Photothermally Detected Stimulated Raman Microscopy towards Ultrasensitive Chemical Imaging

Abstract

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and coupling in a complex system. However, the bond-detection sensitivity of SRS microscopy is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth in either pump or Stokes beam4. Here, to overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index. By probing the refractive index change with a continuous wave beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved on dimethyl sulfide with conserved average power. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. With much improved signal to noise ratio compared to SRS, SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity and minimal water absorption.

Absolute Stimulated Raman Cross Sections of Molecules

“…In fact, for preresonant nitrile-based dyes like MARS, the apparent cross section can be as large as 1.5 × 10 –17 cm 2 , exceeding the infrared number. C–D stretching, another bond commonly used in vibrational imaging, exhibits a σ SRS,apparent of 4 × 10 –22 cm 2 under similar conditions, while its counterpart in infrared is ∼5 × 10 –20 cm 2 . , A common criticism is that Raman scattering cross section is 8 orders of magnitude weaker than infrared absorption, which has often cited as the motivation for the development of mid-infared absorption based microscopy. , Our result here suggests that this criticism does not apply to SRS and even supports the promise of SRS excited photothermal microscopy . This is largely because SRS is in fact an absorptive process, unlike spontaneous Raman, which creates new field modes.…”

“…62,63 Our result here suggests that this criticism does not apply to SRS and even supports the promise of SRS excited photothermal microscopy. 64 This is largely because SRS is in fact an absorptive process, unlike spontaneous Raman, which creates new field modes. In this…”

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

“…Removal of the non-resonant background in the SRS image will significantly improve the sensitivity and signal fidelity. The exploration of alternative modalities like frequency-modulation SRS (56), stimulated Raman photothermal microscopy (57) and mid-infrared photothermal microscopy (58,59) holds promise owing to their high detection sensitivity. The challenges associated with the refractive index mismatch between the sample and the air layer can also be mitigated through the use of photothermal detection regimes.…”

The Duality of Raman Scattering

“…Since σ Raman has been documented over the decades, eq 15a is expected to have practical utility for experimentalists to calculate measurement results such as the signal-to-noise ratio of common samples in microscopy experiments, the level of vibrational saturation especially under high peak power excitation, and also the energy deposition of stimulated Raman photothermal microscopy. 56 Raman duality immediately explains how SRS microscopy can outperform regular Raman microscopy. In the most typical configuration, both the pump and Stokes laser pulses are synchronized (with f rep being the repetition rate) and exhibit the same pulse width (τ pulse being the pulse duration).…”