Method for Lignin Analysis in Wood by Fluorescence Microscopy
Josy Tainara Silva Silva, Silvino Intra Moreira, Jordão Cabral Moulin
TL;DR
This study finds that autofluorescence in wood slides, combined with automated image analysis, reliably measures lignin content in Amazonian species.
Contribution
The study introduces an automated fluorescence microscopy method for lignin analysis in native Amazonian woods.
Findings
Autofluorescence in extractive-free wood slides showed the highest correlation between fluorescence intensity and lignin content.
Automated analysis using ImageJ improved speed, reproducibility, and standardization of lignin fluorescence measurements.
The method achieved a correlation coefficient of r = 0.87 and R² = 76.22% for lignin content estimation.
Abstract
Native Amazonian species present high anatomical variation which is reflected in their lignin content. This study tested different lignin fluorescence treatments and fluorescence intensity processing methods in native Amazonian woods. Validating the fluorescence technique for lignin analysis included relating the total wood lignin content to the fluorescence intensity emitted by lignin in histological sections. Seven native Amazonian species were analyzed. Wood lignin content was obtained by the Klason method. Four treatments were used for fluorescence in the histological sections: autofluorescence (in natura and without extractives), basic fuchsin and Mäule. Images obtained with the fluorescence microscope were processed using ImageJ, applying three different methodologies to obtain fluorescence intensity—two using an integrated density formula and one automatically. Autofluorescence…
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FIGURE 8| Species | Extractive content (%) | Lignin content (%) |
|---|---|---|
| Guaruba ( | 5.91 (0.136) | 32.43 (0.164) |
| Angelim vermelho ( | 14.03 (0.332) | 32.10 (0.166) |
| Angelim pedra ( | 8.60 (0.144) | 30.84 (0.326) |
| Freijó ( | 6.68 (0.089) | 30.40 (0.133) |
| Pequiá ( | 5.46 (0.171) | 30.28 (0.231) |
| Angelim saia ( | 12.15 (0.262) | 28.92 (0.693) |
| Melancieira ( | 9.61 (0.187) | 27.14 (0.633) |
| Treatment | Methods | Regression equation | Coef. determination ( | Pearson correlation coef. ( |
|---|---|---|---|---|
| Autofluorescence in natura slides | 1 | FI = 2.28E+08 − 6,233,801 TL | 49.82% | −0.70 |
| 2 | FI = 3.01E+08 − 7,775,011 TL | 48.50% | −0.69 | |
| 3 | FI = 2.34E+08 − 6,530,456 TL | 52.42% | −0.72 | |
| Autofluorescence on slide without extractive | 1 | FI = −12,528,229 + 741,044 TL | 75.09% | 0.86 |
| 2 | FI = −4,289,370 + 1,095,170 TL | 77.77% | 0.88 | |
| 3 | FI = −23,943,422 + 1,067,387 TL | 76.22% | 0.87 | |
| Mäule | 1 | FI = 1.02E+08–2,562,944 TL | 38.63% | −0.62 |
| 2 | FI = 1.65E+08–3,491,596 TL | 35.46% | −0.59 | |
| 3 | FI = 1.35E+08–3,347,305 TL | 34.67% | −0.58 | |
| Basic fuchsin | 1 | FI = −33,343,615 + 2,571,289 TL | 26.95% | 0.52 |
| 2 | FI = −91,375,719 + 5,787,301 TL | 35.82% | 0.60 | |
| 3 | FI = −1.11E+08 + 5,609,966 TL | 35.16% | 0.59 |
- —Coordenação de Aperfeiçoamento de Pessoal de Nível Superior10.13039/501100002322
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Taxonomy
TopicsLignin and Wood Chemistry · Plant Gene Expression Analysis · Enzyme-mediated dye degradation
Summary
- Results point to autofluorescence on a slide without extractives as the most suitable treatment for studying the relation between fluorescence intensity and lignin content among those analyzed.
- Automatic determination with the subtract tool from ImageJ was the most efficient for processing photomicrographs and determining fluorescence intensity.
Introduction
1
Lignin, one of the main structural components of wood, plays an essential role in its rigidity, impermeability and resistance to attack by microorganisms. Chemically, lignin is a complex aromatic polymer which, associated with cellulose and hemicellulose, confers mechanical and chemical properties to vascular plants (Tribot et al. 2019). Additionally, lignin is highly relevant for industrial processes such as the production of paper, energy, biofuels and composite materials, making it an object of growing interest in scientific research.
In the Amazon Rainforest, wood from native species presents a high diversity of structural and chemical characteristics, including significant variations in lignin content that directly influence wood physical and chemical properties (Ferreira et al. 2020). In this context, fluorescence microscopy has emerged as a promising tool that enables the qualitative and quantitative analysis of lignin in histological wood samples (Gierlinger and Schmidt 2006).
Traditional chemical analyses of wood, with an emphasis on lignin determination, do not allow analyzing lignin at the cellular level. Lignin investigation at the cellular level can be performed using different microscopic techniques and microspectroscopy. One possible methodology is the fluorescence technique which allows, in addition to quantitative analysis, evaluating the spatial distribution of lignin in the wood tissue. Fluorescence in lignin arises from the presence of chromophore and fluorophore groups in its chemical structure (Liu and Zhao 2004). Chromophores are responsible for absorbing light at specific wavelengths, whereas fluorophores re‐emit light after excitation (Donaldson and Radotić 2013).
However, the accuracy of the analyses depends on several factors, including the chemical treatments applied to the samples and the methods for image processing (Garcia et al. 2019). Among the available techniques, autofluorescence microscopy stands out as an efficient method for mapping lignin distribution with greater practicality of analysis. It has been widely used to locate lignin in wood and pulp, investigate the effects of industrial processes, study the photodegradation of wood surfaces and analyze lignin distribution in different woody species (Donaldson 2013).
However, lignin signal can be masked by the presence of other autofluorescent compounds in the plant material. To circumvent this, dyes such as Mäule (Meshitsuka and Nakano 1979), chlorosulfite (Akin and Burdick 1981), basic fuchsin (Dharmawardhana et al. 1992), auramine O (Pesquet et al. 2005), acriflavine (Kutscha and Gray 1972), and phloroglucinol (Vallet et al. 1996; Liljegren 2010; Liebsch et al. 2014) have been developed to specifically highlight lignin. Despite these advances, gaps remain in the application of fluorescence to study lignin, as some fluorescent treatments have yet to be tested in woody tissues or lack more detailed analyses.
Thus, this study relates Klason‐type lignin content with the fluorescence intensity emitted in native Amazonian species, testing different chemical treatments and analysis methods. In this way, we seek to improve the techniques for evaluating lignin at the cellular level and to broaden the understanding of its structure and distribution.
Materials and Methods
2
Collection of Materials
2.1
Wood was collected from sawmills and carpentry shops located in the urban area of the municipality of Altamira, Pará, Brazil. Samples of seven species sold in the region were selected: melancieira (Alexa grandifora Ducke, Fabaceae), angelim saia (Parkia pendula (Willd.) Benth. Ex Walp., Fabaceae), guaruba (Vochysia maxima Ducke, Vochysiaceae), angelim pedra (Hymenolobium petraeum Ducke, Fabaceae), freijó (Cordia sagotii I.M. Johnst., Boraginaceae), pequiá (Caryocar villosum (Aubl.) Pers, Caryocaraceae) and angelim vermelho (Dinizia excelsa Ducke, Fabaceae). Species selection considered variations in lignin content, including species that normally have low, medium and high lignin content.
Analysis Samples
2.2
Part of the wood samples were used for chemical analysis, and part for preparing histological slides for fluorescence intensity analysis. All steps were performed using the same piece of wood to ensure uniformity between analyses.
Wood Lignin Content
2.3
For chemical analysis, the wood was prepared following the TAPPI T 257 cm‐85 standard (1985). Wood samples were crushed in a Willey knife mill and sieved to collect the fraction that passes through the 40 mesh sieve and is retained in the 60 mesh sieve.
Initially, moisture content was determined according to the TAPPI T 210 cm‐93 standard (1993). Duplicate sawdust samples of each species were weighed to obtain the wet mass and subsequently transferred to an oven at a temperature of 103°C ± 2°C for drying until the sample weight was constant. After complete drying, the sample was removed from the oven and weighed again to obtain the dry mass, used to calculate wood moisture.
Extractives were removed following the TAPPI T 204 cm‐17 (2017) standard. Sawdust samples in triplicate were weighed in 2 g, inserted into sintered glass filters with porosity 2, then taken to the soxhlet and coupled to a flask with 150 mL of acetone (CH_3_COCH_3_). After 6 h, counting from the first reflux of the solvent, the extraction bench was turned off for solvent cooling. The second extraction was performed in hot water. After removing the acetone contained in the samples, they were placed in a water bath at 100°C for 1 h and then transferred into an oven at a temperature of 103°C ± 2°C until the sample weight was constant.
Lignin content analysis was of the Klason type, according to the TAPPI T 222 om‐22 (2022) standard and modified following Gomide and Demuner (1986). A total of 0.3 g of the samples in triplicate (free of extractives) was treated with 3 mL of 72% sulfuric acid (H_2_SO_4_) in a water bath at 30°C for 1 h, homogenizing the mixture frequently. Subsequently, the samples solubilized in sulfuric acid were transferred to 100 mL glass bottles with 84 mL of distilled water, which were sealed with a rubber cap and aluminum seal to be subjected to pressure in an autoclave for 60 min. The insoluble lignin was filtered, washed with water and, after drying to constant weight, weighed. Soluble lignin was determined by spectrometry, according to Goldschimid (1971), using the dilution of the filtrate from the procedure to obtain insoluble lignin. Total lignin content was obtained by adding the values of soluble and insoluble lignin.
Preparation of Histological Slides
2.4
Specimens measuring 1.0 × 1.5 × 2.0 cm were prepared in the radial, tangential, and longitudinal directions. Each specimen was softened in boiling water and sectioned in a sliding microtome (Leica SM 2000R) to obtain transverse sections approximately 20 μm thick. Three serial sections were prepared per specimen, and at least 20 non‐overlapping photomicrographs were acquired for each species and treatment.
Sections were rinsed in distilled water and mounted on standard glass slides (26 × 76 mm, 1.0–1.2 mm thickness) and covered with #1.5 coverslips (24 × 32 mm, 0.13–0.17 mm thickness). For in natura fluorescence analysis, sections were mounted directly in glycerol. For extractive‐free analysis and chemical staining treatments (Mäule and basic fuchsin), the same sectioning procedure was adopted prior to reagent application. After mounting, slides were allowed to equilibrate for ~10 min before imaging to ensure stabilization of the mounting medium and fluorescence signal.
Imaging parameters (exposure time, illumination intensity, and camera gain) were kept constant for each fluorescence channel and treatment to ensure comparability among samples. Original files were saved in TIFF format and analyzed in ZEN 3.4 and ImageJ (Fiji).
Fluorescence Microscope
2.4.1
Fluorescence imaging was performed using a Zeiss Axio Scope A1 upright widefield epifluorescence microscope (Zeiss) equipped with transmitted light for brightfield, darkfield, DIC and polarization contrast, and reflected illumination provided by an HBO 50 W mercury vapor lamp. Fluorescence was detected using Zeiss filter sets for DAPI, FITC, and Rhodamine channels. Images were acquired with a ×10/0.25 NA dry objective (×100 total magnification).
Images were captured using an Axiocam MRc5 5‐MP digital camera (2584 × 1936 pixels, 24‐bit, 300 dpi) controlled by ZEN 3.4 (blue edition) software in single‐plane mode (2D; no Z‐stack). Files were saved in TIFF format.
A total of 20 fluorescence micrographs were obtained per species and per treatment. Exposure time ranged between 30 and 200 ms depending on the fluorescence channel and was kept constant within each imaging condition; camera gain and illumination settings were also held constant to ensure comparability among treatments.
Autofluorescence In Natura Slides
2.4.2
Autofluorescence was performed as described by Donaldson et al. (2010). Histological sections of untreated wood tissues were mounted with glycerol and analyzed under a fluorescence microscope with light excitation of 440–490 nm and emission of 490–565 nm.
Autofluorescence on Slides Without Extractives
2.4.3
Since the lignin signal can sometimes be confused by the presence of other autofluorescent compounds in the wood, we adopted the autofluorescence method to remove the extractives. The anatomical sections were submerged in 3.3% (v/v) sodium hypochlorite (NaClO) at a temperature between 30°C and 40°C for 3 min, then washed thrice with distilled water. Subsequently, the sections were analyzed under a fluorescence microscope with light excitation at 440–490 nm and emission at 490–565 nm.
Mäule
2.4.4
Mäule staining reaction was performed as described by Yamashita et al. (2016). The anatomical sections were treated using a 1% (m/v) aqueous solution of potassium permanganate (KMnO_4_) for 5 min and then washed thrice with distilled water, treated with 1 N hydrochloric acid (HCl) for 30 min and washed again with distilled water. To generate the staining reaction 1 M Tris–HCl (Tris‐hydroxymethylaminomethane hydrochloric acid) buffer (pH 8) was added to the sections, which were then mounted on glass slides and covered with coverslips. Light excitation at 440–490 nm and emission at 600–650 nm was performed for microscopic observation of lignin fluorescence.
Basic Fuchsin
2.4.5
Basic fuchsin staining reaction was performed as described by Ursache et al. (2018). Initially, the ClearSee solution was prepared using xylitol (C_5_H_12_O_5_) 10% (m/v), sodium deoxycholate (C_76_H_11_NaO_8_) 15% (m/v), urea (CO(NH_2_)2) 25% (m/v) and water to the desired final volume. The solution was mixed on a magnetic stirrer for 1 h, until the reagents were completely dissolved.
For clearing, the sections were immersed in ClearSee for 30 min followed by immersion in 0.2% basic fuchsin for 5 min. Subsequently, the fuchsin solution was removed and the sections were washed, remaining in ClearSee for 30 min with a solution change, where they remained for another 1 h in ClearSee. The histological slides of the wood tissues were mounted with ClearSee and underwent light excitation in the range between 490 and 565 nm and emission of 600–650 nm for observation under the fluorescence microscope.
Data Processing
2.5
The captured images were processed using ImageJ‐Fiji (Schindelin et al. 2012) to remove non‐fluorescent regions (cell lumen), considered as background, and subsequently quantify fluorescence intensity. The software was calibrated for dimensional recognition to transform pixel distances into micrometers using scale bars as references. The captured regions used to generate photomicrographs for fluorescence measurement have an area of 278 × 208 μm and a resolution of 2584 × 1936 pixels. For greater reliability, the image processing followed three different procedures described below.
- Method 1: Integrated density formula for removing background from non‐fluorescent area of the image (Fernando et al. 2015) employing the following workflow: Convert image to 8 bits → select the total area using a rectangle → access the analyze menu and select the measure option (to measure the signal of the total area and open a pop‐up box with the values) → select an area of 2601.46 μm^2^ inside the vessel to serve as background → access the analyze menu again and select measure (to measure the signal of the area inside the vessel).The fluorescence intensity (FI) in the photomicrograph was measured using Equation (1), described by Fernando et al. (2015). Background intensity was the average of three measurements and the selected area; integrated density was established in relation to the total image size.
- Method 2: Integrated density formula for removing background from non‐fluorescent area of the image with correction according to the analyzed area. Using Equation (1), the small background area was extrapolated to the total image area to obtain the adjusted background (Equation 2), then Equation (3) was applied to obtain the FI.
where IBA_ j _ = Adjusted background intensity; IB = “Background intensity” from Equation (1); AT = total image area; AV = selected area inside the vessel.
- Method 3: Automatic determination of fluorescence intensity by the substract tool. Here, the background is removed directly in the program, leaving only the fluorescence signal, which is subsequently provided by the program after a predetermined command. To find the path for ImageJ analysis, follow the workflow below: Access the process menu and select the math option, then subtract → in subtract select a value that best fits to remove the background, in this case, it was 20 for autofluorescence in natura, Mäule and basic fuchsin, and 25 for autofluorescence without extractive → after removing the background, access the analyze menu and select the measure option (to measure the fluorescence intensity signal and open a pop‐up box with the values).
Statistical Data Analysis
2.6
Statistical analysis methods were conducted for each treatment, adjusting linear regression equations between fluorescence intensity and lignin content. Model adjustment was evaluated using the coefficient of determination (R ^2^) and Pearson's correlation coefficient (r).
Statistical data analysis evaluated the linear relation between wood lignin content and the fluorescence intensity emitted by lignin in the histological sections. Pearson's correlation analysis considered a statistical significance level of 95% (p < 0.05). Additionally, a linear regression analysis was performed between fluorescence intensity and lignin content, evaluating the model fit by the coefficient of determination (R ^2^). Statistics were calculated using the means of each treatment's replicates.
Results
3
Table 1 shows the varying chemical composition of the species, with extractive contents ranging from 5.46% in pequiá ( C. villosum (Aubl.) Pers) to 14.03% in angelim vermelho ( D. excelsa Ducke) and lignin contents ranging from 27.14% in melancieira (A. grandifora Ducke) to 32.43% in guaruba ( V. maxima Ducke).
Figures 1, 2, 3, 4, 5, 6, 7 show images of the species with the treatments used, namely: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; and (d) fluorescence with Mäule. Images of the in natura slides and those without extractives show cells more clearly, whereas the basic fuchsin treatment produced a brighter fluorescence signal in the fiber region, except for guaruba (Figure 5c). A similar increase in fluorescence brightness was also observed in the Mäule treatment, except for guaruba (Figure 5d) and melancieira (Figure 6d).
Photomicrographs of angelim pedra at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Arrows indicate regions of higher brightness in the fibers.
Photomicrographs of angelim saia at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Arrows indicate regions of higher brightness in the fibers.
Photomicrographs of angelim vermelho at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Arrows indicate regions of higher brightness in the fibers.
Photomicrographs of freijó at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Arrows indicate regions of higher brightness in the fibers.
Photomicrographs of guaruba at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Rectangles indicate clear regions in the fibers.
Photomicrographs of melancieira at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Rectangles indicate clear regions in the fibers.
Photomicrographs of pequiá at ×100 magnification. Treatments: (a) autofluorescence of in natura slide; (b) autofluorescence of slide without extractives; (c) fluorescence with basic fuchsin; (d) fluorescence with Mäule. Arrows indicate regions of higher brightness in the fibers.
Interestingly, the Mäule treatment allowed us to observe a difference regarding the types of lignin present in angelim saia, guaruba, melancieira and pequiá through coloration—syringyl lignin presented a red coloration, whereas guaiacyl lignin was colored yellow. The other species showed a greater yellow coloration in the cells, indicating a greater presence of guaiacyl lignin.
Table 2 presents the coefficients of determination (R ^2^) and Pearson's correlation coefficients (r) for each treatment and method combination. Method 2 (integrated density formula for removing background from non‐fluorescent area with correction), applied to the autofluorescence treatment on a slide without extractives, presented the best fit with an R ^2^ of 77.77% and r of 0.88. This indicates that this model significantly explains the variation in fluorescence intensity in relation to lignin content. The autofluorescence treatment on a slide without extractives obtained the highest R ^2^ values for all methods analyzed (75.09%–77.77%), suggesting that this approach is the most appropriate for relating fluorescence intensity and lignin content. Conversely, the Mäule and basic fuchsin treatments presented R ^2^ coefficients below 40%, indicating a weak explanatory capacity of the association between fluorescence intensity and lignin content.
Figure 8 shows the relation between fluorescence intensity and lignin content in the different treatments. Linear regression lines indicate the trends identified by the statistical analyses. Autofluorescence without extractives presents a more defined pattern of positive correlation whereas the Mäule and basic fuchsin treatments show a greater dispersion of points, corroborating the low R ^2^ coefficients found in the numerical analysis. Autofluorescence in natura also exhibits significant variation and an inverse correlation with lignin content, and with less adjustment compared with the version without extractives.
Relation between fluorescence intensity and lignin content in different treatments. (a) Method 1: Integrated density formula for removing background from non‐fluorescent area (Fernando et al. 2015); (b) Method 2: Integrated density formula for removing background from non‐fluorescent area with correction according to the analyzed area; (c) Method 3: Automatic determination of fluorescence intensity by the substract tool.
Discussion
4
The greater brightness observed in the fiber region can be explained by the combination of the reduced lumen size and the concentration of lignin in the fiber cell walls. In species with larger lumens, such as guaruba and melancieira, the microscope was able to focus the structures more precisely, resulting in sharper images with lower apparent brightness. In species with smaller lumens, the visual enhancement of fluorescence can be attributed to the accumulation of lignin in regions such as the cell wall, cell corner, and middle lamella (Donaldson 2001), which form a denser structure and reflect more light (Ban et al. 2018), increasing the visual perception of brightness.
Moreover, each autofluorescence treatment or dye use responds differently to the excitation wavelength used in the fluorescence microscope. The employed colors, red in basic fuchsin and red/yellow in Mäule, appear visually brighter. This may result not only from the lignin fluorescence intensity, but also from the greater effectiveness of the reagent in absorbing the excitation light and emitting it at specific wavelengths.
Removing extractives improves the relation between fluorescence intensity and lignin content. The high correlation coefficient obtained with autofluorescence on slides without extractives suggests that the presence of extractives masks lignin fluorescence, reducing analysis accuracy. Additionally, the low explanatory capacity of the models for the in natura slide, Mäule and basic fuchsin treatments indicates that these methods are the least suitable for correlating fluorescence intensity and lignin content in wood tissues.
Extractives have chromophores and fluorophores in their composition that can emit their own fluorescence and interfere with lignin emission (Lakowicz 2006). This occurred mainly in the autofluorescence of in natura slides where the extractives remained in the structure competing for fluorescence emission with lignin, inverting their correlation with fluorescence intensity. Basic fuchsin and Mäule treatment, even after the tissue cleaning process during histological section preparation, showed no significant correlation. This may be because these reagents, especially basic fuchsin, had not yet been widely applied in studies with wood and therefore may not have been efficient in this case. The dispersion of points in the Mäule and basic fuchsin treatments indicates a lower linear correlation between fluorescence intensity and lignin content, whereas autofluorescence without extractive shows a more predictable behavior and is in line with the adjusted linear model.
In the autofluorescence of slides without extractives, removing the extractives eliminated possible sources of interference and allowed the fluorescence emission to be attributed mainly to the lignin in the cell walls. We also visually confirmed that extractives did not noticeably quench lignin fluorescence in any species, as no increase in fluorescence brightness was observed after extractive removal. This indicates that non‐fluorescent extractives did not significantly interfere with lignin emission under the applied conditions. This resulted in a greater correlation with the lignin content, since the fluorescence measurements became more representative of the real chemical wood composition. In comparing lignin quantification of the Eucalyptus urophylla clone by fluorescence with the conventional bench method, Moulin et al. (2024) found that fluorescence showed a similar trend to the conventional method in indicating the behavior of the lignin present in the wood tissue at the cellular level.
Despite the significant correlation between the autofluorescence treatment without extractives and lignin content, we observed distinct fluorescence intensity results in relation to lignin content mainly for the species freijó (C. sagotii I.M. Johnst.). This may have occurred due to factors intrinsic to the species, including differences in the wood anatomical structure like vessel arrangement, fiber size, and cellular composition, which may influence the fluorescence intensity detected (Donaldson and Radotić 2013).
Lignin is not uniformly distributed within the cell wall matrix and may be more concentrated in specific regions such as middle lamellae or secondary walls (Souza et al. 2019). Similarly, the chemical composition and monomeric units of lignin (guaiacyl, syringyl and p‐hydroxyphenyl) may vary between species (Vanholme et al. 2010) and these differences may have directly affected fluorescence emission, as each type of lignin has different optical and fluorescent properties.
When individually analyzing the performance of the methods used to obtain fluorescence intensity, we observed that Method 1 (integrated density formula for removing the background of the non‐fluorescent area) although reliable, requires greater manual intervention and consequently longer processing time. Additionally, the dependence on manual selection can introduce bias due to the analyst's subjectivity. Method 2 (integrated density formula for removing the background of the non‐fluorescent area with correction according to the area analyzed) presents an improvement in relation to Method 1 regarding data normalization, and had the highest coefficients of determination and correlation; however, it still depends on additional calculations and greater data processing which reduces its practicality for large‐scale analyses.
Method 3 (automatic determination of fluorescence intensity using the substract tool) exhibited the second highest coefficient of determination (R ^2^) and was the most efficient and practical, allowing automatic removal of the background directly in ImageJ. This process significantly reduces analysis time and eliminates possible biases resulting from the analyst's subjectivity. Automation ensures greater reproducibility which is essential for comparative studies. The results obtained by the three methods were consistent, validating their applicability for analyses of this type. However, the automatic method of determining fluorescence intensity is the most recommended, not only for its practicality and efficiency, but also for the standardization of results which is essential for larger‐scale studies or those involving multiple analysts.
Conclusions
5
Based on these findings, we conclude that the most effective treatment for relating fluorescence intensity and lignin content is autofluorescence on a slide without extractives, especially when analyzed by Method 3 (automatic determination of fluorescence intensity by the substract tool). It showed the second highest coefficient of determination (R ^2^ = 76.22%) and Pearson's correlation (r = 0.87) and better performance due to its automation in ImageJ, ensuring speed, reproducibility and standardization of results.
Removing extractives from histological sections produced a greater correlation between fluorescence intensity and lignin content, evincing the interference of these compounds in the autofluorescence treatments in natura, basic fuchsin and Mäule. We recommend removing the extractives to achieve more accurate results in the basic fuchsin and Mäule treatments. Our results confirm the effectiveness of fluorescence microscopy as a tool for lignin characterization, especially when associated with extractive removal and the use of automated methods. This methodology can be applied to other woody species and studies with other objectives, such as investigating lignin in the cell wall.
Author Contributions
Josy Tainara Silva Silva: writing, review and editing. Silvino Intra Moreira: methodology, software. Jordão Cabral Moulin: validation, supervision.
Ethics Statement
This study does not include experiments with human participants or animals.
Conflicts of Interest
The authors declare no conflicts of interest.
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