MPL studies details of photodamage to improve imaging of tissues

29 May 2024

Insight into effects of femtosecond pulses on zebrafish larvae points to enhanced microscopy methods.

One concern in the use of femtosecond lasers for imaging of cells and tissues is the risk of causing photodamage to the delicate living systems being examined, thanks to the heating effects of the laser pulses employed.

Nonlinear techniques have given researchers some freedom to get around this problem, and in ingenious ways.

An example is the 2023 project at France's École Polytechnique studying how two-photon microscopy can reduce photodamage while also allowing deeper imaging with longer wavelength excitation. This showed that the same principle could be extended further to third-order principles, bringing additional benefits.

But the direct mechanisms and effects of ultra-fast laser irradiation on cell viability in vivo have remained largely unexamined, despite this being a vital consideration for label-free and multimodal microscopy approaches.

A project at the Max Planck Institute for the Science of Light (MPL) and Max-Planck-Zentrum für Physik und Medizin (MPZPM) has now studied the mechanisms of photodamage in zebrafish tissue at a cellular level triggered by femtosecond excitation pulses. The findings were published in Communications Physics.

"How do the dynamics of different photodamage mechanisms unfold across a spectrum of femtosecond pulses, particularly at near-infrared (NIR) wavelengths," asked the project. "What are the dynamics behind the photodamage at high peak intensities at NIR, and does the repetition rate of the laser pulses influence the driving mechanism?"

A better grasp of these issues is needed for the successful implementation of cutting-edge nonlinear microscopy techniques for deep-tissue, in vivo use in medical applications, commented MPL, adding that "a comprehensive understanding of the non-invasive, optimal operational parameters and constraints of the imaging system is essential."

Crucial findings for future microscopy techniques

The team applied precisely targeted femtosecond laser pulses to the central nervous system of zebrafish larvae under various irradiation settings. Different cellular dynamics resulting from the pulse-tissue interaction were observed over time, focusing on neural cells and on gauging the responsiveness of macrophages and fibroblasts, according to the team's published paper.

Short-term damage was assessed via loss of tissue integrity, and by monitoring particular neurons and cell types through fluorescent labeling. Long-term damage was studied by observing outright cell death and the response of the animal's immune system.

The results showed that at low repetition rates the damage is relatively confined, due to plasma-based ablation and sudden local temperature rise. At high repetition rates, the damage becomes collateral due to plasma-mediated photochemistry.

"We demonstrated that damage to the central nervous system of zebrafish when irradiated by femtosecond pulses at 1030 nanometers occurs abruptly at the extreme peak intensities required for low-density plasma formation," said Soyeon Jun from MPL.

"This allows for a noninvasive increase in imaging dwell time and photon flux during irradiation at 1030 nm, as long as the peak intensity is below the low-plasma density threshold. This is crucial for nonlinear label-free microscopy."

The results should lead to guidelines for new nonlinear imaging techniques under development at MPL, including femtosecond fieldoscopy, a novel metrology in which molecules are excited by ultrashort pulses and the complex electric field of the transmitted light containing the molecular information is directly measured afterwards.

"These findings significantly contribute to advancements in deep tissue imaging techniques and innovative microscopy techniques like femtosecond fieldoscopy, being developed in my group," commented MPL's Hanieh Fattahi. "This technique allows for the capture of high spatial resolution, label-free images with attosecond temporal resolution."