Intravital two-photon microscopy: focus on speed and time resolved imaging modalities

Area of Science:

• Immunology
• Biophysics
• Microscopy

Background:

• Two-photon microscopy, initially for neurosciences, is now vital for immunological research.
• Existing methods face limitations in image acquisition speed for dynamic biological processes.

Purpose of the Study:

• To describe advanced two-photon microscopy techniques for high-speed imaging.
• To compare novel parallelized excitation methods with conventional single-beam systems.
• To highlight the utility of fluorescence lifetime imaging (FLIM) for cellular physiology analysis.

Main Methods:

• Parallelized excitation using multiple scanning beams simultaneously.
• Detection using sensitive charge-coupled device (CCD)-based line or field detectors.
• Time- and polarization-resolved fluorescence detection, including FLIM.
• Analysis of endogenous fluorophores like NAD(P)H for cellular redox status.

Main Results:

• Achieved significantly higher image acquisition rates compared to state-of-the-art single-beam systems.
• Enabled real-time generation of fluorescence lifetime imaging (FLIM) datasets.
• Provided subcellular resolution for analyzing cellular redox status.

Conclusions:

• High-speed two-photon microscopy, particularly with FLIM, offers powerful new capabilities for immunology.
• This technology allows deeper insights into immune reactions and cellular processes.
• Advancements in data analysis further enhance the real-time applicability of these techniques.