Comparison of Conventional Transmitted Light and Confocal Microscopy for Measuring Wood Cell Dimensions by Image Analysis

Abstract

A comparison was made between conventional transmitted light microscopy and confocallaser scanning microscopy (CLSM) as the source of digital images for the measurement of wood cell dimensions by image analysis. When compared with confocal microscopy, transmitted light microscopy using 20 μm thick, safranin stained sections overestimated wall thickness by up to 50% and underestimated lumen area by up to 4% due to the effects of out-of-focus haze. Confocal microscopy using 20 μm thick, safranin stained sections, was found to produce more accurate images of the wood cells compared to transmitted light microscopy using thick sections. Images obtained by optical sectioning were comparable to the quality that might be obtained by thin sectioning of resin embedded wood. For example bordered pit chambers were easily resolved in single optical sections but were not resolved in transmitted light images. Confocal microscopy can be performed on sections as thick as 120 μm and by acquiring optical sections more than 5 μm below the surface of the section distortion of cells caused by sectioning can be avoided. Image quality declined with depth leading to substantial changes in cell dimensions at depths beyond 10-20 μm and significant errors in cell dimensions beyond 80 μm depth. Image brightness was also found to decline with depth, more rapidly in water than in immersion oil. A comparison of measurements of cell dimensions in water and in immersion oil indicated that wall thickness changes significantly during drying but that other dimensions remain almost the same in dry compared to wet seetions.