A new method for accurate platelet thrombi volume measurement using a confocal microscope

The accuracy of quantitative measurements represents an essential pre-requisite to the characterization and definition of the complex dynamic phenomena occurring in the field of cell biology. In research projects that involve the induction of blood coagulation under flow in microfluidic artificial channels, thrombus volume is an important quantity for estimation as a significant index related to the individual thrombotic risk profile. Concerning its importance in the early diagnosis of cardiovascular diseases, the estimated thrombus volume should reflect and represent reality. In 3D confocal microscopy, systematic errors can arise from distortions of the axial distance, whose accurate calibration remains a challenge. As a result, the 3D reconstructions show a noticeable axial elongation, and the volume measurements are thus overestimated. In this paper, a 400-600 % volume overestimation is demonstrated, and a new easy to use and automatic calibration procedure is outlined for this specific microfluidic and optical context. The adaptive algorithm proposed leads to the automatic compensation of the elongation error and to the accurate thrombus volume measurement. The method has been calibrated using fluorescent beads of known volume, validated with groups of several distinct platelets and finally applied on platelet thrombi.