Label-Free Multiphoton Microscopy: The Origin of Fluorophores and Capabilities for Analyzing Biochemical Processes

Area of Science:

• Biomedical Optics
• Molecular Imaging
• Microscopy

Background:

• Multiphoton microscopy (MPM) enables high-resolution, deep-tissue intravital imaging.
• It utilizes nonlinear optical signals, including multiphoton fluorescence and optical harmonics.
• MPM can also analyze fluorescence decay kinetics for advanced imaging.

Purpose of the Study:

• To review photophysical processes of reporter molecules in MPM.
• To discuss endogenous contrasts for label-free MPM.
• To showcase MPM's capabilities in molecular imaging of biochemical processes.

Main Methods:

• Review of photophysical principles in multiphoton microscopy.
• Analysis of nonlinear optical signal generation (fluorescence, harmonics).
• Examination of fluorescence decay kinetics.

Main Results:

• Detailed description of photophysical processes for endogenous MPM contrast agents.
• Summary of experiments demonstrating label-free MPM for biochemical imaging.
• Highlighting MPM's utility in connective tissue and cellular studies.

Conclusions:

• MPM is a powerful multimodal technique for molecular and intravital imaging.
• Label-free MPM provides significant capabilities for studying biochemical processes in vivo.
• Understanding photophysics is crucial for optimizing MPM applications.