Name: implementation of a nonlinear microscope based on stimulated raman scattering
Uploaded: 2019-07-06T13:00:00
Description: Watch this Scientific Journal Video about Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering at JoVE.com

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>

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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