Author: Crysthal Alvarez
Vitrification is a rapid approach to cryopreserve biological samples without ice formation, which induces mechanical injury to the specimen. This novel approach, however, requires critical cooling and rewarming rates. Biospecimens in droplet scales, 2 to 5 mm in diameter, such as cells and embryos are successfully vitrified at the required critical cooling rates. However, the rewarming stage remains a challenge as conduction warming is slow and leads to devitrification, ice formation during the rewarming stage. To overcome this difficulty, dispersing plasmonic nanomaterials in cryoprotective agent solutions (CPAs) to rapidly convert optical radiation into heat, supplied by a laser beam, has been proposed.
In previous work, we presented titanium nitride (TiN) nanomaterials as an inexpensive, more reliable, and simpler plasmonic alternative to the commonly used gold nanorods (GNRs) to overcome this difficulty. Additionally, we developed a four-laser beam (4LB) system that uses beam splitting to uniformly heat samples from multiple sides and achieve uniform rapid warming rates. The optical system consisted of a collimated 808 nm CW laser beam with an approximate diameter of 2mm. Keeping the concentrations and volume of each nanoparticle solution in a cuvette constant, we demonstrated that using the 4LB setup induced a higher heating rate than only one laser beam at the same laser power. Additionally, we demonstrated TiN nanoparticles and clusters have better heating performance than GNRs as they had an increasing nonlinear behavior of heating rate as the laser power increased and volume of solution decreased, which would be beneficial in rewarming small droplet samples. Furthermore, we demonstrated TiN clusters had similar heating uniformity as GNRs and improved at a higher concentration using the 4LB setup. Later on, we proved TiN nanoparticles can also be used in microdroplet rewarming as we vitrified a droplet and successfully rewarmed it without ice formation, however, no thermal characterization was performed due to droplet size and measurement limitations.
The temperature of CPAs and nanoparticle solutions are measured with thermocouples; however, this only gives information of the localized point. Additionally, measuring the cooling or warming rates in droplets disrupts the field of investigation and can induce ice nucleation. Therefore, to noninvasively measure the rapid cooling and photothermal heating happening inside a droplet, we designed a digital holography interferometry (DHI) system to measure the temperature over time in 2D. As the droplets are being vitrified and rewarmed a HeNe laser passes through the droplet. A CMOS camera records the interference of that beam and the reference beam, resulting in a hologram. Through image processing, the refractive index is calculated from the phase difference maps and is converted into a temperature profile. The results give accurate measurements of temperature within the droplet, which correlate to the cooling/heating rate and temperature distribution. Through these results we can find the optimal cooling and photothermal heating conditions to successfully achieve the required rates to avoid ice formation and devitrification. Additionally, the DHI setup can also be used to determine phase changes during vitrification. Ultimately, we demonstrate DHI as a tool to accurately characterize droplets, and other uL to L scale samples, vitrification phases and rewarming temperatures which can potentially scale up photonic warming and enhance the viability of biological systems.
Figure 3.1. Digital Holography Interferometry (DHI) system.