Two-and Three-Photon Microscopy: Expanding the Depth and Precision of Biological Imaging

The Speakers

Anne Bias, MsC - Pre-doctoral Researcher | Biophysical Analytics Lab in Raluca Niesner's group, Freie Universität Berlin and German Rheumatology Research Center (DRFZ), as well as Berlin University of Applied Sciences

Anne Bias MsC is a PhD researcher in the Biophysical Analytics Lab at FU Berlin & DRFZ. Her work focuses on advanced optical and multiphoton microscopy techniques, particularly in the development and application of three-photon imaging in living tissues. Anne contributes to experimental design, system optimization (for example dispersion compensation), and applying imaging modalities to biological questions, such as immune cell behavior in the musculoskeletal and lymphoid system in vivo. She has co-authored multiple imaging and method papers which bridge technical innovation and biological application.

Ruth Leben, PhD- Postdoctoral Researcher | Biophysical Analytics Lab in Raluca Niesner's group, German Rheumatology Research Center (DRFZ)

Ruth Leben, PhD is a postdoctoral researcher affiliated with the Biophysical Analytics Lab. Her expertise lies in fluorescence lifetime imaging (FLIM), multiphoton microscopy, and data modeling for biological imaging. Ruth has published on vector-analyzed FLIM approaches and works on combining lifetime and intensity modalities to dissect cellular metabolism and function in tissues. Her contributions integrate imaging methods with biological insights in live organisms, particularly in deep-tissue environments.

Summary

Two-photon microscopy (2PM) revolutionized fluorescence imaging by allowing optical sectioning deep within living tissues while minimizing photodamage. Building on this foundation, three-photon microscopy (3PM) extends imaging depth even further by using longer-wavelength infrared excitation, which reduces scattering and improves signal localization. To extend the knowledge about cells in their genuine tissue environment beyond their mere dynamics, fluorescence lifetime imaging (FLIM) of exogeneous and endogenous fluorophores allowed to probe cellular functions and metabolic states in vivo.

We discuss how two-photon (2PM) combined with FLIM and three-photon microscopy (3PM) enable high-resolution dynamic and functional imaging deep within living tissues. While 2PM has become a standard for intravital imaging, 3PM extends optical penetration and allows imaging even in organs inaccessible for 2PM.

Our group has optimized three-photon microscopy for long bone imaging and investigated how tissue properties affect laser-pulse broadening and dispersion, key factors limiting photon density at depth. We present recent data and modeling on tissue-specific pulse broadening and pulse compression approaches that restore efficient excitation for 3PM. Besides, we developed in vivo imaging of metabolic profiles at subcellular resolution and found a higher metabolic diversity in vivo as compared to in vitro conditions, which data will be present as well.  

Learning Objectives:

1. Understand the principles of two-photon and three-photon excitation and how longer wavelength infrared light enables deeper tissue imaging with reduced scattering.
2. Recognize the technical challenges in maintaining photon focus and signal strength at depth, including dispersion and pulse broadening, and learn how adaptive optics and pulse compression can address them.
3. Appreciate current biological applications of multiphoton microscopy for visualizing cellular dynamics and metabolism in living tissues, and how these approaches complement flow and image cytometry.

Who Should Attend:

This webinar is designed for researchers and imaging specialists interested in advanced fluorescence microscopy, particularly two- and three-photon excitation techniques for deep-tissue and intravital imaging.

It will be of special interest to:
- Scientists exploring spatial or intravital microscopy approaches
- Individuals managing or supporting imaging or multiphoton core facilities
- Students and investigators eager to learn about cutting edge, high resolution in vivo imaging and its biological applications.
- Anyone curious about next-generation optical methods and the science driving them will find this session valuable. 

Keywords: Two-Photon Microscopy (2PM), Three-Photon Microscopy (3PM), Intravital Imaging, Deep-Tissue Visualization, Adaptive Optics/Pulse Dispersion

CMLE Credit: 1.0