Our protocol describes how to build a stimulated Raman Scattering Microscopy, which enables the measurement of the vibrational spectrum of the molecules within microseconds. And when applied to imaging, it can provide hyperspectral microscopy to localize and quantify the chemical constituents of the matter in a label-free and non-invasive way. There are several applications of this protocol, mainly in the biological and biomedical sciences.
For example, to image cells, or tissues. There are two important challenges to our approach, the broadband optical source and the detection chain. To address the former, one can purchase an OPR or build one yourself.
Alternatively, one can use the white light supercontinuum generated in bulk crystals or in non-linear optical fibers. The latter challenge can be addressed by sequentially scanning each spectral component with a commercial photodiode and a galvo scanner To begin, extract two microliters from an aqueous suspension of polymethyl methacrylate, or PMMA microbeads, and pour the suspension onto a microscope cover slip. Then, extract two microliters from an aqueous suspension of polystyrene microbeads and combine it with the PMMA suspension on the cover slip.
Gently mix the suspension with a pipette tip and let it dry for 24 hours. A white layer of beads will appear on top of the cover slip when the water dries off. Add 20 microliters of dimethyl sulfoxide and 20 microliters of pure olive oil on top of the cover slip.
And apply nail polish on the edges of the second microscope cover slip. Place the cover slip on the mixture, with the nail polish facing down, and apply enough pressure to seal it. Let it dry.
To optimize the modulation efficiency of the narrow-band Stokes beam. Change the distance between the lenses F1 and F2, and measure the modulated beam with a photodiode. Then, record its profile with an oscilloscope.
Adjust the cavity length of the optical parametric oscillator in such a way that the resulted broadband pump spectrum, together with the narrow-band Stokes at 1040 nanometers, can produce a frequency de-tuning within 2, 800 to 3, 100 inverse centimeters. This spectral range covers the vibrations of the CH stretching region. Send the broadband pump to a prism compressor to compensate for the dispersion effects included by the excitation microscope objective.
Enter the pump into prism A, through its apex, and guide the dispersed pump toward the apex of prism B.Define the amount of negative dispersion necessary and set the distance between the prisms apices accordingly. Use Brewster cut prisms, and ensure that the polarization of the pump beam lies within the triangular planes of the prisms. To optimize the inline balanced detection scheme, set the fast axis of this birefringent crystal vertical, and guide the polarized pump to a YV04 plate with a length of 13.3 millimeters.
Then, use a half-wave plate to set the polarization of the pump beam to 45 degrees. Combine the pump and the Stokes beams with a dichroic mirror and carefully align them with a pair of fluorescent pinholes. Ensure that both the beams are propagating co-linearly.
Attenuate the beams and guide them onto a fast photodiode to temporarily overlap them. After that, remove the photodiode. Next, measure the beam profiles with a calibrated camera, and use an infrared card to estimate the diameters by eye.
Use two telescopes. One for the pump and another for the Stokes beam, and try to match the beam diameters to the back aperture of the excitation objective. Once the stimulated Raman scattering, or SRS signal, is obtained, use the telescope on the pump beam to tweak its diameter, varying it's Rayleigh range, and consequently, the interaction volume at the microscope's focus.
Stop when the maximum SRS is achieved. Use a photodiode to measure the intensity of the pump beam, and with the photodiode's responsivity, calculate the mean power impinging on the active area of the detector. To measure the relative intensity noise of the laser, disconnect the low-pass filter and connect the output of a high-bandwidth photodiode to the input of a lock-in amplifier.
Store the lock-in output in volts over square root of Hertz at different demodulation frequencies and use the photo diodes responsivity to convert volts to Watts. Guide the pump and the Stokes beams to the microscope. Place the sample and find a region without beads to help align the pump beam.
Then, make the excitation and collection objectives confocal. Put a shortpass filter to remove the modulated Stokes and guide the pump beam to the grading. Place a lens after the grading to focus the dispersed beam onto the detectors.
For balanced detection, measure the spectrum of the reference and the signal replicas propagating along the pump beam. Place a small slit, or an iris, between the grading and a polarizing beam splitter, to guarantee the spectral matching between the two photodiode arrays and to filter the dispersed pump spatially. Clip all but one spectral component of the pump replicas to center the transmitted rays on the Nth detector of the reference and signal photodiode arrays.
Use the steering mirrors to adjust the correlation of the different detection channels. To start the SRS microscopy:modulate the Stokes, raster scan the sample, and acquire the modulation transfer on the pump spectrum with its corresponding DC spectrum to get the normalized SRS spectrum from each pixel. Produce three-dimensional matrices, whose rows and columns contain the scanned positions of the sample.
On each vector orthogonal to the XY plane, store an SRS spectrum. Plot the concentration and the spectral profiles to acquire the chemical images and the characteristic spectra of the chemical constituents of the sample. The representative image shows the relative intensity noise spectra of the optical sources used in this protocol.
Shown here is the best spectral region for the SRS experiments. Modulating the Stokes beam at any frequency within this band guarantees that the effects of the laser noise on the SRS signal will be the lowest possible. An exemplary data of the unbalanced and balanced spectra are shown here.
The effects of balanced detection impact the final results of the experiments. Namely, the chemical maps. The composite images in the unbalanced and balanced conditions are shown here.
The representative images show a chemometric analysis of the hyperspectral SRS data. A composite of the concentration maps of the different chemical constituents of the sample and their characteristic SRS spectra are shown here. From this data, the different constituents of the sample, for example, olive oil, DMSO polystyrene, and polymethyl methacrylate can be easily identified.
Currently, our technique can only probe the CH vibration stretching. However, by optimizing the optical source, the same detection chain will allow us to explore the more informative fingerprint region detecting several modes at once. Our detection chain paves the way for the integration of broadband SRS into the clinics, introducing a technology that will complement and enhance the traditional histopathology workflow for tissue diagnosis.
