KEYWORDS: Brain, Deep tissue imaging, Neuroscience, Neuroimaging, In vivo imaging, High speed imaging, High power lasers, Femtosecond pulse shaping, Femtosecond phenomena, Brain imaging
In this work, we customized a multi-photon spinning disc unit with a tunable high-power femtosecond laser to demonstrate the first three-photon spinning disk microscopy, achieving ~100Hz frame rate. We demonstrate its pioneering applications on green fluorescent beads, in vivo Drosophila brains, and fixed mouse brains. Furthermore, our findings from brain tissue imaging reveal that three-photon spinning disk imaging exhibits a significantly lower attenuation rate when compared to its two-photon spinning disk counterpart, paving the way of utilizing three-photon excitation on high-speed deep tissue imaging.
Stimulated Raman scattering (SRS) techniques enable label-free detection of the vibrational modes of molecules with high chemical specificity. However, its practical application to material characterization and bioimaging has been limited by sensitivity accompanied with the low Raman cross-section issue, resulting from typical far electronic resonance excitation. To address this limitation, the electronic pre-resonance (EPR) SRS technique has been developed to enhance Raman signals through bringing the excitation frequency close enough to the absorption peaks of examined molecules. However, a significant weakness of previous demonstrations was the lack of dual-wavelength tunability, restricting EPR-SRS to only a limited number of species in a proof-of-concept experiment. In this study, we present EPR-SRS spectromicroscopy driven by a multiple-plate continuum (MPC) light source. The MPC light source enables the examination of a single vibration mode with independent adjustment of both pump and Stokes wavelengths. As a proof-of-concept experiment, we interrogated the C=C vibration mode of Alexa 635 by continuously scanning the pump-to-absorption frequency detuning across the entire EPR region. The results exhibit a remarkable 150-fold enhancement in SRS signal and demonstrate good agreement with the Albrecht A-term pre-resonance model. Moreover, we observed signal enhancement in EPR-SRS bioimages of Drosophila brains stained with Alexa 635. Leveraging the improved sensitivity and potential to implement hyperspectral measurement, we envision that this technique holds great promise for advancing our understanding of biological systems and facilitating multiplex chemical characterization.
Stimulated Raman scattering (SRS) has attracted significant attention recently for providing high-sensitivity and background-free chemical characterization without exogenous labeling. For the laser system of SRS, the current benchmark is the combination of a pico- or femtosecond mode-locked solid-state oscillator and a synchronously pumped optical parametric oscillator (OPO), offering two central wavelengths λpump and λStokes to match the desired Raman modes. Despite great success, the phase-sensitive nature of degenerate OPO hampers its access to low-frequency Raman shifts, and the inability to independently adjust the second wavelength prevents the OPO source from electronic pre- resonance (EPR) detection for desirable molecules.
In this work, we demonstrated an SRS spectro-microscopy system driven by a multiple-plate continuum (MPC) supercontinuum laser source, whose spectrum spans from 600 nm to 1300 nm, offering capabilities of dual-wavelength tunability across the entire Raman active region (0 to 4000 cm-1). We demonstrated SRS microspectroscopy across the fingerprint, silent, and C-H stretch Raman regions in acetonitrile solution. This novel light source allows significant contrast enhancement through EPR-SRS by tuning the pump wavelengths toward the absorption peak of dye molecules, exemplified by the C=C mode of Alexa 635. Moreover, single-wavelength SRS imaging of the Drosophila brain was presented. We envision that utilizing an MPC light source will substantially enhance the sensitivity and specificity of SRS by implementing EPR mode and spectral multiplexing.
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