Model-based confocal uorescence microscopy measurements of submerged micro geometries

摘要

The challenging environment of in situ micro-geometry measurements in uids (e.g. for laser- or electrochemicalmachining), such as refractive index uctuations, small dimensions and high surface gradients, hinder manyconventional measurement techniques. Confocal microscopy remains most suitable with uncertainties < 1 μm,but complex micro-geometries with edge slopes > 75° often produce unwanted artifacts. To prevent the formationof artifacts, the isotropically emitting uorescence light of a uid layer covering the specimen is measuredinstead. The geometry reconstruction for in situ-relevant uid depths 100 μm is not trivial and requires asignal model that includes the contributions of light absorption and the confocal volume shape. For modelvalidation, the surface position of a reference step-object (nominal height: 250 μm), submerged in a uid layer> 1 mm, is determined using a uorescence signal model that isfitted to the measured data. First experimentsyield a step height uncertainty of 8:8 μm, about one order of magnitude above the requirement. In order toidentify optimization potential, the minimum achievable measurement uncertainty is estimated for both a signalwith experiment-equivalent variance and a shot noise limited signal. The estimated uncertainties are 3:8 μmand 0:1 μm, respectively, and decrease with lower uorophore concentration and uid thickness. The dfferingexperimental and estimated uncertainties result from model simplifications such as the missing contributionof reected light at the specimen surface where the current model assumes that the confocal volume is cutoff. Expanding the model promises to reduce the measurement uncertainty and to converge estimation andexperiment, enabling geometry measurements of complex micro-geometries with different surface reectivitiesunder challenging in situ conditions in uids.