# New Insights in Potato Leaf Freezing by Infrared Thermography
## 3 Results  
Leaves of _S. tuberosum_ were subjected to controlled freezing experiments down to \(-3.0\)\({}^{\circ}\)C. They were either inoculated with ice or not. In half of the leaves ice nucleation was triggered, the other half remained supercooled. While the ice nucleated leaves showed tissue infiltrations and were limp, the supercooled leaves seemed visually unaltered after the low temperature treatment. 4 d afterwards, frost injuries manifested as blackish discolorations in leaves that had been ice nucleated (Figure 1a). Leaves that were exposed to freezing temperatures of \(-3.0\)\({}^{\circ}\)C without ice nucleation showed no symptoms of frost injury on the leaf blade (Figure 1b). When freezing of leaves of \(S\). _tuberosum_ is monitored by DTA, typically one pronounced freezing exotherm is recorded (Figure 2). Under the experimental conditions and with proper thermal insulation of the leaves, the release of heat during freezing lasted for approximately 20 min and exceeded 2.5 K.  
In comparison to DTA, IDTA revealed significantly more details during freezing of the \(S\). _tuberosum_ leaves (Figure 3; Video S1; Video S2). Particularly, the spatio-temporal pattern of freezing is visualized. In IDTA, the temperature pattern immediately before the onset of freezing is subtracted from all the subsequent following images. Therefore, the initial differential image showed a homogenous temperature distribution (Figure 3a). The initial ice wave started from the petiole where ice nucleation occurred under the experimental conditions at \(-1.3\)\({}^{\circ}\)C (Figure 3b). Rapid ice propagation captured the whole leaf blade within 60 s (Figure 3b-h). The spreading of ice occurred via the leaf vascular system, which was immediately followed by freezing throughout the blade. After 78 min a second freezing event on the leaves was recorded by IDTA (Figure 3i-j). This freezing process differed from the first exothermic event. The freezing pattern was diffuse all over the leaf blade without a distinct starting point. The exothermic response lasted distinctly longer than the first rapid event. After controlled  
Figure 1: Leaflets of \(S\). _tuberosum_ leaves subjected to a controlled freezing treatment down to \(-3.0\)\({}^{\circ}\)C either (**a**) inoculated with ice or (**b**) kept supercooled. Digital images were taken 4 d after the low temperature treatment. Figure 2: The DTA (differential thermal analysis) plot determined on leaves of \(S\). _tuberosum_ during a controlled freezing treatment at a rate of \(-3\) K\(\cdot\)h\({}^{-1}\) showed a single significant freezing exotherm. Minute zero indicated the starting point of the freezing event. thawing, the leaves were inspected for frost injuries. As expected, after such treatments the whole leaf blades were frost killed. Figure 3: IDTA (infrared differential thermal analysis) images obtained on a leaf of _S. tuberosum_ during a controlled freezing treatment at a cooling rate of 3 K\(\cdot\)h\({}^{-1}\). Whitening indicates heat released during freezing processes. The time series (**a**–**h**) shows the spatial spreading of ice after ice nucleation in the leaf petiole over the entire leaf blade during the first freezing event (Video S1). The freezing of this first exotherm lasted approximately 1 min. Much later (**i**–**j**) a second freezing event could be detected (Video S2). Time since initial ice nucleation is given in s/min at the top right corner. Leaf temperatures during freezing are indicated in the bottom right corner. With respect to the results of the ice susceptibility test (see Figure 1), and IDTA results, we intended to test the effect of the first freezing event detected in IDTA on cell viability. For this, leaves were monitored by infrared thermography during cooling at a rate of 3 K\(\cdot\)h\({}^{-1}\). Immediately after the first rapid freezing event (triggered by INA bacteria) the leaves were rewarmed at moderate rates to non-freezing temperatures.