Ultra-rapid vitrification and temperature measurement in laser rewarming of vitrified droplets

Successful cryopreservation and revival rely on effective cooling and rewarming strategies. Rewarming processes are more vital, because insufficient rewarming normally leads to devitrification or recrystallization, resulting in irreversible damage to biomaterials. Conventional rewarming methods often fail to achieve the required super-high rates. Laser warming has emerged as a promising approach for achieving ultra rapid and internalized heating. Most studies on laser rewarming rely heavily on simulations and lack experimental validation. Moreover, conventional procedures require the manual transfer of vitrified samples from liquid nitrogen to laser exposure spot, which introduces operational risks. To address these limitations, an integrated platform combining vitrification-based cryopreservation with automated laser rewarming was developed. A fast-response temperature measurement system was incorporated to monitor the real-time thermal changes in samples during laser irradiation. The laser rewarming rate of a 1  μL droplet was measured to be 3.5 × 10 6  K/min, which is far beyond those achieved by conventional water bath methods and significantly higher than the required critical warming rate. Three distinct temperature evolution modes were observed: insufficient, appropriate, and excessive heating. We directly confirmed a temperature overshoot followed by rapid thermal decay immediately after laser exposure, which has never been reported before. By varying the laser energy and beam size, the extent of temperature overshoot and subsequent thermal relaxation were controlled, which enabled the optimization of laser rewarming strategy. The findings can be used for the refinement of numerical models and will contribute to the development of safer and more effective rewarming protocols in the cryopreservation field.