New optical source design simplifies CARS microscopy

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Coherent anti-Stokes Raman spectroscopy (CARS) improves generally small Raman signals so that they are detectable above background noise. Coupled with appropriate optomechanics, CARS provides images of the microscopic chemical distribution within a sample. It requires two simultaneous, superimposed pulsed laser beams, traditionally provided by picosecond optical parametric oscillators (OPOs), which work well like CARS sources, but are expensive and require a dedicated optical lab. Researcher Konstantinos Bourdakos and his colleagues at the University of Southampton (UK) have now replaced the picosecond OPO with a laser diode optical parametric amplifier (OPA), a simpler, smaller and cheaper alternative.1

Molecular identification without labeling

When a photon interacts with a molecule, most often it diffuses without changing wavelength. In Raman scattering, the outgoing light changes wavelength in an exchange of energy that leaves the molecule in a different final vibrational state. If the final vibrational state has a higher energy than the initial, the scattered light is a longer wavelength “Stokes” signal; if the final state has a lower energy, it is an “anti-Stokes” signal of shorter wavelength. The frequencies of the signals depend on the molecular structure, so Raman signals provide molecular signatures without labeling. This is desirable because attachment of reporter molecules is both time consuming and expensive and can potentially alter native molecular configuration.

But Raman signals are much smaller than other scattering and background effects. CARS overcomes the problem by sending two beams through the sample simultaneously. When the energy difference between the two beams – the pump and Stokes beams – matches the energy of a molecular vibration, a significant fraction of the population of molecules is raised to this excited vibrational state. The pump beam then elevates these excited molecules to another virtual state, and when they return to ground state, they emit a much stronger anti-Stokes signal. Spectral filtering further reduces background noise and coupling with the optics of the scanning microscope producing an image whose variations in intensity map the presence of a particular vibrational mode. Repeat the scan at different energies in the “fingerprint region” – a spectral spread of approximately 2845 cm-1—Produces a microscopic map of molecular distribution without any external labeling.

The conceptual simplicity hides some challenges. CARS is a third-order non-linear effect that requires high peak power, which prompts the use of ultra-fast lasers. But shorter pulses have a wider spectral width, which reduces spectral resolution and can increase unwanted non-resonant background noise. Standard CARS microscopy meets these requirements by using a picosecond OPO as the source. However, OPOs are large, expensive, and require a dedicated optical lab to stay at peak performance. Other groups have demonstrated some simplification of the optical design of CARS microscopy;2 now Bourdakos and his colleagues have taken this simplicity even further, replacing the OPO with a picosecond OPA – essentially a single nonlinear crystal – seeded by a laser diode of communication wavelength.

Simple design, powerful imaging

Temperature control allows the laser diode to be set from 1562 to 1568nm. A 1031nm, 80MHz, 2ps pulsed laser is aligned with the seed of the diode beam and the two are coupled into the OPA, where the energy of the pulsed laser is converted into a seed wavelength, spectrally filtered, then doubled in frequency with a second harmonic generation crystal. The output beam has a line width of about 5cm-1, a peak power of 2.1 kW and an adjustable range of 781 to 784 nm. This is the pump of the CARS microscope. A small portion of the original picosecond laser at 1031 nm provides the Stokes beam. Together, they access a spectral range between 3056 cm-1 and 3105 cm-1. Equally important, the resulting anti-Stokes beam is around 630nm, well within the sensitivity of their detector. The researchers demonstrated their simplified source design capable of producing images of polystyrene beads and fatty tissue.

To access the fingerprint region, the researchers replaced only one source from an existing CARS microscope with the continuous wave laser diode OPA. This configuration halves the cost and complexity, while accessing a wide spectral range, placing the anti-Stokes wavelength around 730 nm, again suitable for their detector. They demonstrated the effectiveness of this configuration by imaging animal bone.

“We have demonstrated the ability to perform CARS images at standard frequencies with a much cheaper, more stable system requiring no spectral compression and taking up a quarter or even a fifth of the space,” explains Bourdakos. The team has demonstrated both second harmonic generation and excited two-photon autofluorescence imaging with the source, and is working on extending CARS microscopy to further refinements.

“This is a gradual advance,” adds Bourdakos, “but these types of results using a single picosecond laser and a very simple laser diode source will significantly reduce the costs and optical expertise required to perform the CARS microscopy and, therefore, , open it up to new users. “

THE REFERENCES

1. KN Bourdakos, Proc. SPIE, 11879, 1187903 (2021); doi: 10.1117 / 12.2601570.

2. T. Steinle et al., Sci light. Appl., 5, e16149 (2016); doi: 10.1038 / lsa.2016.149.


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