With the same length of -focus translation, the linear regions in both the upward and downward displacements of trapped particles were determined. However, the difference in the upward and downward graphs of the unattached particle displacement may be due to spherical aberrations, viscous drag force, and mechanical errors. On the other hand, difficulty in the cell experiments is primarily caused by the distortion of particle movement due to repeated cell probing. Overall, when the axial movement of particles is being studied, the defocusing of the particles is observed. This means that the illumination of the particles, image analysis, and the stability of the system are important points to address. As a precaution, the experiments were carried out in series to decrease the probability of changing the illumination level. Then, for every new data set obtained, the image analysis program was recalibrated and rechecked with previous data sets. The ease of use of the system and minimum handling of the setup provided the opportunity to create a stable system, wherein the considerations lie in the mechanical drift of the objective lens and the stage, and the adjustment of laser power.