SEM-EDS image analysis as a tool for scoring the epicuticular wax tube distribution on Pinus sylvestris needles - evaluation using a UV-B field experiment

The wax tube distribution (WTD) of Scots pine (Pinus sylvestris) needles in an UV-B experiment was studied under a scanning electron microscope (SEM) with the help of the image-acquiring, processing and analysing program of a n energy-dispersive (EDS) analyser. To increase the contrast between the ar eas covered by structural wax and amorphous wax deposits, processed (edge d etection, smoothing and thresholding in a binary image) backscattered elect ron images (BEI) were used. WTD was scored as a percentage of the needle su rface area from BEI images of 1300 x magnification. The method was compared to an ocular epicuticular wax classification method, where the wax structu re is scored in terms of distribution classes (I-V). The average wax tube d istribution of developing current-year needles was 21-29% higher when the o cular method was used than that obtained with the image analysis method. Fo r example, the mean WTD in needles collected on 30 June (0.5-month-old need les) was 90% (ambient and supplemental UV-B) by the ocular method, whereas image analysis yielded values around 64% (ambient and supplemental UV-B). O n previous year needles, the difference in the WTD values between these two methods was 5-9%. No statistically significant differences in WTD emerged between the trees exposed to ambient and supplemental UV-B. The image analy sis method is more precise and more objective than the ocular methods commo nly employed. The image analysis method can be used to investigate the wax structure of other conifer species and the response of epicuticular waxes t o different environmental factors. (C) 1999 Elsevier Science B.V. All right s reserved.