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Updated: Jul 7, 2026
Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model
Published on: September 30, 2021
Intravital two-photon microscopy: focus on speed and time resolved imaging modalities
Raluca A Niesner1, Volker Andresen, Matthias Gunzer
1Junior Research Group Immunodynamics, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Insights
Novel two-photon microscopy techniques enhance image acquisition rates for analyzing immune cells. This advanced imaging provides real-time insights into cellular physiology and redox status using fluorescence lifetime imaging (FLIM).
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.
Abstract:
Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.
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