Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

G Pauliat,Rajeev Ranjan,Maria Antonietta Ferrara,C Sibilia,Luigi Sirleto,A Belardini

doi:10.1051/epjconf/202125506003

G Pauliat, Rajeev Ranjan + Show 4 more

Open Access

https://doi.org/10.1051/epjconf/202125506003

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Abstract

In this work, the implementation of a femtosecond Stimulated Raman Scattering microscope, equipped with three femtosecond laser sources: a Titanium-Sapphire (Ti:Sa), an optical parametric oscillator (OPO), and a second harmonic generator (SHG); is presented. Our microscope is designed so that it can cover all the regions of Raman spectra, taking advantage of two possible laser combinations. The first, Ti:Sa and OPO laser beams, which cover the C-H region (>2800 cm-1 ) in stimulated Raman gain (SRG) modality, whereas the second, Ti:Sa and SHG laser beams, covering the C-H region and the fingerprint region in stimulated Raman losses (SRL) modality. The successful realization of the microscope is demonstrated, reporting images of polystyrene beads using both SRL and SRG modalities.

Highlights

In spite of the fact that the fluorescence microscopy represents the fundamental pillars for biological imaging, it shows significant limitations: (i) some molecules and/or biological structures cannot be labeled without changing their biological function, such as lipids; (ii) many labels are cytotoxic, which leads to interference with the biological functionality
The microscope is equipped with three femtosecond laser sources: a Ti:Sapphire (Ti:Sa), a synchronized optical parametric oscillator (SOPO), and a frequency converter for ultrafast lasers, i.e., a second harmonic generator (SHG) optimized for the SOPO, which allows doubling the OPO energy radiation
Ti:Sa and OPO laser combination cover the C-H region in stimulated Raman gain (SRG) modality[17,18,19,20,21,22]. The latter Ti:Sa and SHG laser combination provide the extension of the microscope to the silent region (

Summary

Introduction

In spite of the fact that the fluorescence microscopy represents the fundamental pillars for biological imaging, it shows significant limitations: (i) some molecules and/or biological structures cannot be labeled without changing their biological function, such as lipids; (ii) many labels are cytotoxic, which leads to interference with the biological functionality. Label-free imaging, obtained by microscopy techniques, is highly desirable, affording high chemical selectivity of unlabeled living cells. By exploiting nonlinear optical effects, novel microscopy techniques have been developed, which afford high chemical selectivity of unlabeled living cells and implement real-time threedimensional imaging with high spatial resolution and sensitivity.

Results

Conclusion