Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Abstract

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm−1, is demonstrated.

Broadband stimulated Raman scattering microscopy with wavelength‐scanning detection

“…In coherent anti‐Stokes Raman scattering, the response is read at the anti‐Stokes frequency ω aS = ω p + Ω, whereas in stimulated Raman scattering (SRS), the nonlinear signal is imprinted on the pump/Stokes pulses themselves, in the form of Stokes pulse amplification (stimulated Raman gain, SRG) or pump pulse attenuation (stimulated Raman loss, SRL). Coherent anti‐Stokes Raman scattering suffers from the superposition with a frequency‐independent nonresonant background, which often distorts and masks the resonant signal of interest; SRS, on the other hand, is almost free from nonlinear background and directly measures the resonant signal, making it the CRS microscopy technique of choice …”

“…Liao et al [26] inserted a 12-kHz resonant galvanometric mirror in the Fourier plane of a 4f pulse shaper to measure spectra with pixel dwell times of 83 μs, whereas Alshaykh et al [27] used an acousto-optically programmable dispersive filter to achieve an even higher pixel scan rate of 30 kHz. Laptenok et al [17] employed a dual approach: A narrowband (8 cm −1 ) acousto-optical tunable filter scanned the laser pulse spectral bandwidth (about 150 cm −1 ) for every selected laser central wavelength; subsequently, the laser central wavelength was changed and the scan repeated. In this way, they could cover the entire Raman vibrational region from the fingerprint to the C─H stretch without the need to realign the beam path or to change the optical setup.…”

Imaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues

“…The concept of the Raman stable isotope technique refers to labeling of biomolecules such as DNA, proteins and lipids with stable isotopes. Since biomolecules consist mainly of major biogenic elements such as hydrogen ( 1 H), carbon ( 12 C), nitrogen ( 14 N) and oxygen ( 16 O), stable heavy isotopes ( 2 H, 13 C, 15 N, 18 O) of these elements are used as tracers for probing the isotope incorporation into the cellular biomass [37]. The isotopic substitution of light atoms by heavy atoms of biomolecules leads to a change in the vibrational frequency of Raman peaks, while the Raman intensity remains more or less unaltered.…”

“…Traditionally both techniques only probe one specific Raman vibration, where two lasers need to be coherently matched, so that the CARS and/or SRS signal can be generated. Instrumental developments in lasers and filters during the last decade have made it possible to develop systems covering a broad or even the full spectral range, which opens the way to hyperspectral coherent Raman imaging in a fast manner [18,19].…”

“…Enhancement techniques such as resonance Raman spectroscopy or surfaced-enhanced Raman spectroscopy (SERS) can be employed to significantly improve the Raman signal [14,15]. Furthermore coherent Raman techniques, such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, exploit the non-linear enhancement of the weak Raman effect and offer high specificity and imaging speed [16][17][18]. Although SRS is more complex in the instrumentation, it is often favored over CARS since the signal intensity is directly proportional to the molecular concentration and does not suffer from non-resonant background contributions distorting the typical spectral profile.…”