Real-time Nanoscale Temperature Sensing During Femtosecond Laser Patterning of Protein-Based Bioink

Femtosecond lasers enable microstructure fabrication for biomedical use, but their thermal impact on living systems must be assessed. This study investigates nanoscale photothermal effects during femtosecond direct laser writing (fsDLW) in a protein-based photoresist using nano-thermometry. NaYF4:Yb3+/Er3+ luminescent nanoparticles served as temperature sensors, tracking heat variations under different laser parameters. Writing was performed at 755 nm, with temperature monitored via a 976 nm laser. Results show scan speed, laser power, and photosensitizer concentration (methylene blue) strongly affect heating. Faster scans reduced thermal buildup, while higher laser power increased temperature spikes. At 20 µm/s, heating remained at ΔT ≈ 11 °C in a sub-femtoliter focal volume, supporting safe fsDLW for biological applications. These insights help optimize femtosecond laser settings for precise, biocompatible microfabrication and enable real-time thermal monitoring for safer laser-based bioprinting.