## 4 Conclusions  
In this study, for the first time, the possible size-dependent effects of 100 mg/L AlNPs (2000 nm and 200 nm) on seed germination were observed using bOCT. The higher internal activity within the seed was observed in biospeckle OCT images for AlNPs compared to the control after 24 h following exposure. Moreover, the highest increment of internal biological activity was observed for the 200 nm AlNPs exposure group compared to the 2000 nm AlNPs exposure and the control group, thus emphasizing a possible size-dependent positive effect. In addition, seed germination percentage, root length, shoot length, root fresh weight, and shoot fresh weight were also observed to compare with the bOCT results. The results are in agreement with the bOCT results. However, the conventional measurements took a minimum of 40 h to exhibit a significant effect, while the same effect was observed within 6 h of exposure using bOCT, thus emphasizing its greater reliability and observation speed.  
Figure 6: Effects of different alumina treatments (control, 2000 nm, and 200 nm alumina nanoparticles) on different conventional growth parameters in 7 day old lentil seedlings. Data reflect mean \(\pm\) SD (n = 3), * denotes a significant increment of growth parameters compared to the control (* \(p\) < 0.05, **_* p_ < 0.01).  
Hence, our results imply the possible size-dependent positive impact of AlNPs on seed germination due to the enhancement of energy metabolism. The findings presented in this paper reveal the positive effect of AlNPs on lentils. The effect is more significant when the particle size decreases. This is believed to be due to the enhancement of energy metabolism under the exposure to AlNPs. Further investigations are required on the biospeckles to determine the effect of AlNPs on seed germination.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13169203/s1](https://www.mdpi.com/article/10.3390/app13169203/s1)]([https://www.mdpi.com/article/10.3390/app13169203/s1](https://www.mdpi.com/article/10.3390/app13169203/s1)), Figure S1: The average OCT structural image obtained by averaging over 100 OCT (x-z) scans with six regions of interest (ROI) indicated by rectangles. Each rectangle corresponds to 512 \(\times\) 25 pixels; Figure S2: Effects of different alumina treatments (control, 2000 nm, and 200 nm alumina nanoparticles) on root dry weight of 7-day-old lentil seedlings; Figure S3: Effects of different alumina treatments (control, 2000 nm, and 200 nm alumina nanoparticles) on shoot dry weight of 7-day-old lentil seedlings.  
Conceptualization, Y.S.K.D.S. and H.K.; methodology, H.K. and U.M.R.; software, H.K.; validation, Y.S.K.D.S.; formal analysis, Y.S.K.D.S.; investigation, Y.S.K.D.S.; resources, Y.S.K.D.S.; data curation, Y.S.K.D.S.; writing--original draft preparation, Y.S.K.D.S.; writing--review and editing, Y.S.K.D.S., U.M.R. and H.K.; visualization, Y.S.K.D.S.; supervision, H.K.; project administration, H.K.; funding acquisition, H.K. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the JSPS Grant-in-Aid for Scientific Research (B) 19H04289 of the Ministry of Education, Culture, Sports, Science, and Technology in Japan.  
Not applicable.  
The data presented in this study are available on request from the corresponding author.  
The authors declare no conflict of interest.