to OPO for the path in between M6-M7&#160;(follow Figure 1).</li>
	</ol>
	</li>
	<li>Temporal synchronization of the beams
	<ol>
		<li>Use of the fast photodiode plus oscilloscope:
		<ol>
			<li>Stop propagation of the Ti:Sa and OPO beams and place a fast detector in front of the OPO beam (in between M6 and M7).</li>
			<li>Connect the trigger signal provided by the Ti:Sa laser box with an oscilloscope in channel 2.</li>
			<li>Connect the detector cable with the oscilloscope in channel 1 and visualize the OPO temporal profile.</li>
			<li>Record the time (abscissa) measured by the oscilloscope corresponding to its maximum value, namely t1.</li>
			<li>Stop the OPO beam and release the Ti:Sa beam.</li>
			<li>Visualize the Ti:Sa temporal profile and record the time (abscissa) corresponding to its maximum value, namely t2.</li>
			<li>Minimize the difference between t1-t2 using the delay line to overlap the two beams. In our case, the minimum measurable difference is 10 ps.</li>
			<li>Remove the fast detector in between M6 and M7.</li>
		</ol>
		</li>
		<li>Use of the autocorrelator: 
		NOTE: In the schematic diagram shown in Figure 1, an autocorrelator is installed without interfering with optical paths of the beams. In addition, an additional mirror is introduced and mounted on a flip-flop mount (referred to as FFM/AM) in between M6 and M7 to divert the beam into the autocorrelator.
		<ol>
			<li>Flip the AM to direct the beam into the autocorrelator.</li>
			<li>Stop the Ti:Sa and release the OPO.</li>
			<li>Set the beam distance adjustment screw micrometer of the autocorrelator to Normal position (8.35 mm).</li>
			<li>Power on the autocorrelator controller and start the software application on the personal computer controlling it.</li>
			<li>Project the OPO beam from FFM/AM to the input mirror into the autocorrelator.</li>
			<li>Control the reflection spot (using a paper detector card) of the beam on the alignment window of the autocorrelator.</li>
			<li>In the case of no-beam or low-beam intensity, adjust the position and orientation of FFM/AM to the optimum extent, and try to adjust the input mirror (mounted on the autocorrelator) to maximize the laser pulse signal. The autocorrelator signal is obtained as shown in Figure 5a.</li>
			<li>Stop the OPO and project&#160;of the Ti:Sa beam from FFM/AM to input mirror into the autocorrelator. Repeat steps 3.2.2.6 and 3.2.2.7. The autocorrelator signal is obtained as shown in the Figure 5b.</li>
			<li>Set the beam distance adjustment screw micrometer to Cross position (7.30 mm).</li>
			<li>Release both beams.</li>
			<li>Scan the delay line to obtain the two beams OPO and Ti:Sa overlapped. The cross-correlator signal is obtained as shown in the Figure 6.</li>
			<li>Flip the mirror FFM/AM so that the beams can reach M7 and scan head of the microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Microscope alignment
	<ol>
		<li>Perform white light microscopic observation: 
		NOTE: Prior to microscopic observation, ensure that the microscope is properly aligned.
		<ol>
			<li>Prepare the test sample, which consists of a phosphate buffer solution in which polystyrene beads with diameters of 3 &#956;m are dispersed. The solution is placed inside a sandwich of two glass slides.</li>
			<li>Turn on the microscope and power supply of white light. Follow the manual for observation under white light.</li>
			<li>Use a condenser to illuminate the sample. Use objective of 60x to collect light. Place the sample onto the stage. Optimize the focal position of the 60x microscope objective.</li>
			<li>Select the FOV of interest. Take a CCD image of the sample (Figure 7).</li>
			<li>Turn off the power supply of white light.</li>
		</ol>
		</li>
		<li>Microscope alignment with femtosecond laser beams: OPO and Ti:Sa
		<ol>
			<li>Remove the condenser using the escape button to temporarily retract the 60x microscope objective lens. Move the 60x microscope objective lens off the optical path, rotating the nosepiece.</li>
			<li>Mount the detector to the upper part of microscope using the external mechanical mount. Connect the detector output through a low-pass filter of 50&#937; to oscilloscope and monitor the OPO signal.</li>
			<li>Turn on the processor that controls the scanner head. Project the OPO beam into the scanner head of the microscope.</li>
			<li>Check the position of the beam inside the microscope, make sure that the location of the beam is in the center or near the center.</li>
			<li>Check that the position of the beam inside the head of PD is in the center.</li>
			<li>Maximize the power measured by the detector using an x-y translator.</li>
			<li>Switch the beam from OPO to Ti:Sa and verify that a maximum signal is also obtained for the titanium-sapphire laser. THis indicates that both beams are well-aligned.</li>
			<li>Finalize the beam alignment, introducing the 60x microscope objective lens and rotating back the nosepiece.</li>
			<li>Use the refocus button on the microscope to regain the finalized focus to the 60x microscope objective lens.</li>
			<li>Place the objective with magnification 40x in place of the condenser without touching or disturbing the sample.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Optimization of spatial and temporal synchronizations of beams
	<ol>
		<li>Temporal synchronization
		<ol>
			<li>Set the power of Ti:Sa and OPO measured before the microscope to 30 mW for both beams. Set the wavelength of OPO 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.</li>
			<li>Release both beams (Ti:Sa and OPO) so that they enter the microscope.</li>
			<li>Run the scanning delay line computerized translator and record the measured intensity by LIA for each position of the delay line. Wait until the delay line scanning is complete. The obtained temporal profile is visualized in Figure 8a.</li>
			<li>Set the wavelength of OPO to 1076 nm again so that pump and probe are in resonance with the vibrational frequency of the beads. Repeat step 3.4.1.3 (the obtained temporal profile is visualized in Figure 8b).</li>
			<li>Set the obtained overlap beam position in the delay line to acquire SRS images.</li>
		</ol>
		</li>
		<li>Spatial synchronization of the beams 
		NOTE: The transmission images are useful to optimize the FOV, illumination, and focal position of the microscope objectives, and to check if the two beams are spatially overlapped.
		<ol>
			<li>Transmission image acquisition of OPO
			<ol>
				<li>Stop the Ti:Sa beam and reduce the OPO power to 8 mW.</li>
				<li>Connect the detector readout to the data acquisition card.</li>
				<li>Run the data acquisition program along with microscope scanning console.</li>
				<li>Save the file and process the data to get the image. The raw image appears as shown in Figure 9a.</li>
			</ol>
			</li>
			<li>Transmission image acquisition of Ti:Sa
			<ol>
				<li>Stop the OPO beam and reduce the Ti:Sa power to 2.5&#8211;4.5 mW.</li>
				<li>Connect the detector with LIA and LIA readouts with the data acquisition card.</li>
				<li>Repeat steps 3.4.2.1.3 and 3.4.2.1.4. The raw image appears as shown in Figure 9b.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. SRS image acquisition
NOTE: A dedicated algorithm has been realized in order to store data. It supports the following image formats: 512 px x 512 px and 256 px x 256 px, with acquisition times of 16 s, 8 s, 4 s, and 2 s.
<ol>
	<li>Introduce a stack of filters in between the 40x objective and PD to remove the pump pulses (Ti:Sa) and acquire only the Stokes signal (OPO).</li>
	<li>Set the pump signal to 810 nm with a focused power of 8 mW and the probe signal to 1076 nm with a focused power of 8 mW to investigate a typical C-H bond of polystyrene (Raman shift of 3054 cm&#8722;1).</li>
	<li>Connect the detector with the LIA and LIA readout to the data acquisition card.</li>
	<li>Set the image acquisition pixel format as per requirement and set the acquisition time.</li>
	<li>Run the program that controls the microscope controller.</li>
	<li>Run the dedicated algorithm program that acts as synchronization between the microscope controller, detection system, and DAQ (see Figure 4).</li>
	<li>Save the matrix file once the acquisition is completed.</li>
	<li>Import the raw data file and save the image in the required format (typically saved in .tif format) using ImageJ software. The image is shown in the Figure 10.</li>
</ol>

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The authors declare no conflicts of interest.

SRS microscopy has taken label-free imaging to new heights, especially in studies of complex biological structures such as lipids, which are fundamental to cells and cellular architecture. Lipids are involved in multiple physiological pathways such as production of biological membranes, and they serve as biosynthetic precursors and signal transducers10. Lipids are packaged into specialized intracellular organelles, also called lipid droplets (LDs). Their diameters vary from few tens of nanometers ...

In life science applications, SRS microscopy has emerged as powerful tool for label-free imaging. The basic idea of SRS microscopy is to combine the strength of vibrational contrast and its ability to acquire images in a few seconds.
SRS is a process in which the frequency difference between two laser beams frequencies (pump signal and stokes signal at different frequencies) matches the molecular vibration of an investigated sample, causing stimulated Raman scattering and a significant increase in the Stokes signal. Unlike linear Raman spectroscopy, SRS exhibits a nonlinear dependence on the incoming light fields and produces coherent radiation. SRS has two fundamental advantages: 1) speed, which makes images less sensitive to artefacts arising from sample movement or degradation, and 2) an excellent signal-to-noise ratio (SNR). In addition, SRS exhibits a spectrum identical to the spontaneous Raman, and the SRS signal is linearly proportional to the concentration of the chemical bond excited1,2,3,4,5.
In our microscope, a femtosecond (fs) SRS experimental set-up is integrated with an inverted optical microscope equipped with a fast mirrors scanning unit (Figure 1)6,7,8. Two pulsed laser sources are used to implement this microscope. The first is a fs-Ti:Sa with a pulse duration of approximately 140 fs, repetition rate of 80 MHz, and emission wavelengths in the range of 680-1080 nm. The second, used as probe beam and pumped by Ti:Sa, is a femtosecond synchronized optical parametric oscillator (SOPO), with a pulse duration of approximately 200 fs, repetition rate of 80 MHz, and emission wavelengths in the range of 1000-1600 nm. It should be noted that the minimum photon energy difference between the Ti:Sa and SOPO beam is 2500 cm-1. Therefore, using this combination of laser systems, only the high frequency C-H region (2800-3200 cm-1) of Raman spectra can be explored6,7,8.
In order to set up a&#160;SRS microscope, there are three crucial issues to consider, which are described in the successive paragraphs. The first is the implementation of a high-frequency modulation transfer method (see Figure 2 and step 2.1 of the protocol for a description). In a&#160;SRS experimental investigation, a crucial parameter is the sensitivity of the system. A&#160;SRS signal is detected as a small change in the intensity of excitation beams; therefore, it can be corrupted by laser intensity noise and shot noise. This issue can be overcome by integrating this system with a high-frequency modulation transfer method (see Figure 2 and step 2.1 of the protocol for details). In this method, an electro-optic modulator (EOM) is used to modulate the pump. The modulation transferred to the probe beam can then be detected by a PD after blocking the pump beam with a stack of optical filters [stimulated Raman gain (SRG) detection mode]. The PD output is connected by a low pass filter to a lock-in amplifier (LIA), which demodulates the measured signal. By increasing the modulation frequency of the beam to frequencies above 1 MHz, the intrinsic limit of PDs can be obtained.
The second issue to consider is the installation of a mechanical mount which permits to carry out forward detection and at the same time to preserve microscope observation in brightfield. In addition, it has to reduce the noise due to mechanical vibration during the generation of images and to allow the precise repositioning of detection system (see Figure 3 and step 2.2 of the protocol).
The third is the synchronization of the signal acquired by the phase-sensitive detection scheme, with the beam positioned onto the sample monitored by the scan head of the microscope. In order to realize images, the SMs require three TTL signals that are made available by the microscope controller connected to the scan head unit: pixel clock, line sync, and frame sync. The synchronization is achieved by controlling using a PCI card, the three TTL signals, and the acquisition of a voltage signal at the output channel of LIA6,7,8. A homemade software has been developed and described previously6,7,8, while the hardware of the synchronization system is reported in Figure 4.
A fundamental procedure when carrying out SRS imaging is microscope alignment. It is realized over the course of four steps, which are described in the successive paragraphs. The first is the spatial overlap of two beams (see step 3.1 of the protocol). In this experimental set-up, the two beams were spatially collinearly combined by a dichroic mirror. The preliminary step is the alignment of OPO and Ti:Sa so that each reaches the microscope. Then, considering OPO as a reference beam and taking advantage of a position sensitive detector, the Ti:Sa is spatially overlapped to OPO.
The second crucial aspect is the temporal overlap of two beams (see step 3.2 of the protocol). Even if the pump and OPO beams are perfectly synchronized9, since they follow slightly different beam paths inside the OPO housing, at the OPO exit they have a time delay of about 5 ns and spatial difference of 5 cm. Therefore, Ti:Sa and OPO require being re-timed optically to ensure temporal overlap at the sample. This is typically accomplished with a finely tunable optical delay line, which in this case is inserted between the Ti:Sa and microscope (see Figure 1). In order to obtain the temporal overlap of two beams, two techniques are used. The first is carried out using a fast PD and oscilloscope, while the second is based on auto- and cross-optical correlations. Using the first technique, a rough overlap of two beams is obtained (uncertainty of 10 ps), while an accurate temporal overlap of two beams is obtained using a cross-correlator (resolution of 1 fs).
The third crucial aspect is alignment of the two beams inside the microscope (see step 3.3 of the protocol). A preliminary white light observation of sample allows to individuate the desired field of view (FOV). Afterwards, laser beams, entering the microscope by a side port of microscope, are aligned in order to reach the PD mounted on the upper part (Figure 3). However, for a correct image acquisition, setting a number of parameters is required (for example, pixel dimension and pixel dwell time). The sampling frequency must respect the constraint imposed by Nyquist&#8217;s theorem in order to preserve all information in an image, while for a correct correspondence between the spatial coordinates of pixels and SRS value measured in each pixel, the integration time of LIA should be equal or comparable to the pixel dwell time.
In the final step of microscope alignment, numerous tests are carried out to optimize the spatial and temporal alignment (see step 3.4 of the protocol). A number of transmission images (TI) for both Ti:Sa and OPO are acquired in order to optimize spatial overlap. In a TI, a single beam is used, and the transmitted beam intensity from the sample is measured by a PD. In the case of TI realized by OPO, the PD output signal is directly connected to PCI card, while in the case of TI realized by Ti:Sa, the PD output signal is connected to LIA and analog output of LIA is connected to PCI card. The transmission images are very useful to optimize the FOV, the illumination, the focal position of microscope objectives and to check if the two beams are spatially overlapped6,7,8.
The optimization of the pump and probe beam&#8217;s temporal overlap is obtained by scanning the delay line with steps of 0.001 mm corresponding to a 3.3 fs time-shift and carrying out a SRS measurement in a single point of a polystyrene bead sample 3 &#181;m in diameter. The amplitude of an SRS signal measures values from LIA, as a function of the probe-pump delay, and provides a maximum corresponding with exact temporal overlap of the two beams6,7,8. Before concluding, it should be noted that all discussed steps are mandatory to obtain a high quality image.

<table>
	<tbody>
		<tr>
			<td>Acquisation tool</td>
			<td>Nikon</td>
			<td>Nikon C2Tool</td>
			<td>Acquisation supported tool</td>
		</tr>
		<tr>
			<td>APE Pulse link control software</td>
			<td>APE-</td>
			<td>APE Pulse link control software</td>
			<td>software control</td>
		</tr>
		<tr>
			<td>Autocorrelator</td>
			<td>APE</td>
			<td>APE PulseCheck USB 50</td>
			<td>Autocorrelator</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Thorlabs</td>
			<td>Thorlabs DET10A</td>
			<td>Photodiode</td>
		</tr>
		<tr>
			<td>Detector card</td>
			<td>Thorlabs</td>
			<td>Thorlabs VRC</td>
			<td>IR detector Card</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>Semrock FF875-Di01-25X36</td>
			<td>Dichroic mirror</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>FF875-Di01-25x36</td>
			<td>Dichroic mirror</td>
		</tr>
		<tr>
			<td>EOM</td>
			<td>Conoptics</td>
			<td>(EOM CONOPTICS 3350-160 KD*P).</td>
			<td>Pockels cell</td>
		</tr>
		<tr>
			<td>Fast detector</td>
			<td>Thorlabs</td>
			<td>Thorlabs DET025AL/M</td>
			<td>Photodiode</td>
		</tr>
		<tr>
			<td>Fast mirror scanning unit</td>
			<td>Nikon</td>
			<td>C2</td>
			<td>Microscpe scanning head</td>
		</tr>
		<tr>
			<td>Femtosecond laser Ti:SA</td>
			<td>Coherent</td>
			<td>Coherent Chameleon Ultra II</td>
			<td>Chameleon Ultra II</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>TTi</td>
			<td>TG5011 AIM &#8211; TTi</td>
			<td>Function generator</td>
		</tr>
		<tr>
			<td>Inverted optical microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE-2000-E, Nikon</td>
			<td>Eclipse TE-2000-E, Nikon</td>
		</tr>
		<tr>
			<td>Lock-in Amplifier</td>
			<td>Standford Research System</td>
			<td>SR844-200 MHz dual phase</td>
			<td>A lock-in amplifier from Stanford Research Systems</td>
		</tr>
		<tr>
			<td>Notch filter,</td>
			<td>Semrock</td>
			<td>NF03-808E-25</td>
			<td>Notch filter</td>
		</tr>
		<tr>
			<td>Optical delay line</td>
			<td>Newport</td>
			<td>Newport M-ILS200CC</td>
			<td>Tunable optical delay line</td>
		</tr>
		<tr>
			<td>Optical Parametric Oscillator</td>
			<td>Coherent</td>
			<td>Coherent Compact OPO</td>
			<td>Coherent Compact OPO</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>WaveRunner</td>
			<td>640Zi 4GHz OSC/LeCroy</td>
			<td>Digital Oscilloscope</td>
		</tr>
		<tr>
			<td>PCI Card</td>
			<td>National instrument</td>
			<td>NI PCIe 6363</td>
			<td>Data acquisation card</td>
		</tr>
		<tr>
			<td>Position Sensors Detectors</td>
			<td>Newport</td>
			<td>Newport Conex PSD9</td>
			<td>Position detector sensor</td>
		</tr>
		<tr>
			<td>Power meter head</td>
			<td>Coherent</td>
			<td>PowerMax PM10,</td>
			<td>Laser power detector</td>
		</tr>
		<tr>
			<td>Translation Stages</td>
			<td>Thorlabs</td>
			<td>Thorlabs PT1/M</td>
			<td>Meachnical Translation Stage with Standard Micrometer</td>
		</tr>
	</tbody>
</table>

implementation of a nonlinear microscope based on stimulated raman scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

An example of SRS measurement (i.e., SRS measurement in a single point of the sample) is reported in Figure 7. When the beams are&#160;not overlapped in time or space, the obtained result is reported in Figure 8a. In off-resonance, the amplitude of signal measured by LIA is zero, while the phase of signal measured by LIA jumps between negative and positive values. Whereas, when the beams are overlapped in space, moving the delay line in an appropriate ran...

Watch this Scientific Journal Video about Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering at JoVE.com

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

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7.5K Views.  National Research Council, Institute for Microelectronics and Microsystems, Naples Unit, Italy. In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Video: Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Automation of Mode Locking in a Nonlinear Polarization Rotation Fiber Laser through Output Polarization Measurements

When a laser is mode-locked, it emits a train of ultra-short pulses at a repetition rate determined by the laser cavity length. This article outlines a new and inexpensive procedure to force mode locking in a pre-adjusted nonlinear polarization rotation fiber laser. This procedure is based on the detection of a sudden change in the output polarization state when mode locking occurs. This change is used to command the alignment of the intra-cavity polarization controller in order to find mode-locking conditions. More specifically, the value of the first Stokes parameter varies when the angle of the polarization controller is swept and, moreover, it undergoes an abrupt variation when the laser enters the mode-locked state. Monitoring this abrupt variation provides a practical easy-to-detect signal that can be used to command the alignment of the polarization controller and drive the laser towards mode locking. This monitoring is achieved by feeding a small portion of the signal to a polarization analyzer measuring the first Stokes parameter. A sudden change in the read out of this parameter from the analyzer will occur when the laser enters the mode-locked state. At this moment, the required angle of the polarization controller is kept fixed. The alignment is completed. This procedure provides an alternate way to existing automating procedures that use equipment such as an optical spectrum analyzer, an RF spectrum analyzer, a photodiode connected to an electronic pulse-counter or a nonlinear detecting scheme based on two-photon absorption or second harmonic generation. It is suitable for lasers mode locked by nonlinear polarization rotation. It is relatively easy to implement, it requires inexpensive means, especially at a wavelength of 1550 nm, and it lowers the production and operation costs incurred in comparison to the above-mentioned techniques.

A protocol to detect and automate mode locking in a pre-adjusted nonlinear polarization rotation fiber laser is presented. The detection of a sudden change in the output polarization state when mode locking occurs is used to command the alignment of an intra-cavity polarization controller in order to find mode-locking conditions.

When a laser is mode-locked, it emits a train of ultra-short pulses at a repetition rate determined by the laser cavity length. This article outlines a ...

Stimulated Stokes and Antistokes Raman Scattering in Microspherical Whispering Gallery Mode Resonators

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres can be thought as a store of energy with a huge variety of applications: compact laser sources, highly sensitive biochemical sensors and nonlinear phenomena. A protocol for the fabrication of both the microspheres and coupling system is given. The couplers described here are tapered fibers. Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS). A proof of the cavity-enhanced phenomenon is given by the lack of correlation among the pump, signal and idler: a resonant mode has to exist in order to obtain the pair of signal and idler. In the case of hyperparametric oscillations (four wave mixing and stimulated anti-stokes Raman scattering), the modes must fulfill the energy and momentum conservation and, last but not least, have a good spatial overlap.

Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS).

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Experimental Implementation of a New Composite Fabrication Method: Exposing Bare Fibers on the Composite Surface by the Soft Layer Method

The bipolar plate is a key component in proton exchange membrane fuel cells (PEMFCs) and vanadium redox flow batteries (VRFBs). It is a multi-functional component that should have high electrical conductivity, high mechanical properties, and high productivity.
In this regard, a carbon fiber/epoxy resin composite can be an ideal material to replace the conventional graphite bipolar plate, which often leads to the catastrophic failure of the entire system because of its inherent brittleness. Though the carbon/epoxy composite has high mechanical properties and is easy to manufacture, the electrical conductivity in the through-thickness direction is poor because of the resin-rich layer that forms on its surface. Therefore, an expanded graphite coating was adopted to solve the electrical conductivity issue. However, the expanded graphite coating not only increases the manufacturing costs but also has poor mechanical properties.
In this study, a method to expose fibers on the composite surface is demonstrated. There are currently many methods that can expose fibers by surface treatment after the fabrication of the composite. This new method, however, does not require surface treatment because the fibers are exposed during the manufacture of the composite. By exposing bare carbon fibers on the surface, the electrical conductivity and mechanical strength of the composite are increased drastically.

A protocol to expose bare fibers on the composite surface by eliminating resin rich area is presented. The fibers are exposed during fabrication of the composites, not by the post surface treatment. The exposed carbon composites exhibit high electrical conductivity in the through-thickness direction and high mechanical property.

The bipolar plate is a key component in proton exchange membrane fuel cells (PEMFCs) and vanadium redox flow batteries (VRFBs). It is a multi-functional ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

Predicting Catalyst Extrudate Breakage Based on the Modulus of Rupture

The mechanical strength of extruded catalysts and their natural or forced breakage by either collision against a surface or by a compressive load in a fixed bed are important phenomena in catalyst technology. The mechanical strength of the catalyst is measured here by its bending strength or flexural strength. This technique is relatively new from the perspective of applying it to commercial catalysts of typical sizes used in the industry. Catalyst breakage by collision against a surface is measured after a fall of the extrudates through the ambient air in a vertical pipe. Quantifying the impact force is done theoretically by applying Newton's second law. Measurement of catalyst breakage due to stress in a fixed bed is done following the standard procedure of the bulk crush strength test. Novel here is the focus on measuring the reduction in the length to diameter ratio of the extrudates as a function of the stress.

Here we present a protocol to measure the modulus of rupture of an extruded catalyst and the breakage of said catalyst extrudates by collision against a surface or by compression in a fixed bed.

The mechanical strength of extruded catalysts and their natural or forced breakage by either collision against a surface or by a compressive load in a ...