Optical microscopy imaging and image-analysis issues in laser cleaning

In earlier work we characterised single-pulse laser cleaning of medium-density (areal coverage 10%-20%) alumina particles from glass surfaces with three different lasers. The method of measuring particle-removal efficiency involves optical microscopy imaging (digital) and subsequent image analysis of the area covered by, and/or the number of, particles before and after the single-pulse laser cleaning treatment. The sample-preparation technique used leads to both single particles and agglomerates on the surface. The issues of depth of focus in the imaging and grey-scale thresholding in the image analysis of such samples, with a range of particle and agglomerate sizes, has been systematically investigated. A protocol for optimum imaging and illumination of such samples is described herein. It has been developed based on the results of a systematic investigation of the effect of the image focal plane position relative to the surface also described herein. The image analysis to quantify the particles on the surface involves a judgement of the best threshold grey-scale level in the image to define the boundary between particles and background substrate. A quantitative appraisal of the impact on the laser cleaning efficiency results of a threshold grey-scale level that is set too high or too low, and how these results compare with those obtained for the "best-judgement" threshold grey-scale level, has been completed.