Frame-Stacking Method for Dark Digital Holographic Microscopy to Acquire 3D Profiles in a Low-Power Laser Environment

Digital Holographic Microscopy (DHM) is a method of converting hologram images into three-dimensional (3D) images by image processing, which enables us to obtain the detailed shapes of the objects to be observed. Three-dimensional imaging of the microscopic objects by DHM can contribute to the early diagnosis and the detection of the diseases in the medical field by observing the shape of the cells. DHM requires several experimental components. One of them is the laser, which is a problem because its high power may cause the deformation and the destruction of the cells and the death of the microorganisms. Since the greatest advantage of DHM is the detailed geometrical information of the object by 3D measurement, the loss of such information is a serious problem. To solve this problem, a Neutral Density (ND) filter has been used to reduce power after the laser irradiation. However, the image acquired by the image sensor becomes too dark to obtain sufficient information, and the effect of noise increased due to the decrease in the amount of light. Therefore, in this paper, we propose the Frame-Stacking Method (FSM) for dark DHM for reproducing 3D profiles that enable us to observe the shape of the objects from the images taken in low-power environments when the power is reduced. The proposed method realizes highly accurate 3D profiles by the frame decomposition of the low-power videos into images and superimposing and rescaling the obtained low-power images. On the other hand, the continuous irradiation of the laser beam for a long period may destroy the shape of the cells and the death of the microorganisms. Therefore, we conducted experiments to investigate the relationship between the number of superimposed images corresponding to the irradiation time and the 3D profile, as well as the characteristics of the power and the 3D profile.