Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

Area of Science:

• Spectroscopy and Microscopy
• Biophotonics
• Chemical Imaging

Background:

• Spontaneous Raman imaging offers rich chemical information but is often limited by slow acquisition speeds.
• Existing Raman microscopy techniques struggle to achieve high spatial and spectral resolution simultaneously with rapid 3D imaging.
• Light-sheet microscopy provides rapid 3D imaging capabilities but typically lacks detailed chemical specificity.

Purpose of the Study:

• To develop a rapid spontaneous Raman 2D imaging technique compatible with light-sheet microscopy.
• To achieve high-speed spectral data acquisition for 3D chemical imaging of biological samples.
• To demonstrate the utility of the technique for analyzing complex biological and material samples.

Main Methods:

• Utilized continuous wave lasers and interferometric tunable filters for spectral scanning in light-sheet microscopy.
• Employed a spectral knife-edge technique by angularly tuning filter edges to scan Stokes wavelengths.
• Retrieved pixel-wise Raman spectra through differentiation of recorded image stacks, preserving 3D resolution.

Main Results:

• Demonstrated rapid 2D Raman imaging on solvent solutions and composites of polystyrene beads and lipid droplets.
• Successfully imaged the C-H vibrational band region (2800-3100 cm⁻¹) in a *C. elegans* worm.
• Achieved image acquisition speeds four orders of magnitude faster than conventional confocal point scanning Raman systems.

Conclusions:

• The developed technique significantly enhances the speed of spontaneous Raman 3D imaging in light-sheet microscopy.
• This advancement opens possibilities for fast, high-resolution chemical analysis of dynamic biological processes in vivo.
• The method provides a powerful new tool for multimodal imaging, combining structural and chemical information.