Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don’t need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.
A super-resolution quantitative phase-contrast imaging method using high refractive index microsphere is developed to overcome the diffraction limit of optical field, which is produced by the object in the digital holographic microscopy. A microsphere placed on the surface of the object can collect the underlying near-field information, which appears as the evanescent waves and transforms them into propagating waves. Due to the spherical symmetry provided by the microsphere, the super-resolution of the imaging system can be realized in all directions at the same time with one-shot recording. The experiments are carried out for a cosine grating with the line width of 255 nm as the object, which confirms that the lateral resolution can be less than λ/2. Meanwhile, the quantitative phase-contrast image is experimentally obtained. The reconstructed complex field distribution provides the great flexibility with the digital processing for the microscope imaging, such as the ability of refocusing and numerical reconstruction.
The microsphere has great potential to improve the resolution of the system. The high frequency information of the object can be collected by the microsphere to enhance the imaging resolution. However, the unavoidable chromatic dispersion exists in the microsphere incoherence imaging, which reduces the image quality. In this paper, the microsphere microscopy system is designed with the coherent light. The polystyrene (PS) microspheres with the diameter of 50μm and 90μm are applied, and their refractive index is 1.59. The object is a transmission grating with a cycle of 1.2μm and line spacing of 600 nm. The result indicates that the grating can be clearly detected, and the magnification of the microsphere with the diameter of 50μm and 90μm is 2.33 and 1.96 respectively. Comparing with the traditional white light microscopy, the imaging contrast has been improved though the speckle noise is introduced for coherent light. Therefore the microsphere has the potential to improve the resolution of the phase-contrast imaging.
Digital holographic microscopy has been widely used for the imaging of micro-objects and biological samples. Lensless in-line digital holographic microscopy is capable of wide field-of-view imaging. However the spatial resolution of the reconstructed images is limited by the pixel size of the detector. The relative position shift between the sample and the detector can effectively improve the resolution in the traditional sub-pixel shifting method, but it requires a high precision of translation stage. To overcome this problem, we propose a method based on the point source scanning to realize sub-pixel shifting. High precision sub-pixel shifting is achieved easily by using the geometric between point source and detector. Through moving the point source, multiple holograms with sub-pixel shifts are captured. These holograms are merged together to obtained a high resolution hologram by a synthesizing algorithm. Then, the high resolution reconstructed image of the object can be obtained by the angular spectrum algorithm. The feasibility of the proposed method is demonstrated by simulation and experiments. A USAF resolution test target was used as the object. Compared with the traditional digital holography, a higher resolution reconstructed image is obtained by our method. The proposed method has the advantages of simple recording setup and lower precision requirement of the translation stage. It can achieve the wide field-of-view and high resolution imaging.
The imaging character of microsphere is determined by its refractive index and size. In this paper, it is demonstrated that the spherical aberration(S) and numerical aperture (NA) of the microsphere are different with different refractive index or different radius. The relationship between the focal length and refractive index is obtained by numerical simulation. It is shown that the focal length decreases with the increase of the refractive index. The focal length and diameter possesses a linear relation，which is matched with the theoretical result. It is also found that the photonic nano-jet may appear when the refraction index and size of the microsphere are chosen properly. The microsphere has great potential in the laser processing of surfaces materials and super-resolution detection of nano structure etc. So the study on the microsphere imaging has great significance.
A phase shifting digital holography with pre-magnification is designed. In order to fully utilize the bandwidth of the camera, a four-step phase-shifting digital holography is adopted to retrieve the complex distribution of the object. To further enhance the resolution of the reconstructed image without phase aberration, two microscope objectives (MOs) are placed in front of the object and the reference mirror. The MO in the reference arm provides parallel beam at the PZT plane thus improve the precision of the phase shifting. A 1951 USAF negative resolution target is used as the sample. Experiment result demonstrates the feasibility of the proposed method.