Multiphoton microscopy enables three-dimensional imaging of biological tissue. Scanning two-photon (2P) microscopy in particular has become firmly established in medicine. However, the technique has its limitations: scattering effects within the sample degrade image quality, and the frame rates of point-scanning systems are insufficient to capture very rapid biological processes.

Infrared wavelength for a cost-efficient laser system

This is where the UTOPI3 project comes in. The consortium is advancing three-photon (3P) microscopy, in which three photons simultaneously excite a fluorescent molecule instead of two. To achieve this, the team is working at a wavelength of 1,840 nanometers. Since this wavelength range is currently not reliably or cost-efficiently covered by commercially available laser systems, the LZH is developing and researching a new femtosecond source within the UTOPI3-Laser sub-project. The source is designed to generate pulses shorter than 250 fs with pulse energies of up to 1 µJ across this wavelength range. The laser system will be engineered for straightforward integration with advanced imaging modalities, while also being user-friendly and financially accessible — enabling broad adoption of the improved technique in clinical settings.

Three Instead of Two: Greater Depth Through Three-Photon Excitation

Multiphoton microscopy relies on a distinctive excitation principle: rather than being excited by a single short-wavelength, high-energy photon, fluorophores (fluorescent molecules found in biological tissue) can be excited by multiple longer-wavelength, lower-energy photons. In 3P microscopy, three photons must reach the fluorophore simultaneously, each carrying one-third of the energy of an equivalent short-wavelength photon. This is achieved using laser light with a wavelength of 1.8 µm.

Using long-wavelength excitation significantly reduces light scattering, even in dense biological tissue. This results in a penetration depth that can be up to twice as great as with conventional approaches. The lower-energy light also minimizes phototoxic effects, making the technique considerably gentler on tissue.

To achieve the shared goal of fast, efficient 3P microscopy at maximum penetration depth, the consortium combines the 3P laser source with wide-field excitation and high-speed scanning techniques. Rather than scanning point by point, the system acquires data frame by frame — enabling substantially higher imaging rates.

About UTOPI3

The UTOPI3-Laser sub-project is part of the innovation consortium "Ultrashort-Pulse Fiber Laser-Based Wide-Field Microscopy with 3-Photon Excitation" (UTOPI3). It is funded by the Investment and Development Bank of Lower Saxony (NBank) using resources from the European Regional Development Fund. Project partners include Leibniz University Hannover, Emden/Leer University of Applied Sciences, and Laser Zentrum Hannover e.V.