# Method for Lignin Analysis in Wood by Fluorescence Microscopy

## 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.

## Key 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 was the most effective treatment for relating fluorescence intensity and lignin content on a slide without extractive, especially when analyzed automatically (Method 3). It showed the second highest coefficient of determination (R
2 = 76.22%) and correlation (r = 0.87), and better performance due to its automation in ImageJ, ensuring speed, reproducibility and result standardization. Results indicate the reliability and safety of fluorescence analysis for studying lignin at the cellular level.

Autofluorescence in extractive‐free wood slides is a promising approach for investigating the relationship between fluorescence intensity and lignin content, particularly when combined with automated photomicrograph analysis using ImageJ.