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...

<ol>
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G., Contreras-Rojas, L. R., Xie, X. S., Guy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Drug+Delivery+to+Skin+with+Stimulated+Raman+Scattering+Microscopy.">Imaging Drug Delivery to Skin with Stimulated Raman Scattering Microscopy.</a> Molecular Pharmaceutics. 8 (3), 969-975 (2011).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+chemical+imaging+by+stimulated+Raman+projection+microscopy+and+tomography.">Volumetric chemical imaging by stimulated Raman projection microscopy and tomography.</a> Nature Communications. 8, 15117 (2017).</li><li>Ferrara, M. A., Filograna, A., Ranjan, R., Corda, D., Valente, C., Sirleto, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threedimensional+label-free+imaging+throughout+adipocyte+differentiation+by+stimulated+Raman+microscopy.">Threedimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy.</a> PLoS ONE. 14 (5), e0216811 (2019).</li></ol>

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. ...

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.

implementation-nonlinear-microscope-based-on-stimulated-raman

Research

JoVE Journal

Engineering

7.4K ビュー.  National Research Council, Institute for Microelectronics and Microsystems, Naples Unit, Italy. このプロトコルは、非線形顕微鏡に興味を持つ科学者が、刺激されたラマン散乱に基づく顕微鏡の主要な構成要素、セットアップ、およびアライメント手順を理解するのに役立ちます。SRS顕微鏡の主な利点は、振動コントラストに基づいてラベルフリーイメージングを実行し、数秒で画像を取得する能力です。SRS顕微鏡は、細胞や細胞アーキテクチャの基本である脂質?...