Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging

Area of Science:

• Bioimaging
• Biomedical Engineering
• Microscopy

Background:

• Imaging vital, thick, and complex tissues in real-time and non-invasively presents a significant challenge in bioimaging.
• Nonlinear optical (NLO) multi-photon microscopy provides a solution for label-free, non-destructive investigation of live biological samples.

Purpose of the Study:

• To review the fundamentals of NLO multi-photon microscopy.
• To discuss NLO techniques suitable for biological applications.
• To highlight recent advancements and challenges in NLO imaging for biomedical applications.

Main Methods:

• Discussion of NLO microscopy principles.
• Overview of techniques including two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS).
• Presentation of recent NLO imaging examples for in vitro and in vivo diagnostics.

Main Results:

• NLO multi-photon microscopy enables label-free, non-destructive investigation of physio-pathological processes.
• Techniques like TPEF, SHG-THG, and CRS allow sub-cellular spatial resolution in live samples.
• NLO imaging serves as a diagnostic instrument for monitoring biological specimens in their unperturbed state.

Conclusions:

• Multi-modal, multi-photon NLO microscopy offers significant technological advantages for bioimaging.
• NLO imaging is a powerful tool for studying cellular functions and disease mechanisms.
• Outstanding challenges remain in the biomedical engineering applications of NLO microscopy.