Phase measuring deflectometry based microscopy for shape visualization and thickness quantification

Shape profiling and thickness information of living cells can provide critical insights about the cells, which can help in their identification and characterization. Most living cells are difficult to image as they are very small and almost transparent. In conventional bright-field microscopy, this issue is resolved by using staining agents, but they can potentially disrupt a cell’s natural life cycle. Generally, to address this issue, quantitative phase contrast imaging, such as digital holography is employed, as it provides direct phase information. However, in some practical applications, employing the digital holography technique can become challenging due to stringent optical requirements and its high sensitivity to thickness change. Here, we present phase measuring deflectometry as a microscopy technique by employing a four-step phase-shifting method for shape visualization and thickness measurement of transparent micro-objects. The technique provides phase that is proportional to the deflection angle, which, in turn, depends on the gradient of the optical thickness of the sample. So, the system was calibrated using a 5 μm diameter transparent polystyrene microsphere. A scaling factor was determined by the calibration process, which was then tested by measuring the thickness of a 15 μm diameter transparent polystyrene microsphere. This result obtained with phase measuring deflectometry agrees with the digital holographic microscopy measurement. The proposed technique was further used for visualization and thickness measurement of the red blood cells (RBCs). Based on the available information, the presented technique and algorithm have not been previously exploited for shape visualization and thickness measurement of transparent micro-objects.