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 &amp;ndash; 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...

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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. ...

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

Research

JoVE Journal

Engineering

7.5K Views.  National Research Council, Institute for Microelectronics and Microsystems, Naples Unit, Italy. Dit protocol kan wetenschappers die geïnteresseerd zijn in niet-lineaire microscopie helpen om de belangrijkste componenten, het opzetten en de uitlijningsprocedure van een microscoop op basis van gestimuleerde Raman-verstrooiing te begrijpen. De belangrijkste voordelen van SRS-microscopie zijn de mogelijkheden om labelvrije beeldvorming uit te voeren op basis van trillingscontrast en om binnen enkele seconden een beeld te verwerven. SRS microscopie heeft genomen label-vrije beeldvorming naa...

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

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

Bioengineering

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

Chemistry

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Biology

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

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