Nanoscopic and Correlative Porosity Analysis by Electron Microscopy of Biobased Porous Materials

Nanoscopic and Correlative Porosity Analysis by Electron Microscopy of Biobased Porous Materials | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nanoscopic and Correlative Porosity Analysis by Electron Microscopy of Biobased Porous Materials Kai Shen, Liwei Xia, Zhi Wang, Boka Xiang, Fanda Pan, Hua Huang, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8102710/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Biobased porous materials such as porous biochar or plant-based porous fiber are excellent candidates for applications in the fields of catalysts, energy, environment, etc. Porosity is the pivotal microstructural characteristic of these biomaterials, as it governs not only the capacity to accommodate metal-based active components, but also regulates the diffusion of target molecules. Currently, due to the lack of advanced characterization techniques and statistical analysis algorithms, comprehensive analysis of the nanoscopic porosity of biomaterials and the correlation between porosity and attributes like growth location and origin is often lacking. This results in a limited understanding of the pore structures in these materials. This study takes tobacco biomass as an example to reveal the correlation between microstructure and properties through electron microscopy and mercury intrusion methods, coupled with principal component analysis. The results reveal consistent pore structures across different bake tobacco samples, with an uneven distribution of pore sizes. Bake tobacco from upper and middle plant parts exhibit higher density compared to lower parts, and variations of porosity exist among bake tobaccos from different regions. The rich porous microstructure of bake tobacco based biomaterials has been systematically revealed. This research provides valuable insights for understanding microstructures of biobased porous materials, facilitating improvements of macroproperties. Furthermore, it establishes a foundation for interpreting the microstructure and macroproperties, paving a way for novel design of biobased porous materials for a broad range of applications. Biobased porous materials Electron microscopy Mercury porosimetry Pore structures Principal component analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Biobased porous materials have advantages for a wide range of applications due to their characteristics of large surface area, tunable pore volume and pore size distribution.(Gabhane, et al. 2020 ; Yang, et al. 2023 ) For the applicaions of biobased porous materials, it’s crucial to conduct struc- tural and compositional analysis, especially the nanoscopic and correlative porosity. However, a comprehensive analysis of nanoscopic porosity for these biobased porous materials, particularly one that incorporates statistical and correlative perspectives among macroscopic and microscopic physical parameters, as well as other attributes such as species and places of origin, is often lacking. The absence of advanced character- ization tools and algorithms for statistical analysis has led to a limited understanding of porosity in these biomaterials. As typical biobased porous materials, bake tobacco based porous materials have not yet been abundantly and advancedly investigated. The characterization of tobacco’s structure and compositions has mainly been investigated by employing conventional methods. For example, Tian et al. used the thermogravi- metric analysis (TGA) technique to study the pyrolysis characteristics and kinetics of waste tobacco leaves and stems in air and inert atmospheres at different heating rates, and determined a reaction model for the pyrolysis process of the leaves and stems, leading to an assessment of the potential for using waste tobacco leaves and stems as bioenergy feedstocks, providing a macro-scale pyrolysis information.(Tian, et al. 2023 ) Besides, Fourier-transform infrared spectroscopy ( FTIR), (Barontini, et al. 2013 ; Wang, et al. 2021 )[ X-ray diffraction (XRD), (Dallé, et al. 2021 ; Zhao, et al. 2017 ) nitrogen gas adsorption-desorption, (Yin, et al. 2015 ) mercury porosimetry, (Zi, et al. 2019 ) gas-liquid chromatography, (Huang, et al. 2018 ; Jacob, et al. 2011 ; Stepanov, et al. 2010 ) mass spectrometry,(Hossain and Salehuddin 2013 ; Zhang, et al. 2013 ) nuclear magnetic resonance, (Wei, et al. 2018 ; Zhu, et al. 2014 ) and other techniques have also been used for research on tobacco. However, in general, these characterization tools have primarily concentrated on developing processing pathways, optimizing parameters, and designing equipment ,(Stephenson, et al. 2020 ) lacking emphasis on a systematic, in-depth understanding of tobacco microstructure. Recent advancements in electron microscopy (EM) provide exciting opportunities to intuitively explore the microstructure of biomaterials. EM’s intricate details at the microscale substantially enhance our understanding of fundamental structures, lay- ing a robust foundation for explaining macroscopic properties. There are two primary characterization modes for EM, namely transmission electron microscopy (TEM) and scanning electron microscopy ( SEM) .(Dudkiewicz, et al. 2011 ) TEM and SEM differ in imaging principles, resolution, and applications, suiting them for distinct research and uses.(Akhtar, et al. 2018 ; Inkson 2016 ) TEM is ideal for internal structures and nanoscale details, while SEM excels in observing surface morphology. (Inkson 2016 ) In TEM, the electron beam transmits nanoscale-thick sam- ples, enabling high-resolution observation of internal morphology and composition. In SEM, a convergent electron beam scans the surface, providing structural and com- positional information. Zechmann used SEM to analyse tobacco surfaces, revealing presence of stomata. Alkhatib et al. used SEM to assess tobacco leaf surfaces with different lead (Pb) concentrations, exploring lead toxicity effects on gas exchange, chlorophyll, fluorescence, chloroplast ultrastructure, and stomatal aperture. (Alkhatib, et al. 2012 ) In a study by Yu et al., SEM was used to assess tobacco biochar’s absorption capacity in cadmium-contaminated soil. SEM imaging visualized biochar before and after cad- mium adsorption, revealing the remarkable cadmium adsorption capability.(Yu, et al. 2021 ) Sha et al. showcased the feasibility of preparing nitrogen-enriched porous carbon from tobacco waste through pretreatment by SEM analysis. (Sha, et al. 2015 ) Gao et al. used SEM to analyse the information of cell area and cell perimeter on the surface of tobacco leaves in different zones. (Gao and Bao 2023 ) Up to now, SEM has served as a bridge that connects the microscopic and macroscopic aspects of matter, complementing other characterization techniques. (Hao, et al. 2018 ) However, due to the fact that using TEM for tobacco characterization has to overcome the limit that the electron beam may damage samples, especially bio- logical ones, TEM studies for tobacco based biomaterials are quite rare.(Zechmann 2023 ) For this reason, nanoscopic and correlative porosity analysis for tobacco based porous materials has not been realized. In this study, shortcomings were addressed by using low-dose electron microscopy to image tobacco with minimal electron doses, thereby preventing radiation damage under the electron microscope. Tobacco samples from various growth regions of China were subjected to TEM and SEM characterization to compare their microstructural differences, focusing on the porosity analysis. Furthermore, various parts of individual tobacco plants were studied. Techniques like mercury porosimetry were used to anal- yse microscopic structures and visualize microchannels. Conclusions on tobacco pore size from these techniques align closely with EM observations. This comprehensive approach played a vital role in establishing a fundamental connection between tobacco microstructure and its macroscopic properties for a wide range of applications . 2 Materials and Methods The information regarding the year, source, category, and characteristic parts of the samples used in the experiment is detailed in Supplementary Table 1. Before characterization, the samples need to undergo pre-treatment, and the specific operations are as follows: As shown in Supplementary Fig. 1, before TEM characterization, each tobacco sample undergoes the following pre-treatment steps to ensure proper dispersion on the copper grid for electron microscope observation: Firstly, select appropriately shaped and regular tobacco samples, then grind them in a mortar and pestle, ensuring thorough grinding. Subsequently, disperse a small amount of finely ground sample in an ethanol solution; after ultrasonic dispersion, drop the ethanol supernatant onto the copper grid. After natural evaporation of ethanol, the sample achieves good dispersion on the copper grid. To prevent the accumulation which may hinder the observation of morphology and microstructure under the electron microscope, the concentration of the supernatant should be kept relatively dilute. For SEM testing, samples with a relatively flat shape are selected, cut into an appropriate size, and then fixed on conductive adhesive to ensure stability when the tobacco enters the sample chamber. Before mercury intrusion porosimetry characterization, ensure there are no other impurities in the tobacco sample, and the sample size is suitable for the sample chamber of the mercury intrusion porosimeter. The morphology and nanostructure were investigated using field emission SEM. The low-magnification chemical composition analysis was conducted by using energy dispersive X-ray spectrometer (EDX) equipped in the SEM. The high-resolution transmission electron microscopy was obtained at 300 kV on a transmission electron microscope. The experimental procedure for the mercury intrusion porosimetry is as follows: Place the prepared sample in the centre of the sample chamber of the mercury intrusion porosimeter to ensure uniform pressure distribution. Before testing, evacuate the system to eliminate air and other impurities, inject mercury (Hg) into the sample, gradually increasing the pressure. As the pressure increases, mercury enters the pores of the sample, filling them. Real-time record the injection pressure of mercury and the corresponding volume change. Stop the mercury intrusion process when the mercury enters the pores and reaches an equilibrium state, filling the pores in the sample. Then, release the residual mercury, extract it from the sample until the pressure drops to ze-ro. Using the recorded pressure and volume data, parameters such as the sample's pore volume, pore size distribution, and porosity can be calculated. 3 Results and discussion This study focuses on nanoscopic and correlative porosity analysis for biobased porous materials, taking tobacco as a typical example. First of all, electron microscopy characterization was conducted on tobacco samples obtained from different cities within the same province, as well as from different districts within the same city. Compared to mercury porosimetry, electron microscopy not only allows for the measurement of pore size but also provides detailed information about pore shape, distribution, and internal structure. This is crucial for understanding the microstructure of materials at nanoscale. Additionally, electron microscopy enables localized elemental analysis, facilitating the rapid determination of chemical composition distribution. In contrast, mercury porosimetry provide overall information about the sample. Tobacco has also been studied by previous researchers using TEM, but generally at a lower resolution, thus preventing a more in-depth study of the microstructure, as it is not possible to better correlate microscopic and macroscopic studies.(Baliga et al., 2003; Adeel et al., 2021) For the analysis of multiple datasets, applying principal component analysis (PCA) to the research involves first providing a brief introduction to the core idea of PCA. The primary goal of PCA is to find a space onto which the original data can be projected, thus achieving dimensionality reduction. The reduced-dimensional vectors (principal components) are expected to capture the main information of the original data. This space should meet two requirements: first, it should retain the most important components as much as possible; second, the retained components should have minimal correlation with each other. From a mathematical and informational perspective, retaining the main components means preserving data with larger variance, as greater variance indicates higher uncertainty, higher entropy, and therefore more information. Minimizing the correlation between components means that the covariance between the components should be small; otherwise, the information represented by two principal components would be highly similar, leading to wasted space. The PCA flowchart is shown in Supplementary Fig. 2. For multiple sets of data, the PCA algorithm can identify the most relevant subsets, which can greatly aid in data analysis. Firstly, as shown in Fig. 1 a-b, The pore regions in TEM images were delineated using contrast, the pore area and size were quantified, and the Average Feret diameter, Median Feret diameter, and Porosity were obtained. Combined with the data obtained from mercury intrusion porosimetry, including Bulk density, Apparent (skeletal) density, Porosity, and Pore volume, PCA was conducted on these datasets to explore the correlation between the two characterization methods. The PCA analysis results are presented in Supplementary Tables 2–3, where it is found that the first principal component (PC1) explained 38.5% of the variance, while the second principal component (PC2) explained 22.1% of the variance. As shown in Fig. 1 c, the Scree Plot displays the eigenvalues of each principal component, aiding in determining how many principal components should be retained. The Scree Plot indicated that the eigenvalues of the first two principal components were higher than the others, suggesting that they contained the most important information in the data. Together, these two principal components explained over 60% of the data variance, effectively compressing infor-mation for dimensionality reduction. The primary contributing factor to PC1 was the porosity obtained from mercury intrusion porosimetry, reflecting the spatial characteristics of the internal structure of tobacco samples under macroscopic testing conditions. On the other hand, the primary contributing factor to PC2 was the mean Feret diameter obtained from TEM, indicating the physical size and internal structure of tobacco samples under microscopic conditions. In the Score Plot (Fig. 1 d), points of different colors represent structures from different parts of the tobacco samples (A for upper part in black, B for middle part in red, C for lower part in green). The distribution of these points reflects the positions of different samples on PC1 and PC2. It can be observed from the plot that tobacco samples from different parts and regions exhibit distributions on PC1 and PC2, but the separation is more significant on PC1, suggesting that variables on PC1 (porosity) provide better discrimination among different parts of tobacco. Moreover, the minimal overlap of the 95% confidence ellipses indicates a high degree of discrimination between categories. The 95% confidence ellipses also show that there is less overlap between upper structure A and middle B and lower C, especially on PC1, indicating that porosity is a key parameter for different parts of tobacco structures. Furthermore, as shown in Fig. 1 e- 1 f, the Biplot illustrates the loadings of the original variables in the principal component space. It can be observed from the plot that the vectors for pore volume and porosity are in the same direction, indicating a positive correlation between these two variables. In conclusion, the PCA analysis confirmed the consistency between the characterization by mercury intrusion porosimetry and TEM. Tobacco samples from different parts exhibit distinct characteristics in porosity and mean Feret diameter, which may be related to the physiological structure and function of different parts of tobacco. These findings provide a basis for further investigation into the physical and chemical properties of tobacco parts and hold potential value for improving the functionality for nanozyme applications. The PCA results has indicated that porosity is a key parameter for different parts of tobacco structures. Except for porosity, the morphology and elemental information are also important for practical applications. To gain the morphology and elemental information, SEM observations were conducted on the surface and cross-section of three sets of tobacco samples from Yuxi, Tonghai, Yunnan. This is because the upper, middle, and lower parts of tobacco in that region have very distinct differences. As shown in Fig. 2 , in all three sets, the tobacco samples exhibited surfaces characterized by distinct roughness, compactness, and a high density of microscale pores. The surfaces of the tobacco samples exhibited shallow, small, hemispherical pores. The pores distribution appeared relatively uniform, with the pores in the upper part being smaller and denser, while those in the middle and lower parts were some-what larger. In the cross-sections of the tobacco, cylindrical and slit-like pores were observed (Supplementary Fig. 3). Moreover, the SEM images of the cross-sections clearly indicated that the upper tobacco leaves had a relatively denser structure compared to the middle and lower leaves, a conclusion that aligns consistently with the TEM and mercury porosimetry findings (Fig. 3 ). The data on the morphology and size of tobacco pores can be linked to the properties as nanozyme chemosensors. This enables us to allocate tobacco samples from the upper, middle, and lower parts, as well as from different regions, to their respective applications more reasonably. The rapid and intuitive electron microscopy technology provides a strong guarantee for the further development of the tobacco based porous biomaterials for nanozyme chemosensors. On the other hand, tobacco elemental analysis can help determine the content of certain essential elements for rational design of nanozymes. Energy dispersive X-ray spectroscopy (EDX) analysis was utilized for elemental determination in tobacco, with one of its advantages being a characterization method for localized elemental distribution, allowing elemental distribution information to be obtained both on the surface and within cross-sections at nanoscale. Herein, it is found that there are various metallic and non-metallic elements present at the surface of tobacco leaves, and their composition and content exhibit differences depending on the growth location of to-bacco and variations between the surface and cross-section. As shown in Fig. 2 , sur-face EDX analysis reveals that the distribution of carbon (C) and oxygen (O) elements at the surface of the upper, middle, and lower parts of tobacco leaves is relatively uniform. Additionally, there are trace amounts of both metallic and non-metallic elements present in all three regions (Supplementary Table 4). Simultaneously, EDX analysis of the cross-sections showed that there are almost no other elements except C and H (Supplementary Fig. 4 and Supplementary Table 4), suggesting that the foreign elements are mainly distributed at the surface of tobatoo, which is not absorbed into the inner parts. Furthermore, the elemental ratios of carbon and oxygen in the cross section is larger than that on the sur-face(Supplementary Table 4), this may be attributed to the fact that the surface of tobacco leaves is likely exposed to the atmosphere, where it can be influenced by the presence of oxygen, resulting in the formation of an oxidative layer on the surface. Additionally, the surface of tobacco leaves is typically more exposed to light, and therefore, it may be affected by varying light conditions, which can influence the rate of photosynthesis and the distribution of carbon. Taken together, these factors contribute to the differences in the carbon-to-oxygen ratio between the surface and cross-section. However, whether on the surface or in the cross-section, samples from different parts show little variation in the content of carbon (C) and oxygen (O) elements (Supplementary Table 4). This indicates that the differences in density of pores from different parts are not primarily due to elemental variations. The above results reflect the morphology and properties of the substance from the most fundamental and microscopic aspects of tobacco. These results can be applied to rational design of various kinds of products by regulating the porosity, morphology and elements. Additionally,an electron sensitivity test was conducted on the tobacco samples, revealing that when the electron dose rate reached 2e − /Å 2 s, electron beam damage occurred in the tobacco. The tobacco samples exhibited unavoidable distortion and compression, with some structures starting to disappear. The internal material showed significant signs of degradation (Supplementary Fig. 5). In our electron microscopy characterization experiments of tobacco, we rigorously control the electron dose rate to prevent electron radiation damage. Furthermore, the diffraction patterns of tobacco indicate a distinct non-crystalline structure, in line with the characteristic features of tobacco (Supplementary Fig. 5). Based on the above analysis, a systematic characterization of porosity was conducted for various tobacco samples from different regions, aiming to expand understanding of the influence of plant parts and geographical regions on porosity. Correlative analysis is performed by using TEM and mercury intrusion porosimetry. TEM characterization offers the advantage of being convenient, quick, and visually intuitive, while mercury intrusion porosimetry provides further complementary evidence, supporting the microstructural design of nanozymes. TEM images reveal that all the tobacco leaf samples exhibit distinct cylindrical and crack-like pore structures, with the presence of some pore-free dense substances. Tobacco leaves can be categorized into upper leaves, middle leaves, and lower leaves based on their growth position. Tobaccos with distinct parts exhibit varying structures, necessitating the microstructural characterization of tobacco leaves for their upper, middle, and lower parts. The sampling positions of different parts of tobacco are shown in Supplementary Fig. 6. Importantly, noticeable variations in pore structures are observed among samples obtained from different parts, even within the same growth region. This distinction is particularly prominent in samples collected from Jingdong, Puer, Yunnan, and Tonghai, Yuxi, Yunnan. TEM images from these two regions depict that the tobacco samples of upper part exhibit the densest microstructure, followed by the middle leaves, whereas the lower leaf tissues display larger pores and a relatively looser arrangement (Fig. 3 a and Fig. 3 b). These observations consistently align with the results obtained from the analysis con-ducted using mercury porosimetry (Fig. 3 d, Fig. 3 e, Fig. 3 f and Fig. 3 g), thus demonstrating a robust agreement between the two techniques. One plausible explanation for this phenomenon is that the upper leaves experience longer growth time, and generally receive a greater amount of sunlight and photosynthetic energy, necessitating an increased presence of chloroplasts and organelles to facilitate efficient photosynthesis. As a consequence, the upper leaves develop a denser tissue structure, enabling them to effectively capture and utilize light energy. (Sarlikioti et al., 2011 ) Conversely, the lower leaves, shaded by the upper leaves, receive comparatively less sunlight, resulting in a looser or more open tissue structure. Furthermore, data pertaining to the median pore diameter (volume), median pore diameter (area), and porosity of tobacco leaves from various regions consistently exhibit the same pattern across the upper, middle, and lower parts (Fig. 3 c). As a result, the observed structural characteristics of tobacco leaves from different positions on the same plant within a specific region appear to be a universally recurring phenome-non. Apparently, he phenomenon of tobacco leaves becoming progressively looser in microstructure from top to bottom is very intuitively represented in TEM images. Meanwhile, a comparison was conducted between tobacco samples from the same part and tobacco grown in different regions. Tobacco samples from some regions exhibited slightly larger pores compared to others. Overall, the pore structures in upper samples from different regions exhibited minimal differences, a fact supported by pore size distribution, volume median pore size, and porosity parameters obtained through mercury porosimetry (Fig. 4 k, Fig. 4 l and Fig. 4 m). In addition, as mentioned earlier, for upper tobacco leaves from different provinces, while the stomatal structure remains generally consistent overall, subtle differences exist. Tobacco samples from Hunan Province notably exhibit larger stomata compared to other provinces, making them more conducive to the exchange of substances with the external environment. This may be attributed to the moderate temperatures and adequate rainfall during the tobacco growth period in Hunan Province.(Tang et al., 2020 ) Further analysis of the differences between the volume median pore size and the area median pore size reveals that, there are still certain distinctions in the upper to-bacco samples from Henan province. For instance, in TEM images as shown in Fig. 4 c, Fig. 4 f, and Fig. 4 i, the pore sizes appear larger compared to other groups. This conclusion is in good agreement with the median pore size (volume) conclusions obtained from mercury porosimetry data (Fig. 4 m), suggesting tobacco from different regions exhibits differences in pore density. These conclusions were consistent with tobacco samples of upper part from Yunnan Province, where, among six sample groups, the overall differences in pore structures were relatively small. However, samples from Tonghai, Yuxi in Yunnan exhibited larger pore sizes and looser textures com-pared to other samples, reaffirming some regional variations in the microstructure of tobacco of the upper parts. Subsequently, a statistical analysis was conducted on the electron microscopy characterization of the middle part of tobacco samples. When compared with the visual inspection of samples from the upper part, the samples of middle part exhibited significantly larger pore sizes (Fig. 5 a-f), a fact supported by the median pore diameter (volume) data obtained through mercury intrusion porosimetry and pore size distribution charts (Fig. 5 g-i). The tobacco samples of middle part also displayed a similar pattern to the samples of upper part, with generally consistent pore sizes but slight regional variations. It is worth mentioning that samples from Tonghai, Yuxi, exhibit greater compactness compared to other regions. This could be due to the tobacco growth cycle coinciding with the rainy season in this region, which increases leaf density. The variation of porosity among different plant parts and geographical regions implies that tabacco based biomaterials possess rich porous microstructure, providing an important platform for products design by regulating the key parameters such as pore size, pore topology, active species, etc.. 4 Conclusion In summary, electron microscopy and mercury intrusion porosimetry were utilized on biobased porous materials, focusing on diverse tobacco samples. Generally, all samples exhibited relatively consistent pore structures and nonuniform size distributions. The PCA results scientifically confirmed the correlation between the conclusions drawn from mercury intrusion porosimetry and TEM. Both characterizations demonstrated variations of porosity among different plant parts and geographical regions of the tobacco samples; the upper parts appeared denser, potentially due to sunlight exposure and longer growth time. Additionally, the geoclimatic conditions play an important role in the variations of pore size. Surface and crosssection C/O ratio differences were observed, linking to oxygen contact. These findings are crucial for rational design of various kinds of producst, offering valuable insights for improving the performance in a wide range of applications. Declarations 5 Additional Requirements No new data were created or analyzed in this study. Data sharing is not applicable to this article. 6 Conflict of Int erest There are no conflicts to declare. 7 Author Contributio ns Conceptualization, K.S. and T.S.; methodology, L.X. and Z.W.; software, L.X. and Y.L.; validation, L.X., Z.W. and Y.L.; formal analysis, L.X., Z.W. and Y.L.; investigation, L.X., Z.W. and Y.L.; resources, H.F. and T.S.; data curation, B.X., F.P., H.H., Y.D.S., K.J., X.G.1, C.X., Z.Z., X.G.2, C.L., P.S., Y.L., B.D.; writing—original draft preparation, K.S. and L.X.; writing—review and editing, K.S., L.X., L.Z. and T.S.; visualization, Y.L.; supervision, H.F. and T.S.; project administration, K.S, H.F. and T.L.S.; funding acquisition, K.S. All authors have read and agreed to the published version of the manuscript. 8 Funding This research was funded by the Science Foundation of China Tobacco Zhejiang Industrial (Grant No. ZJZY2021A032). References Akhtar K, Khan SA, Khan SB, Asiri A (2018) M.Scanning electron microscopy: Principle and applications in nanomaterials characterization. Handb Mater Charact, 113–145 Alkhatib R, Maruthavanan J, Ghoshroy S, Steiner R, Sterling T (2012) Creamer, R.Physiological and ultrastructural effects of lead on tobacco. Biol Plant 56:711–716 Barontini F, Tugnoli A, Cozzani V, Tetteh J, Jarriault M, Zinovik (2013) I.Volatile products formed in the thermal decomposition of a tobacco substrate. Ind Eng Chem Res 52(42):14984–14997 Dallé D, Hansen B, Zattera AJ, Francisquetti EL, Catto AL (2021) Borsoi, C.Kinetic evaluation of tobacco stalk waste exposed to alkaline surface treatment under different conditions. Cellulose 28:2053–2073 Dudkiewicz A, Tiede K, Loeschner K, Jensen LHS, Jensen E, Wierzbicki R, Boxall ABA, Molhave (2011) K.Characterization of nanomaterials in food by electron microscopy. TRAC Trends Anal Chem 30(1):28–43 Gabhane JW, Bhange VP, Patil PD, Bankar ST, Kumar (2020) S.Recent trends in biochar production methods and its application as a soil health conditioner: A review. SN Appl Sci 2(7):1307 Gao R, Bao (2023) S.Advances in the application of nano-enzymes in the electrochemical detection of reactive oxygen species: A review. Chemosensors 11(8):440 Hammud HH, Alotaibi N, Otaibi A, Aljaafari N, Ahmed A, Azam F (2021) Prakasam, T.Hierarchical porous carbon cobalt nanocomposites-based sensor for fructose. Chemosensors 9(1):6 Hao Y, Yuan W, Ma C, White JC, Zhang Z, Adeel M, Zhou T, Rui Y (2018) Xing, B.Engineered nanomaterials suppress Turnip mosaic virus infection in tobacco (Nicotiana benthamiana). Environ Science: Nano 5(7):1685–1693 Hossain AM, Salehuddin S (2013) M.Analytical determination of nicotine in tobacco leaves by gas chromatography-mass spectrometry. Arab J Chem 6(3):275–278 Huang Z, Bi Y-J, Sha Y-F, Xie W-Y, Wu D, Liu B- (2018) Z.Separation and analysis of sucrose esters in tobacco by online liquid chromatography–gas chromatography/mass spectrometry. Anal Sci 34(8):887–891 Inkson B (2016) J.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for materials characterization. In: Hübschen G, Altpeter I, Tschuncky R, Herrmann H-G (eds) Materials Characterization Using Nondestructive Evaluation (NDE) Methods. Woodhead Publishing, pp 17–43 Jacob P, Yu L, Duan M, Ramos L, Yturralde O, Benowitz N (2011) L.Determination of the nicotine metabolites cotinine and trans-3-hydroxycotinine in biologic fluids using LC–MS/MS. J Chromatogr B 879(3):267–276 Khan I, Akhtar N, Khan S (2020) A.Palynological investigation of some selected species of family Fabaceae from Pakistan, using light and scanning electron microscopy techniques. Microsc Res Tech 83(3):223–231 Li C, Ahmed W, Li D, Yu L, Xu L, Xu T, Zhao (2022) Z.Biochar suppresses bacterial wilt disease of flue-cured tobacco by improving soil health and microbial diversity. Appl Soil Ecol 171:104314 Liu J, Niu (2022) X.Rational design of nanozymes enables advanced biochemical sensing. Chemosensors 10(10):386 Sarlikioti V, de Visser PHB, Marcelis LF (2011) M.Exploring spatial distribution of light interception and photosynthesis of canopies by a functional-structural plant model. Ann Botany 107(5):5 Sha Y, Lou J, Bai S, Wu D, Liu B, Ling (2015) Y.Facile preparation of nitrogen-doped porous carbon from waste tobacco and application in electrochemical capacitors and CO capture. Mater Res Bull 64:327–332 Sophanodorn K, Unpaprom Y, Whangchai K, Homdoung N, Dussadee N, Ramaraj R (2022) .Environmental management and valorization of cultivated tobacco stalks by combined pretreatment for potential bioethanol production. Biomass Convers Biorefinery 12(5):1627–1637 Stepanov I, Villalta PW, Knezevich A, Jensen J, Hatsukami D, Hecht (2010) S.Analysis of 23 polycyclic aromatic hydrocarbons in smokeless tobacco by GC–MS. Chem Res Toxicol 23(1):66–73 Stephenson MJ, Reed J, Patron NJ, Lomonossoff GP, Osbourn (2020) A.Engineering tobacco for plant natural product production, vol 6. Comprehensive Natural Products III, Elsevier, pp 244–262. 1 Tang Z, Chen L, Chen Z, Fu Y, Sun X, Wang B, Xia (2020) T.Climatic factors determine the yield and quality of Honghe flue-cured tobacco. Sci Rep 10(1):19868 Tian W-W, Xu F, Xing S-J, Wu R, Yuan (2023) Z.-Y.Comprehensive study on the thermal decomposition process of waste tobacco leaves and stems to investigate their bioenergy potential: Kinetic, thermodynamic, and biochar analysis. Thermochimica acta 723:179473 Wang A, Cai B, Fu L, Liang M, Shi X, Wang B, Deng N, Li (2021) B.Analysis of pyrolysis characteristics and kinetics of cigar tobacco and flue-cured tobacco by TG-FTIR. Beitr zur Tabakforschung Int / Contrib Tob Res 30(1):29–43 Wang Y, Fu B, Pan L, Chen L, Fu X, Li K (2013) .Overexpression of Arabidopsis Dof1, GS1 and GS2 enhances nitrogen assimilation in transgenic tobacco under low-nitrogen conditions. Plant Mol Biology Report 31(4):886–900 Wei S, Tian B-Q, Jia H-F, Zhang H-Y, He F, Song (2018) Z.-P.Investigation on water distribution and state in tobacco leaves with stalks during curing by LF-NMR and MRI. Drying Technol 36(12):1515–1522 Xie W-Q, Gong Y-X, Huang S- (2021) W.High-throughput gas chromatographic analysis of soluble carbon content in tobacco-related products enabled by phase-conversion reaction. J Sep Sci 4(2):86–91 Xiong J, Yang Y, Wang L, Chen S, Du Y, Song (2022) Y.Electrochemical sensors based on metal–porous carbon nanozymes for dopamine, uric acid and furazolidone. Chemosensors 10(11):86–91 Yang Y, Ye C, Zhang W, Zhu X, Li H, Yang D, Ahmed W, Zhao (2023) Z.Elucidating the impact of biochar with different carbon/nitrogen ratios on soil biochemical properties and rhizosphere bacterial communities of flue-cured tobacco plants. Front Plant Sci 14:1250669 Yin C, Xu Z, Shu J, Li Y, Sun W, Zhou Z, Chen M, Zhong (2015) F.Influence of physicochemical characteristics on the effective moisture diffusivity in tobacco. Int J Food Prop 18(3):690–698 Yu X, Zhou H, Ye X, Wang H (2021) From hazardous agricultural waste to hazardous metal scavenger: Tobacco stalk biochar-mediated sequestration of Cd leads to enhanced tobacco productivity. J Hazard Mater 413:125303 Zahmouli N, Marini S, Guediri M, Ben Mansour N, Hjiri M, Mir E, Espro L, Neri C, Leonardi G (2018) G.Nanostructured nickel on porous carbon–silica matrix as an efficient electrocatalytic material for non-enzymatic glucose sensing. Chemosensors 6(4):54 Zechmann B (2023) Different imaging techniques for 2D and 3D characterization of plant cell ultrastructure in the SEM and TEM. Microsc Microanal 29(Supplement 1):874–875 Zhang K, Zhang K, Cao Y, Pan W-P (2013) .Co-combustion characteristics and blending optimization of tobacco stem and high-sulfur bituminous coal based on thermogravimetric and mass spectrometry analyses. Bioresour Technol 131:325–332 Zhao D, Yang F, Dai Y, Tao F, Shen Y, Duan W, Zhou X, Ma H, Tang L, Li (2017) J.Exploring crystalline structural variations of cellulose during pulp beating of tobacco stems. Carbohydr Polym 174:146–153 Zi W, Chen Y, Pan Y, Zhang Y, He Y, Wang Q (2019) .Pyrolysis, morphology and microwave absorption properties of tobacco stem materials. Sci Total Environ 683:341–350 Zhu X, Liu B, Zheng S, Gao Y (2014) Quantitative and structural analysis of pectin in tobacco by 13C CP/MAS NMR spectroscopy. Carbohydr Polym 6(16):6407–6413 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8102710","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":558263005,"identity":"6ca3d0c6-400c-4fb6-927b-fe7a7e303453","order_by":0,"name":"Kai Shen","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Shen","suffix":""},{"id":558263007,"identity":"fad1dee0-9f77-4f07-8524-d97286ea33b0","order_by":1,"name":"Liwei Xia","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Liwei","middleName":"","lastName":"Xia","suffix":""},{"id":558263008,"identity":"19a40d20-51b3-4e81-924d-6938422cd694","order_by":2,"name":"Zhi Wang","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Wang","suffix":""},{"id":558263009,"identity":"8aad2cde-3a54-4aa0-ba55-8c2e29133273","order_by":3,"name":"Boka Xiang","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Boka","middleName":"","lastName":"Xiang","suffix":""},{"id":558263012,"identity":"162f1492-3592-49af-bf15-e7fb8172c511","order_by":4,"name":"Fanda Pan","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Fanda","middleName":"","lastName":"Pan","suffix":""},{"id":558263014,"identity":"6d18484a-e4b7-4486-901f-5446e11b655e","order_by":5,"name":"Hua Huang","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Huang","suffix":""},{"id":558263017,"identity":"856c569f-adc5-4693-a817-0ac7f0a4c0bf","order_by":6,"name":"Yuedian Shou","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yuedian","middleName":"","lastName":"Shou","suffix":""},{"id":558263019,"identity":"4e85d787-7c62-474e-92b4-fb7266a9aa98","order_by":7,"name":"Kaixuan Jiao","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kaixuan","middleName":"","lastName":"Jiao","suffix":""},{"id":558263021,"identity":"77b111e6-7bf2-4c06-bd9c-bae09323366b","order_by":8,"name":"Xuefeng Gao","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xuefeng","middleName":"","lastName":"Gao","suffix":""},{"id":558263022,"identity":"d00e0f95-0568-45fb-9ca4-8db70c8a83e2","order_by":9,"name":"Chen Xia","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Xia","suffix":""},{"id":558263038,"identity":"ec8c9138-8107-49d8-803c-f0d623254517","order_by":10,"name":"Zhangnan Zhong","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhangnan","middleName":"","lastName":"Zhong","suffix":""},{"id":558263041,"identity":"ee5d94f7-845f-4850-859b-aeeb27d84c02","order_by":11,"name":"Xinruiang Jiang","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinruiang","middleName":"","lastName":"Jiang","suffix":""},{"id":558263044,"identity":"c24f0d73-bf36-40df-9d89-aadf80014d04","order_by":12,"name":"Xiaoyuan Gao","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Gao","suffix":""},{"id":558263046,"identity":"32815f82-271f-4bfe-9769-acb3038e4ce6","order_by":13,"name":"Jingru Gao","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jingru","middleName":"","lastName":"Gao","suffix":""},{"id":558263047,"identity":"cba1ebd0-80f4-466e-8398-895c1f28cf22","order_by":14,"name":"Cuiyu Li","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Cuiyu","middleName":"","lastName":"Li","suffix":""},{"id":558263048,"identity":"300fb222-4894-4c41-834a-c1ab7c26992a","order_by":15,"name":"Ping Sun","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Sun","suffix":""},{"id":558263050,"identity":"7454faf3-5066-41b1-b8b7-6cdbfc91407d","order_by":16,"name":"Yikuan Liu","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yikuan","middleName":"","lastName":"Liu","suffix":""},{"id":558263051,"identity":"0be155b5-c619-4b2b-bb1b-ed54669dd7e3","order_by":17,"name":"Bin Deng","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Deng","suffix":""},{"id":558263052,"identity":"5be12bc7-43a6-4039-a3c5-2a55ae1c030e","order_by":18,"name":"Luyi Zhou","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Luyi","middleName":"","lastName":"Zhou","suffix":""},{"id":558263053,"identity":"47af7848-7746-446d-9618-6dfe865ae2a4","order_by":19,"name":"Hu Fan","email":"","orcid":"","institution":"China Tobacco Zhejiang Industrial Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"Fan","suffix":""},{"id":558263054,"identity":"c9ccf2b2-1276-4a1d-9cbe-6651df743dd1","order_by":20,"name":"Tulai Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3RMYoCMRSA4TcEkuapbSTgXiEyMI0DXuWJMLWwjZ0DC3qFETzEHGEkhY0HGLHRRhs7Gysx2coqplzY/EUC4X0kEIBY7C8m3DKXAy5K0L8nzQeBzK37PO1hYwmFkmRZTNaVGw8hYyYuasZNUh+up+/OAwbdlpL7zHsLZqpCw/SRKEWCtN8SU5WfcIXScEsaRyZ1S5yhl9iHoTaoD9vSkUUAgUwhFbJfMXCE9EdiMB1hk+seFjDcFHK43p9/lI+I1ep87DzlYil2F33L86/ubrq9+8h73P6+tHtSBgIAdgoejcVisX/VCz4NQQ5kgPpsAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Tulai","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-11-13 07:23:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8102710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8102710/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98423297,"identity":"9058b405-8edc-4a49-b013-520ecc53d42f","added_by":"auto","created_at":"2025-12-17 16:32:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":520966,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/45aed746e1c527025b0612aa.docx"},{"id":98422971,"identity":"a4a7e58c-c320-4696-bc67-31ca42eeb3ba","added_by":"auto","created_at":"2025-12-17 16:31:42","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18703,"visible":true,"origin":"","legend":"","description":"","filename":"d6626200afb748969e997819969fa8f5.json","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/791c1f0a82a092abebfa0ff0.json"},{"id":97970121,"identity":"819d1986-ad65-495e-a22b-10518b35b17b","added_by":"auto","created_at":"2025-12-11 10:34:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":982833,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/acca538df804e944b0a74cc2.docx"},{"id":98422923,"identity":"86d6dab0-57e2-4025-91aa-b63ac0e6602e","added_by":"auto","created_at":"2025-12-17 16:31:39","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100030,"visible":true,"origin":"","legend":"","description":"","filename":"d6626200afb748969e997819969fa8f51enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/443e7cff8a40f92cf783e151.xml"},{"id":97970124,"identity":"faf14e3c-09f3-4e90-ae6b-60dcf98fb931","added_by":"auto","created_at":"2025-12-11 10:34:58","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82808,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/9adaba31d96e962a8805a087.png"},{"id":98423039,"identity":"f8c6f535-7b27-49e0-8932-402a81d8fbc1","added_by":"auto","created_at":"2025-12-17 16:31:46","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36696,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/ff6d2662f7c5de3abf7d3782.png"},{"id":97970120,"identity":"46f5934c-c597-485c-a94d-ef14c9b1d3cc","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78251,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/6556a2988599267907ee69be.png"},{"id":97970129,"identity":"0300681f-9e6e-4234-a615-f8716ef300a3","added_by":"auto","created_at":"2025-12-11 10:34:58","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97002,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/0a62e7dbdc7d7a80141758cd.png"},{"id":97970126,"identity":"2022fe77-d45f-4f36-a601-32b194fe07d0","added_by":"auto","created_at":"2025-12-11 10:34:58","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82916,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/5770df7d2ac549fd68623434.png"},{"id":98423036,"identity":"f3e22026-c0d0-4f94-8d49-af406dcd6139","added_by":"auto","created_at":"2025-12-17 16:31:46","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98630,"visible":true,"origin":"","legend":"","description":"","filename":"d6626200afb748969e997819969fa8f51structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/e40a9d8fc164edbf18e404d0.xml"},{"id":97970128,"identity":"7029350f-6932-4a90-b5b2-b59475023f85","added_by":"auto","created_at":"2025-12-11 10:34:58","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106389,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/bca47db229e590e4651229f0.html"},{"id":97970114,"identity":"996e3688-7447-4078-a743-8dcb2cc34991","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106031,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of pore size data obtained from TEM and PCA of pore size data for each group of tobacco combined with mercury intrusion porosimetry. (a) Representation of pores in TEM images based on contrast (green lines indicate pore outlines, using the sample from Luoning, Luoyang, Henan as an example). (b) Pro-cessing performed on all samples as in (a). Principal component analysis of (c) Scree Plot, (d) Score Plot (tobacco samples in the upper part are in black, in the middle are in red, and in the lower part are in green), (e) Biplot, and (f) Joint plotting of Score Plot and Biplot. (f) PCA flow chart.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/665493fd48413717f32dc25c.jpeg"},{"id":97970113,"identity":"9ab75bf8-187a-4f2e-83ae-60c52030f71a","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39607,"visible":true,"origin":"","legend":"\u003cp\u003eEDX mapping results for surface of tobacco from different parts ((a)top, (b)middle and (c)lower) within the same region, using Tonghai County, Yuxi City, Yunnan Province, as an example.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/cd2e8c2b5c3052fe2a79adf0.jpeg"},{"id":97970115,"identity":"3030d049-6e65-4a22-a9bc-748ee9b504dd","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":91807,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images from different parts within the same region of tobacco samples from (a) Puer, Jingdong, Yunnan and (b) Tonghai, Yuxi, Yunnan. (c) Cumulative intru-sion vs pore size curves and (d) differential intrusion vs pore size curves obtained through mercury intrusion porosimetry for Puer, Jingdong, Yunnan tobacco samples. (e) Cumulative intrusion vs pore size curves and (f) differential intrusion vs pore size curves obtained through mercury intrusion porosimetry for Tonghai, Yuxi, Yunnan tobacco samples. (g) Median pore diameter(volume) of these regions obtained by mercury injection method.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/cf2350773cca33118e32e345.jpeg"},{"id":97970119,"identity":"f2c6b8f0-bd5c-48df-8c79-dd823f73ee0f","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116549,"visible":true,"origin":"","legend":"\u003cp\u003e(a-j) TEM images of same part in tobaccos from different growth regions in Henan province (the tobacco come from (a) Neixiang County, Nanyang City, (b) Sheqi County, Nanyang City, (c) Dengzhou County, Nanyang City, (d) Fangcheng County, Nanyang City, (e) Xixia County, Nanyang City, (f) Luoning County, Luoyang City, (g) Songxian County, Luoyang City, (h) Lingbao County, Sanmenxia City, (i) Lushi County, Sanmenxia City, (j) Biyang County, Zhumadian City). (k) cumulative intrusion vs pore size curves and (l) differential intrusion vs pore size curves obtained through mercury intrusion porosimetry. Along with corresponding (m)Median pore diameter(volume) of these regions obtained by mercury injection method.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/f182a61cddc42cb69032d58e.jpeg"},{"id":98423175,"identity":"381257b7-7dec-4592-a14b-18f941954f1e","added_by":"auto","created_at":"2025-12-17 16:31:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100970,"visible":true,"origin":"","legend":"\u003cp\u003e(a-f) TEM images of same part in tobacoos from different growth regions in Yunnan province (the tobacco come from (a) Xiangyun County, Dali City, (b) Luxi County, Honghe City, (c) Qiubei County, Wenshan City, (d) Luliang County, Qujing City, (e) Jingdong County, Puer City, (f) Tonghai County, Yuxi City. Along with corre-sponding (g) cumulative intrusion vs pore size curves and (h) differential intrusion vs pore size curves obtained through mercury intrusion porosimetry. (i) Median pore diameter of (volume) these regions obtained by mercury injection method.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/b966c7a3e7b07435c1c00720.jpeg"},{"id":102298607,"identity":"cb3f5c6d-b994-4254-a67b-4e1e31e4edce","added_by":"auto","created_at":"2026-02-10 10:52:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":909600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/5ddb8730-f5d2-4708-b7e8-14beee52913e.pdf"},{"id":97970117,"identity":"ccbcd18f-8f26-4387-990c-40c290c78d89","added_by":"auto","created_at":"2025-12-11 10:34:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":982833,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8102710/v1/bf932d13778d946811c94d53.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanoscopic and Correlative Porosity Analysis by Electron Microscopy of Biobased Porous Materials","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBiobased porous materials have advantages for a wide range of applications due to their characteristics of large surface area, tunable pore volume and pore size distribution.(Gabhane, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFor the applicaions of biobased porous materials, it\u0026rsquo;s crucial to conduct struc- tural and compositional analysis, especially the nanoscopic and correlative porosity. However, a comprehensive analysis of nanoscopic porosity for these biobased porous materials, particularly one that incorporates statistical and correlative perspectives among macroscopic and microscopic physical parameters, as well as other attributes such as species and places of origin, is often lacking. The absence of advanced character- ization tools and algorithms for statistical analysis has led to a limited understanding of porosity in these biomaterials. As typical biobased porous materials, bake tobacco based porous materials have not yet been abundantly and advancedly investigated. The characterization of tobacco\u0026rsquo;s structure and compositions has mainly been investigated by employing conventional methods. For example, Tian et al. used the thermogravi- metric analysis (TGA) technique to study the pyrolysis characteristics and kinetics of waste tobacco leaves and stems in air and inert atmospheres at different heating rates, and determined a reaction model for the pyrolysis process of the leaves and stems, leading to an assessment of the potential for using waste tobacco leaves and stems as bioenergy feedstocks, providing a macro-scale pyrolysis information.(Tian, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Besides, Fourier-transform infrared spectroscopy ( FTIR), (Barontini, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)[ X-ray diffraction (XRD), (Dall\u0026eacute;, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) nitrogen gas adsorption-desorption, (Yin, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) mercury porosimetry, (Zi, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) gas-liquid chromatography, (Huang, et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jacob, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stepanov, et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) mass spectrometry,(Hossain and Salehuddin \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) nuclear magnetic resonance, (Wei, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhu, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and other techniques have also been used for research on tobacco. However, in general, these characterization tools have primarily concentrated on developing processing pathways, optimizing parameters, and designing equipment ,(Stephenson, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) lacking emphasis on a systematic, in-depth understanding of tobacco microstructure.\u003c/p\u003e\u003cp\u003eRecent advancements in electron microscopy (EM) provide exciting opportunities to intuitively explore the microstructure of biomaterials. EM\u0026rsquo;s intricate details at the microscale substantially enhance our understanding of fundamental structures, lay- ing a robust foundation for explaining macroscopic properties. There are two primary characterization modes for EM, namely transmission electron microscopy (TEM) and scanning electron microscopy ( SEM) .(Dudkiewicz, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) TEM and SEM differ in imaging principles, resolution, and applications, suiting them for distinct research and uses.(Akhtar, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Inkson \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) TEM is ideal for internal structures and nanoscale details, while SEM excels in observing surface morphology. (Inkson \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) In TEM, the electron beam transmits nanoscale-thick sam- ples, enabling high-resolution observation of internal morphology and composition. In SEM, a convergent electron beam scans the surface, providing structural and com- positional information. Zechmann used SEM to analyse tobacco surfaces, revealing presence of stomata. Alkhatib et al. used SEM to assess tobacco leaf surfaces with different lead (Pb) concentrations, exploring lead toxicity effects on gas exchange, chlorophyll, fluorescence, chloroplast ultrastructure, and stomatal aperture. (Alkhatib, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) In a study by Yu et al., SEM was used to assess tobacco biochar\u0026rsquo;s absorption capacity in cadmium-contaminated soil. SEM imaging visualized biochar before and after cad- mium adsorption, revealing the remarkable cadmium adsorption capability.(Yu, et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Sha et al. showcased the feasibility of preparing nitrogen-enriched porous carbon from tobacco waste through pretreatment by SEM analysis. (Sha, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Gao et al. used SEM to analyse the information of cell area and cell perimeter on the surface of tobacco leaves in different zones. (Gao and Bao \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Up to now, SEM has served as a bridge that connects the microscopic and macroscopic aspects of matter, complementing other characterization techniques. (Hao, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) However, due to the fact that using TEM for tobacco characterization has to overcome the limit that the electron beam may damage samples, especially bio- logical ones, TEM studies for tobacco based biomaterials are quite rare.(Zechmann \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) For this reason, nanoscopic and correlative porosity analysis for tobacco based porous materials has not been realized.\u003c/p\u003e\u003cp\u003eIn this study, shortcomings were addressed by using low-dose electron microscopy to image tobacco with minimal electron doses, thereby preventing radiation damage under the electron microscope. Tobacco samples from various growth regions of China were subjected to TEM and SEM characterization to compare their microstructural differences, focusing on the porosity analysis. Furthermore, various parts of individual tobacco plants were studied. Techniques like mercury porosimetry were used to anal- yse microscopic structures and visualize microchannels. Conclusions on tobacco pore size from these techniques align closely with EM observations. This comprehensive approach played a vital role in establishing a fundamental connection between tobacco microstructure and its macroscopic properties for a wide range of applications .\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003eThe information regarding the year, source, category, and characteristic parts of the samples used in the experiment is detailed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eBefore characterization, the samples need to undergo pre-treatment, and the specific operations are as follows: As shown in Supplementary Fig.\u0026nbsp;1, before TEM characterization, each tobacco sample undergoes the following pre-treatment steps to ensure proper dispersion on the copper grid for electron microscope observation: Firstly, select appropriately shaped and regular tobacco samples, then grind them in a mortar and pestle, ensuring thorough grinding. Subsequently, disperse a small amount of finely ground sample in an ethanol solution; after ultrasonic dispersion, drop the ethanol supernatant onto the copper grid. After natural evaporation of ethanol, the sample achieves good dispersion on the copper grid. To prevent the accumulation which may hinder the observation of morphology and microstructure under the electron microscope, the concentration of the supernatant should be kept relatively dilute. For SEM testing, samples with a relatively flat shape are selected, cut into an appropriate size, and then fixed on conductive adhesive to ensure stability when the tobacco enters the sample chamber. Before mercury intrusion porosimetry characterization, ensure there are no other impurities in the tobacco sample, and the sample size is suitable for the sample chamber of the mercury intrusion porosimeter. The morphology and nanostructure were investigated using field emission SEM. The low-magnification chemical composition analysis was conducted by using energy dispersive X-ray spectrometer (EDX) equipped in the SEM. The high-resolution transmission electron microscopy was obtained at 300 kV on a transmission electron microscope.\u003c/p\u003e\u003cp\u003eThe experimental procedure for the mercury intrusion porosimetry is as follows: Place the prepared sample in the centre of the sample chamber of the mercury intrusion porosimeter to ensure uniform pressure distribution. Before testing, evacuate the system to eliminate air and other impurities, inject mercury (Hg) into the sample, gradually increasing the pressure. As the pressure increases, mercury enters the pores of the sample, filling them. Real-time record the injection pressure of mercury and the corresponding volume change. Stop the mercury intrusion process when the mercury enters the pores and reaches an equilibrium state, filling the pores in the sample. Then, release the residual mercury, extract it from the sample until the pressure drops to ze-ro. Using the recorded pressure and volume data, parameters such as the sample's pore volume, pore size distribution, and porosity can be calculated.\u003c/p\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003eThis study focuses on nanoscopic and correlative porosity analysis for biobased porous materials, taking tobacco as a typical example. First of all, electron microscopy characterization was conducted on tobacco samples obtained from different cities within the same province, as well as from different districts within the same city. Compared to mercury porosimetry, electron microscopy not only allows for the measurement of pore size but also provides detailed information about pore shape, distribution, and internal structure. This is crucial for understanding the microstructure of materials at nanoscale. Additionally, electron microscopy enables localized elemental analysis, facilitating the rapid determination of chemical composition distribution. In contrast, mercury porosimetry provide overall information about the sample. Tobacco has also been studied by previous researchers using TEM, but generally at a lower resolution, thus preventing a more in-depth study of the microstructure, as it is not possible to better correlate microscopic and macroscopic studies.(Baliga et al., 2003; Adeel et al., 2021)\u003c/p\u003e\u003cp\u003eFor the analysis of multiple datasets, applying principal component analysis (PCA) to the research involves first providing a brief introduction to the core idea of PCA. The primary goal of PCA is to find a space onto which the original data can be projected, thus achieving dimensionality reduction. The reduced-dimensional vectors (principal components) are expected to capture the main information of the original data. This space should meet two requirements: first, it should retain the most important components as much as possible; second, the retained components should have minimal correlation with each other. From a mathematical and informational perspective, retaining the main components means preserving data with larger variance, as greater variance indicates higher uncertainty, higher entropy, and therefore more information. Minimizing the correlation between components means that the covariance between the components should be small; otherwise, the information represented by two principal components would be highly similar, leading to wasted space. The PCA flowchart is shown in Supplementary Fig.\u0026nbsp;2. For multiple sets of data, the PCA algorithm can identify the most relevant subsets, which can greatly aid in data analysis.\u003c/p\u003e\u003cp\u003eFirstly, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, The pore regions in TEM images were delineated using contrast, the pore area and size were quantified, and the Average Feret diameter, Median Feret diameter, and Porosity were obtained. Combined with the data obtained from mercury intrusion porosimetry, including Bulk density, Apparent (skeletal) density, Porosity, and Pore volume, PCA was conducted on these datasets to explore the correlation between the two characterization methods. The PCA analysis results are presented in Supplementary Tables\u0026nbsp;2\u0026ndash;3, where it is found that the first principal component (PC1) explained 38.5% of the variance, while the second principal component (PC2) explained 22.1% of the variance. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, the Scree Plot displays the eigenvalues of each principal component, aiding in determining how many principal components should be retained. The Scree Plot indicated that the eigenvalues of the first two principal components were higher than the others, suggesting that they contained the most important information in the data. Together, these two principal components explained over 60% of the data variance, effectively compressing infor-mation for dimensionality reduction. The primary contributing factor to PC1 was the porosity obtained from mercury intrusion porosimetry, reflecting the spatial characteristics of the internal structure of tobacco samples under macroscopic testing conditions. On the other hand, the primary contributing factor to PC2 was the mean Feret diameter obtained from TEM, indicating the physical size and internal structure of tobacco samples under microscopic conditions. In the Score Plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), points of different colors represent structures from different parts of the tobacco samples (A for upper part in black, B for middle part in red, C for lower part in green). The distribution of these points reflects the positions of different samples on PC1 and PC2. It can be observed from the plot that tobacco samples from different parts and regions exhibit distributions on PC1 and PC2, but the separation is more significant on PC1, suggesting that variables on PC1 (porosity) provide better discrimination among different parts of tobacco. Moreover, the minimal overlap of the 95% confidence ellipses indicates a high degree of discrimination between categories. The 95% confidence ellipses also show that there is less overlap between upper structure A and middle B and lower C, especially on PC1, indicating that porosity is a key parameter for different parts of tobacco structures. Furthermore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, the Biplot illustrates the loadings of the original variables in the principal component space. It can be observed from the plot that the vectors for pore volume and porosity are in the same direction, indicating a positive correlation between these two variables. In conclusion, the PCA analysis confirmed the consistency between the characterization by mercury intrusion porosimetry and TEM. Tobacco samples from different parts exhibit distinct characteristics in porosity and mean Feret diameter, which may be related to the physiological structure and function of different parts of tobacco. These findings provide a basis for further investigation into the physical and chemical properties of tobacco parts and hold potential value for improving the functionality for nanozyme applications.\u003c/p\u003e\u003cp\u003eThe PCA results has indicated that porosity is a key parameter for different parts of tobacco structures. Except for porosity, the morphology and elemental information are also important for practical applications. To gain the morphology and elemental information, SEM observations were conducted on the surface and cross-section of three sets of tobacco samples from Yuxi, Tonghai, Yunnan. This is because the upper, middle, and lower parts of tobacco in that region have very distinct differences. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, in all three sets, the tobacco samples exhibited surfaces characterized by distinct roughness, compactness, and a high density of microscale pores.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe surfaces of the tobacco samples exhibited shallow, small, hemispherical pores. The pores distribution appeared relatively uniform, with the pores in the upper part being smaller and denser, while those in the middle and lower parts were some-what larger. In the cross-sections of the tobacco, cylindrical and slit-like pores were observed (Supplementary Fig.\u0026nbsp;3). Moreover, the SEM images of the cross-sections clearly indicated that the upper tobacco leaves had a relatively denser structure compared to the middle and lower leaves, a conclusion that aligns consistently with the TEM and mercury porosimetry findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The data on the morphology and size of tobacco pores can be linked to the properties as nanozyme chemosensors. This enables us to allocate tobacco samples from the upper, middle, and lower parts, as well as from different regions, to their respective applications more reasonably. The rapid and intuitive electron microscopy technology provides a strong guarantee for the further development of the tobacco based porous biomaterials for nanozyme chemosensors.\u003c/p\u003e\u003cp\u003eOn the other hand, tobacco elemental analysis can help determine the content of certain essential elements for rational design of nanozymes. Energy dispersive X-ray spectroscopy (EDX) analysis was utilized for elemental determination in tobacco, with one of its advantages being a characterization method for localized elemental distribution, allowing elemental distribution information to be obtained both on the surface and within cross-sections at nanoscale. Herein, it is found that there are various metallic and non-metallic elements present at the surface of tobacco leaves, and their composition and content exhibit differences depending on the growth location of to-bacco and variations between the surface and cross-section. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, sur-face EDX analysis reveals that the distribution of carbon (C) and oxygen (O) elements at the surface of the upper, middle, and lower parts of tobacco leaves is relatively uniform. Additionally, there are trace amounts of both metallic and non-metallic elements present in all three regions (Supplementary Table\u0026nbsp;4). Simultaneously, EDX analysis of the cross-sections showed that there are almost no other elements except C and H (Supplementary Fig.\u0026nbsp;4 and Supplementary Table\u0026nbsp;4), suggesting that the foreign elements are mainly distributed at the surface of tobatoo, which is not absorbed into the inner parts. Furthermore, the elemental ratios of carbon and oxygen in the cross section is larger than that on the sur-face(Supplementary Table\u0026nbsp;4), this may be attributed to the fact that the surface of tobacco leaves is likely exposed to the atmosphere, where it can be influenced by the presence of oxygen, resulting in the formation of an oxidative layer on the surface. Additionally, the surface of tobacco leaves is typically more exposed to light, and therefore, it may be affected by varying light conditions, which can influence the rate of photosynthesis and the distribution of carbon. Taken together, these factors contribute to the differences in the carbon-to-oxygen ratio between the surface and cross-section. However, whether on the surface or in the cross-section, samples from different parts show little variation in the content of carbon (C) and oxygen (O) elements (Supplementary Table\u0026nbsp;4). This indicates that the differences in density of pores from different parts are not primarily due to elemental variations. The above results reflect the morphology and properties of the substance from the most fundamental and microscopic aspects of tobacco. These results can be applied to rational design of various kinds of products by regulating the porosity, morphology and elements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally,an electron sensitivity test was conducted on the tobacco samples, revealing that when the electron dose rate reached 2e\u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003es, electron beam damage occurred in the tobacco. The tobacco samples exhibited unavoidable distortion and compression, with some structures starting to disappear. The internal material showed significant signs of degradation (Supplementary Fig.\u0026nbsp;5). In our electron microscopy characterization experiments of tobacco, we rigorously control the electron dose rate to prevent electron radiation damage. Furthermore, the diffraction patterns of tobacco indicate a distinct non-crystalline structure, in line with the characteristic features of tobacco (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eBased on the above analysis, a systematic characterization of porosity was conducted for various tobacco samples from different regions, aiming to expand understanding of the influence of plant parts and geographical regions on porosity. Correlative analysis is performed by using TEM and mercury intrusion porosimetry. TEM characterization offers the advantage of being convenient, quick, and visually intuitive, while mercury intrusion porosimetry provides further complementary evidence, supporting the microstructural design of nanozymes. TEM images reveal that all the tobacco leaf samples exhibit distinct cylindrical and crack-like pore structures, with the presence of some pore-free dense substances. Tobacco leaves can be categorized into upper leaves, middle leaves, and lower leaves based on their growth position. Tobaccos with distinct parts exhibit varying structures, necessitating the microstructural characterization of tobacco leaves for their upper, middle, and lower parts. The sampling positions of different parts of tobacco are shown in Supplementary Fig.\u0026nbsp;6. Importantly, noticeable variations in pore structures are observed among samples obtained from different parts, even within the same growth region. This distinction is particularly prominent in samples collected from Jingdong, Puer, Yunnan, and Tonghai, Yuxi, Yunnan. TEM images from these two regions depict that the tobacco samples of upper part exhibit the densest microstructure, followed by the middle leaves, whereas the lower leaf tissues display larger pores and a relatively looser arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These observations consistently align with the results obtained from the analysis con-ducted using mercury porosimetry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), thus demonstrating a robust agreement between the two techniques. One plausible explanation for this phenomenon is that the upper leaves experience longer growth time, and generally receive a greater amount of sunlight and photosynthetic energy, necessitating an increased presence of chloroplasts and organelles to facilitate efficient photosynthesis. As a consequence, the upper leaves develop a denser tissue structure, enabling them to effectively capture and utilize light energy. (Sarlikioti et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) Conversely, the lower leaves, shaded by the upper leaves, receive comparatively less sunlight, resulting in a looser or more open tissue structure. Furthermore, data pertaining to the median pore diameter (volume), median pore diameter (area), and porosity of tobacco leaves from various regions consistently exhibit the same pattern across the upper, middle, and lower parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). As a result, the observed structural characteristics of tobacco leaves from different positions on the same plant within a specific region appear to be a universally recurring phenome-non. Apparently, he phenomenon of tobacco leaves becoming progressively looser in microstructure from top to bottom is very intuitively represented in TEM images.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMeanwhile, a comparison was conducted between tobacco samples from the same part and tobacco grown in different regions. Tobacco samples from some regions exhibited slightly larger pores compared to others. Overall, the pore structures in upper samples from different regions exhibited minimal differences, a fact supported by pore size distribution, volume median pore size, and porosity parameters obtained through mercury porosimetry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em). In addition, as mentioned earlier, for upper tobacco leaves from different provinces, while the stomatal structure remains generally consistent overall, subtle differences exist. Tobacco samples from Hunan Province notably exhibit larger stomata compared to other provinces, making them more conducive to the exchange of substances with the external environment. This may be attributed to the moderate temperatures and adequate rainfall during the tobacco growth period in Hunan Province.(Tang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFurther analysis of the differences between the volume median pore size and the area median pore size reveals that, there are still certain distinctions in the upper to-bacco samples from Henan province. For instance, in TEM images as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, the pore sizes appear larger compared to other groups. This conclusion is in good agreement with the median pore size (volume) conclusions obtained from mercury porosimetry data (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em), suggesting tobacco from different regions exhibits differences in pore density. These conclusions were consistent with tobacco samples of upper part from Yunnan Province, where, among six sample groups, the overall differences in pore structures were relatively small. However, samples from Tonghai, Yuxi in Yunnan exhibited larger pore sizes and looser textures com-pared to other samples, reaffirming some regional variations in the microstructure of tobacco of the upper parts.\u003c/p\u003e\u003cp\u003eSubsequently, a statistical analysis was conducted on the electron microscopy characterization of the middle part of tobacco samples. When compared with the visual inspection of samples from the upper part, the samples of middle part exhibited significantly larger pore sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-f), a fact supported by the median pore diameter (volume) data obtained through mercury intrusion porosimetry and pore size distribution charts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-i). The tobacco samples of middle part also displayed a similar pattern to the samples of upper part, with generally consistent pore sizes but slight regional variations. It is worth mentioning that samples from Tonghai, Yuxi, exhibit greater compactness compared to other regions. This could be due to the tobacco growth cycle coinciding with the rainy season in this region, which increases leaf density.\u003c/p\u003e\u003cp\u003eThe variation of porosity among different plant parts and geographical regions implies that tabacco based biomaterials possess rich porous microstructure, providing an important platform for products design by regulating the key parameters such as pore size, pore topology, active species, etc..\u003c/p\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn summary, electron microscopy and mercury intrusion porosimetry were utilized on biobased porous materials, focusing on diverse tobacco samples. Generally, all samples exhibited relatively consistent pore structures and nonuniform size distributions. The PCA results scientifically confirmed the correlation between the conclusions drawn from mercury intrusion porosimetry and TEM. Both characterizations demonstrated variations of porosity among different plant parts and geographical regions of the tobacco samples; the upper parts appeared denser, potentially due to sunlight exposure and longer growth time. Additionally, the geoclimatic conditions play an important role in the variations of pore size. Surface and crosssection C/O ratio differences were observed, linking to oxygen contact. These findings are crucial for rational design of various kinds of producst, offering valuable insights for improving the performance in a wide range of applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003eAdditional Requirements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo new data were created or analyzed in this study. Data sharing is not applicable to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6 Conflict of\u003c/strong\u003e\u003cstrong\u003eInt\u003c/strong\u003e\u003cstrong\u003eerest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003cstrong\u003eAuthor Contributio\u003c/strong\u003e\u003cstrong\u003ens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, K.S. and T.S.; methodology, L.X. and Z.W.; software, L.X. and Y.L.; validation, L.X., Z.W. and Y.L.; formal analysis, L.X., Z.W. and Y.L.; investigation, L.X., Z.W. and Y.L.; resources, H.F. and T.S.; data curation, B.X., F.P., H.H., Y.D.S., K.J., X.G.1, C.X., Z.Z., X.G.2, C.L., P.S., Y.L., B.D.; writing—original draft preparation, K.S. and L.X.; writing—review and editing, K.S., L.X., L.Z. and T.S.; visualization, Y.L.; supervision, H.F. and T.S.; project administration, K.S, H.F. and T.L.S.; funding acquisition, K.S. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Science Foundation of China Tobacco Zhejiang Industrial (Grant No. ZJZY2021A032).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkhtar K, Khan SA, Khan SB, Asiri A (2018) M.Scanning electron microscopy: Principle and applications in nanomaterials characterization. Handb Mater Charact, 113\u0026ndash;145\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlkhatib R, Maruthavanan J, Ghoshroy S, Steiner R, Sterling T (2012) Creamer, R.Physiological and ultrastructural effects of lead on tobacco. Biol Plant 56:711\u0026ndash;716\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarontini F, Tugnoli A, Cozzani V, Tetteh J, Jarriault M, Zinovik (2013) I.Volatile products formed in the thermal decomposition of a tobacco substrate. Ind Eng Chem Res 52(42):14984\u0026ndash;14997\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDall\u0026eacute; D, Hansen B, Zattera AJ, Francisquetti EL, Catto AL (2021) Borsoi, C.Kinetic evaluation of tobacco stalk waste exposed to alkaline surface treatment under different conditions. Cellulose 28:2053\u0026ndash;2073\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDudkiewicz A, Tiede K, Loeschner K, Jensen LHS, Jensen E, Wierzbicki R, Boxall ABA, Molhave (2011) K.Characterization of nanomaterials in food by electron microscopy. TRAC Trends Anal Chem 30(1):28\u0026ndash;43\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGabhane JW, Bhange VP, Patil PD, Bankar ST, Kumar (2020) S.Recent trends in biochar production methods and its application as a soil health conditioner: A review. SN Appl Sci 2(7):1307\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao R, Bao (2023) S.Advances in the application of nano-enzymes in the electrochemical detection of reactive oxygen species: A review. Chemosensors 11(8):440\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHammud HH, Alotaibi N, Otaibi A, Aljaafari N, Ahmed A, Azam F (2021) Prakasam, T.Hierarchical porous carbon cobalt nanocomposites-based sensor for fructose. Chemosensors 9(1):6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHao Y, Yuan W, Ma C, White JC, Zhang Z, Adeel M, Zhou T, Rui Y (2018) Xing, B.Engineered nanomaterials suppress Turnip mosaic virus infection in tobacco (Nicotiana benthamiana). Environ Science: Nano 5(7):1685\u0026ndash;1693\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain AM, Salehuddin S (2013) M.Analytical determination of nicotine in tobacco leaves by gas chromatography-mass spectrometry. Arab J Chem 6(3):275\u0026ndash;278\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Z, Bi Y-J, Sha Y-F, Xie W-Y, Wu D, Liu B- (2018) Z.Separation and analysis of sucrose esters in tobacco by online liquid chromatography\u0026ndash;gas chromatography/mass spectrometry. Anal Sci 34(8):887\u0026ndash;891\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInkson B (2016) J.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for materials characterization. In: H\u0026uuml;bschen G, Altpeter I, Tschuncky R, Herrmann H-G (eds) Materials Characterization Using Nondestructive Evaluation (NDE) Methods. Woodhead Publishing, pp 17\u0026ndash;43\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJacob P, Yu L, Duan M, Ramos L, Yturralde O, Benowitz N (2011) L.Determination of the nicotine metabolites cotinine and trans-3-hydroxycotinine in biologic fluids using LC\u0026ndash;MS/MS. J Chromatogr B 879(3):267\u0026ndash;276\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan I, Akhtar N, Khan S (2020) A.Palynological investigation of some selected species of family Fabaceae from Pakistan, using light and scanning electron microscopy techniques. Microsc Res Tech 83(3):223\u0026ndash;231\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi C, Ahmed W, Li D, Yu L, Xu L, Xu T, Zhao (2022) Z.Biochar suppresses bacterial wilt disease of flue-cured tobacco by improving soil health and microbial diversity. Appl Soil Ecol 171:104314\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Niu (2022) X.Rational design of nanozymes enables advanced biochemical sensing. Chemosensors 10(10):386\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarlikioti V, de Visser PHB, Marcelis LF (2011) M.Exploring spatial distribution of light interception and photosynthesis of canopies by a functional-structural plant model. Ann Botany 107(5):5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSha Y, Lou J, Bai S, Wu D, Liu B, Ling (2015) Y.Facile preparation of nitrogen-doped porous carbon from waste tobacco and application in electrochemical capacitors and CO capture. Mater Res Bull 64:327\u0026ndash;332\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSophanodorn K, Unpaprom Y, Whangchai K, Homdoung N, Dussadee N, Ramaraj R (2022) .Environmental management and valorization of cultivated tobacco stalks by combined pretreatment for potential bioethanol production. Biomass Convers Biorefinery 12(5):1627\u0026ndash;1637\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStepanov I, Villalta PW, Knezevich A, Jensen J, Hatsukami D, Hecht (2010) S.Analysis of 23 polycyclic aromatic hydrocarbons in smokeless tobacco by GC\u0026ndash;MS. Chem Res Toxicol 23(1):66\u0026ndash;73\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStephenson MJ, Reed J, Patron NJ, Lomonossoff GP, Osbourn (2020) A.Engineering tobacco for plant natural product production, vol 6. Comprehensive Natural Products III, Elsevier, pp 244\u0026ndash;262. 1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang Z, Chen L, Chen Z, Fu Y, Sun X, Wang B, Xia (2020) T.Climatic factors determine the yield and quality of Honghe flue-cured tobacco. Sci Rep 10(1):19868\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTian W-W, Xu F, Xing S-J, Wu R, Yuan (2023) Z.-Y.Comprehensive study on the thermal decomposition process of waste tobacco leaves and stems to investigate their bioenergy potential: Kinetic, thermodynamic, and biochar analysis. Thermochimica acta 723:179473\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang A, Cai B, Fu L, Liang M, Shi X, Wang B, Deng N, Li (2021) B.Analysis of pyrolysis characteristics and kinetics of cigar tobacco and flue-cured tobacco by TG-FTIR. Beitr zur Tabakforschung Int / Contrib Tob Res 30(1):29\u0026ndash;43\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Fu B, Pan L, Chen L, Fu X, Li K (2013) .Overexpression of Arabidopsis Dof1, GS1 and GS2 enhances nitrogen assimilation in transgenic tobacco under low-nitrogen conditions. Plant Mol Biology Report 31(4):886\u0026ndash;900\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei S, Tian B-Q, Jia H-F, Zhang H-Y, He F, Song (2018) Z.-P.Investigation on water distribution and state in tobacco leaves with stalks during curing by LF-NMR and MRI. Drying Technol 36(12):1515\u0026ndash;1522\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie W-Q, Gong Y-X, Huang S- (2021) W.High-throughput gas chromatographic analysis of soluble carbon content in tobacco-related products enabled by phase-conversion reaction. J Sep Sci 4(2):86\u0026ndash;91\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiong J, Yang Y, Wang L, Chen S, Du Y, Song (2022) Y.Electrochemical sensors based on metal\u0026ndash;porous carbon nanozymes for dopamine, uric acid and furazolidone. Chemosensors 10(11):86\u0026ndash;91\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Ye C, Zhang W, Zhu X, Li H, Yang D, Ahmed W, Zhao (2023) Z.Elucidating the impact of biochar with different carbon/nitrogen ratios on soil biochemical properties and rhizosphere bacterial communities of flue-cured tobacco plants. Front Plant Sci 14:1250669\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin C, Xu Z, Shu J, Li Y, Sun W, Zhou Z, Chen M, Zhong (2015) F.Influence of physicochemical characteristics on the effective moisture diffusivity in tobacco. Int J Food Prop 18(3):690\u0026ndash;698\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu X, Zhou H, Ye X, Wang H (2021) From hazardous agricultural waste to hazardous metal scavenger: Tobacco stalk biochar-mediated sequestration of Cd leads to enhanced tobacco productivity. J Hazard Mater 413:125303\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZahmouli N, Marini S, Guediri M, Ben Mansour N, Hjiri M, Mir E, Espro L, Neri C, Leonardi G (2018) G.Nanostructured nickel on porous carbon\u0026ndash;silica matrix as an efficient electrocatalytic material for non-enzymatic glucose sensing. Chemosensors 6(4):54\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZechmann B (2023) Different imaging techniques for 2D and 3D characterization of plant cell ultrastructure in the SEM and TEM. Microsc Microanal 29(Supplement 1):874\u0026ndash;875\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang K, Zhang K, Cao Y, Pan W-P (2013) .Co-combustion characteristics and blending optimization of tobacco stem and high-sulfur bituminous coal based on thermogravimetric and mass spectrometry analyses. Bioresour Technol 131:325\u0026ndash;332\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao D, Yang F, Dai Y, Tao F, Shen Y, Duan W, Zhou X, Ma H, Tang L, Li (2017) J.Exploring crystalline structural variations of cellulose during pulp beating of tobacco stems. Carbohydr Polym 174:146\u0026ndash;153\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZi W, Chen Y, Pan Y, Zhang Y, He Y, Wang Q (2019) .Pyrolysis, morphology and microwave absorption properties of tobacco stem materials. Sci Total Environ 683:341\u0026ndash;350\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu X, Liu B, Zheng S, Gao Y (2014) Quantitative and structural analysis of pectin in tobacco by 13C CP/MAS NMR spectroscopy. Carbohydr Polym 6(16):6407\u0026ndash;6413\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biobased porous materials, Electron microscopy, Mercury porosimetry, Pore structures, Principal component analysis","lastPublishedDoi":"10.21203/rs.3.rs-8102710/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8102710/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiobased porous materials such as porous biochar or plant-based porous fiber are excellent candidates for applications in the fields of catalysts, energy, environment, etc. Porosity is the pivotal microstructural characteristic of these biomaterials, as it governs not only the capacity to accommodate metal-based active components, but also regulates the diffusion of target molecules. Currently, due to the lack of advanced characterization techniques and statistical analysis algorithms, comprehensive analysis of the nanoscopic porosity of biomaterials and the correlation between porosity and attributes like growth location and origin is often lacking. This results in a limited understanding of the pore structures in these materials. This study takes tobacco biomass as an example to reveal the correlation between microstructure and properties through electron microscopy and mercury intrusion methods, coupled with principal component analysis. The results reveal consistent pore structures across different bake tobacco samples, with an uneven distribution of pore sizes. Bake tobacco from upper and middle plant parts exhibit higher density compared to lower parts, and variations of porosity exist among bake tobaccos from different regions. The rich porous microstructure of bake tobacco based biomaterials has been systematically revealed. This research provides valuable insights for understanding microstructures of biobased porous materials, facilitating improvements of macroproperties. Furthermore, it establishes a foundation for interpreting the microstructure and macroproperties, paving a way for novel design of biobased porous materials for a broad range of applications.\u003c/p\u003e","manuscriptTitle":"Nanoscopic and Correlative Porosity Analysis by Electron Microscopy of Biobased Porous Materials","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 10:34:52","doi":"10.21203/rs.3.rs-8102710/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"817e16b1-0730-477d-9b74-eac1b00b8be5","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T04:55:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 10:34:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8102710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8102710","identity":"rs-8102710","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}