La. Donaldson et Mjf. Lausberg, COMPARISON OF CONVENTIONAL TRANSMITTED LIGHT AND CONFOCAL MICROSCOPY FOR MEASURING WOOD CELL DIMENSIONS BY IMAGE-ANALYSIS, IAWA journal, 19(3), 1998, pp. 321-336
A comparison was made between conventional transmitted light microscop
y and confocal laser scanning microscopy (CLSM) as the source of digit
al images for the measurement of wood cell dimensions by image analysi
s. When compared with confocal microscopy, transmitted light microscop
y using 20 mu m thick, safranin stained sections overestimated wall th
ickness by up to 50% and underestimated lumen area by up to 3% due to
the effects of out-of-focus haze. Confocal microscopy using 20 mu m th
ick, safranin stained sections, was found to produce more accurate ima
ges of the wood cells compared to transmitted light microscopy using t
hick sections. Images obtained by optical sectioning were comparable t
o the quality that might be obtained by thin sectioning of resin embed
ded wood. For example bordered pit chambers were easily resolved in si
ngle optical sections but were not resolved in transmitted light image
s. Confocal microscopy can be performed on sections as thick as 120 mu
m and by acquiring optical sections more than 5 mu m below the surfac
e of the section distortion of cells caused by sectioning can be avoid
ed. Image quality declined with depth leading to substantial changes i
n cell dimensions at depths beyond 10-20 mu m and significant errors i
n cell dimensions beyond 80 mu m depth. Image brightness was also foun
d to decline with depth, more rapidly in water than in immersion oil.
A comparison of measurements of cell dimensions in water and in immers
ion oil indicated that wall thickness changes significantly during dry
ing but that other dimensions remain almost the same in dry compared t
o wet sections.