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.

Linking intra-tree-ring wood density variations and tracheid anatomical characteristics in Douglas fir (Pseudotsuga menziesii(Mirb.) Franco)

“…A section thickness of 12 µm was chosen because it compromise, according to Park [29] and Donaldson et al [11], readability vs. conservation of structural identity. Sections were stained ensuring uniform colours crucial for image analysis [7,21].…”

Relationships between the intra-ring wood density assessed by X-ray densitometry and optical anatomical measurements in conifers. Consequences for the cell wall apparent density determination

“…A correct focus during the acquisition of the images is thus essential. For Donaldson [6], there can be an effect of "out-of-focus" due to the thickness of the sections and ray cells and pits may be filled by the inclusion of lateral walls on the deepness of the image.…”

“…-During drying, sections are subjected to shrinkage and this latest would be responsible of a reduction [6,7,30] or of an increase [19] of the cell wall proportion. Intra-ring density and anatomy in conifers 261 -The moisture content of a mounted section is not known with accuracy and according to Boutelje [2] the observed shrinkage varies with the used drying technique.…”

New anatomical method of grain angles measurement using confocal microscopy and image cross-correlation

“…A 30-μm-thick transverse section was prepared and mounted with Canada balsam without staining. Staining agents such as safranin are useful for observing a xylem tissue sections with a conventional light microscope and there are some reports that safranin is also effective for observation using a confocal microscope (Donaldson and Lauberg 1998;Kitin et al 2000Kitin et al , 2003. Safranin is disadvantageous for the present purpose, however, due to unequal staining and the problem of absorption of fluorescence from itself, compared with nonstaining.…”

“…Confocal microscopy provides optical sections of tree xylem Fig. 1 Diagrams on image cross-correlation analysis explain a the principle and b the way to calculate cross-correlation function through Fast Fourier Transform tissue; i.e., images sampled mainly from arbitrary focal planes in a single section (Donaldson and Lauberg 1998;Kitin et al 2000Kitin et al , 2003. When cell orientation is inclined, i.e., not right angles with the transverse section, there is a transverse lag between two optical sections at different depths from a single transverse section.…”