## 3 Results and Discussion
### Quantitative Analysis and Normalized bOCT Contrast  
Figure 4 shows a quantitative comparison of bOCT contrast images over the treatments. The quantitative analysis was performed using six ROIs to clearly observe the possible size-dependent effect of alumina as shown in Figure 3a (Figure S1). The ROIs were selected close to the seed coat since bOCT data are more reliable for a few micron depths, and seed biological activities are also dominant close to the seed coat. Then, the average normalized contrast was calculated using the contrast of each ROI as a function of time to compare the effect of AlNPs quantitatively among the treatments. For reasons related to brevity, a brief description of the ANC and the calculation procedure are given in this paper, and a detailed explanation of the calculation can be found in previous publications [12; 13]. In this study, a normalization process was used to eliminate the individual variation across the samples. First, the average contrasts across the six different ROIs were calculated before and after 24 h of exposure. Then, the normalized contrasts were calculated by taking the ratio of the above two steps. Finally, the Averaged Normalized Contrast (ANC) was calculated by taking an average of six different seed samples. Interestingly, a significant difference from  
Figure 3: Region of interest (ROI) and comparison of bOCT images of lentil seed under exposure to AlNPs of two sizes, 200 nm and 2000 nm, at 24 h (**a**). The horizontal and vertical scale bars represent 0.4 mm and 0.06 mm, respectively, and average speckle contrast within ROIs of bOCT images (**b**). Data reflect mean \(\pm\) SD (n = 3), * denotes a significant increment of average bOCT contrast compared to the control (* \(p\) < 0.05). the average bOCT contrast was observed after only 6 h of exposure. During the experiment, the bOCT observations were taken after 0 h, 6 h, and 24 h following the exposures, since the average germination rate of the lentil seed was estimated as 24 h. Therefore, the bOCT observations were taken before the germination to observe the significant effect at an early stage before the germination. A clear significant increment (\(p<0.05\)) of ANC could be seen for both alumina treatments after only 6 h of exposure, compared to the control. For the 2000 nm AlNPs exposures, the increment rates of ANCs compared to that of the control were 4.7% and 9.2% at 6 h and 24 h, respectively. The same tendency could be observed for the 200 nm AlNPs exposures, for which the increment rates compared to the control were 9.3% and 17.9% at 6 h and 24 h, respectively. This result clearly emphasizes the positive impact of AlNPs on lentil seed germination and seedling growth. Interestingly, the positive effect was enhanced at smaller particle sizes. Therefore, alumina has a possible size-dependent positive effect on lentil seed germination. This is believed to be due to the enhancement of energy metabolism while under exposure to AlNPs. The results of this study agree with the research conducted by [7; 8], where they observed a positive effect after 2 and 7 days. However, using the bOCT technique, the effect on lentil seed germination was observed after only 6 h of exposure. Moreover, the internal activity of bOCT contrast of seeds for all the tested concentrations were gradually enhanced over time, as shown in Figure 4. However, the ANC at 0 h to 6 h rapidly increased compared to the time interval of 6 h to 24 h. In general, when seeds are exposed to water or any other treatments, in the first few hours, the seeds start to absorb water in order to activate basic metabolic processes, thus enhancing their internal activity. Furthermore, the diffusion of water within the lentil seed reached a maximum at around 6 h, and then plateaued [20]. The enlargement of the seed could be observed due to the enlargement of internal cells that occurred as a result of rapid water absorption. Therefore, during this process, the rapid increment of internal activity could be observed compared to the subsequent time intervals.