Lamina 3

MitoSox Red (Molecular Probes, Eugene, OR) was dissolved in a 1:1 mixture of dimethylsulfoxide (DMSO) and saline to a final concentration of 33 μM. Under isoflurane anesthesia, 10 μl of MitoSox was injected intrathecally by using a direct transcutaneous intrathecal injection method described previously. Approximately 23 hrs after MitoSox injection, mice received an i.d. injection of either capsaicin (0.5 %, 25 μl) or the same volume of vehicle on both the plantar and dorsal surfaces of the left hind foot. Mice remained under 1.5 % isoflurane anesthesia for 30 min to suppress any side effects induced by capsaicin. Mice were perfused through the aorta with saline followed by fixative containing 4% paraformaldehyde 1 hr after capsaicin injection and the L4-L5 spinal cord segments were removed. The cord was postfixed 4-15 hr in the perfusion fixative, equilibrated in 30% sucrose, cryosectioned at 30 μm, and mounted on gelatin coated slides. The sections were examined under a fluorescent microscope with a rhodamine filter. Two different regions of the dorsal horn were photographed from 10 randomly selected sections from each animal: the lateral part of laminae I -II and laminae III-V. Photographs were taken with a Q-Imaging Retiga 2000R digital camera attached to an Olympus BX50 microscope using a 63x oil objective lens and saved as digital image files. The number of MitoSox positive cellular profiles with distinctive nuclei (dark oval shaped space surrounded by red granules) was counted from these pictures.

Rapid cooling and laser warming were performed as described by Khosla et al.^{6 (link)}, with small modifications as shown in Figure 2. Instead of a zebrafish embryo, microliter-sized droplets with varying CPA and GNR concentrations were pipetted onto the 3.0 × 2.0 × 0.08 mm blade of a modified cryotop (Fig. 6a). A specially designed automated system was used to rapidly cool the droplets by plunging them into liquid nitrogen for at least 10 seconds to allow for equilibration to liquid nitrogen temperature (Fig. S4). For rewarming, a 1064nm Nd: YAG laser (iWeld 980 Series, 120 joule, LaserStar Technologies, FL, USA) was used to provide a high energy singular millisecond pulse. This laser is equipped with a stereomicroscope with an eyepiece crosshair reticule to allow for visualization and alignment of the specimen within the laser chamber, along with a phototube to record high speed video. Once the jig raises the cryotop into the laser’s focus, the laser is automatically fired. The energy provided by a single pulse can be varied by changing the input voltage and pulse time. A laser calibration table was generated using a laser power meter (Nova II, Ophir, Jerusalem, Israel) to determine the amount of energy in joules produced by the laser at a given voltage and pulse time (See Figure S3).
This entire process of cooling and laser warming droplets is captured by the camera. There are two distinct time points in the video which are used to observe ice formation during this process. The first is “prewarming” wherein the droplet is quickly raised into the laser’s focus from the liquid nitrogen; this transition takes less than 0.3s. For our purposes, a vitrified droplet appears transparent without any white spots, whereas ice formation in the droplet appears white or cloudy (either partially or completely). For example, a vitrified 1µL droplet of 2M PG and 1M Trehalose can be seen in Fig. 6d, and a crystalized 1µL droplet of 2M PG can be seen in Fig. 6e.
The second observation point is “post-warming,” wherein the droplet is seen immediately after the laser is fired. It should be noted that the video recording during the millisecond(s) of laser warming is blocked by a protective filter due to possible damage to the camera. Our physical definition of success is that the droplet remains clear, i.e. without the appearance of ice post laser warming. A first warming failure mode we term crystallization exists when the droplet shows ice (white spots) due to underheating because the laser energy was too low to completely rewarm the droplet to its melting temperature. A second failure mode exists if the droplet disappears or get damaged if the laser energy exceeds the rewarming threshold. Examples of all three cases can be seen in Figures 7a–c. For each laser warming case, the voltage and pulse width were varied until there was no ice formation within the droplet during rewarming (n=5). For example, a laser pulse with voltage of 250V and pulse width of 1.6ms successfully rewarmed a vitrified 1µL droplet with 2M PG, 1M Trehalose and 1.26×10¹⁷ nps/m³ GNR. This technique was used to obtain the laser energy conditions for physical success at different GNR concentrations, CPA concentrations, and droplet volumes.