Tian Peng, Yan Wei, Li Fanxing, et al. Biology microscopy using well-distributed sphere digital in-line holography[J]. Opto-Electronic Engineering, 2019, 46(1): 180110. doi: 10.12086/oee.2019.180110
Citation: Tian Peng, Yan Wei, Li Fanxing, et al. Biology microscopy using well-distributed sphere digital in-line holography[J]. Opto-Electronic Engineering, 2019, 46(1): 180110. doi: 10.12086/oee.2019.180110

Biology microscopy using well-distributed sphere digital in-line holography

    Fund Project: Supported by Department of Science and Technology of Sichuan Province (2015JQ0009) and the National Natural Science Foundation of China (61705232)
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  • Traditional pinhole spherical wave digital in-line holography has proved to be powerful imaging tools. Image quality is affected by uncertain round of pinhole. Here, we propose a well-distributed sphere wave generation method and it demonstrates wide field of view and high resolution microscopy. The laser focuses into an infinitesimal spot through laser beam expander and microscope objective. Pinhole permutation with different sizes is utilized to match the focal point, and emerges an ideal spherical wave. Interference fringes pattern, formed by reference sphere wave and scattered sphere wave of object, is collected by large area image sensor. The influence of dirty in image sensor and parasitic light is eliminated through subtraction with and without object. Fresnel inverse transformation reconstruction algorithm presents the object information. Biology microscopy experiments demonstrate that the proposed techniques increase the flexibility in producing well-distributed point light source and improve the image quality. Field of view is 3.22 mm×3.22 mm and resolution is 5.09 μm. Furthermore, adjustable field of view with magnification, fast, no-contact make it to be a promising tool in optical element measurement, material identification, biology and medicine.
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  • Overview: Digital in-line holography (DIH) with spherical wave, originally proposed by Gabor, is the simplest way in realizing holographic. The object light and reference light are coaxial, and interference fringes pattern are recorded digitally by image sensor. Complex amplitude distribution of object are displayed through reconstruction algorithm. In visible light range, although the resolution is micron, it provides wide field of view. Moreover, the characters of fast, real-time, non-contact make it be a promising tool in material identification, biology microscopy, lab-on-a-chip applications and particle track.

    The quality of point source spherical wave, emerged from pinhole, has an important impact on the imaging. However, the size and uncertain round of pinhole cannot be eliminated refer to fabrication error. Although researchers have been capable of manufacturing nanometer accuracy pinhole, the cost is expensive extremely far more than optical elements. On the other hand, wetting films, pixel super-resolution, differential-interference-contrast are applied efficiently to improve image quality, field-of-view, and resolution, but they require sophisticated operation steps far beyond the simplicity of the spherical wave digital in-line geometry.

    Diffraction is much better in condition of pinhole diameter matching for incident light wavelength. And the actual size of pinhole is determined by parameters and distance of image sensor. It makes heavy demands on manufacturing accuracy. We first consider obtaining the well-distributed spherical wave. Laser focuses into an infinitesimal spot through laser beam expander and microscope objective in turn. Altering axis distance between pinhole array and microscope can obtain suitable focal spot. Matching with the pinhole, an ideal spherical wave is generated. The influence of uncertain round of pinhole can be shifted to the edge of image sensor that is negligible. Then reconstruction algorithm simplify the computational process, which presents the object information. Finally, as a proof-of-concept, biology experiments demonstrate the proposed techniques.

    As shown in mosquito eggs microscopy. Figure (a) is reconstruction result without any image processing, the field of view is 3.22 mm×3.22 mm and the resolution is 5.09 μm. Figure (b) and (c) are magnified digitally four times to display single egg. The difference of reconstruction distance, the result is changing. It is called digital focusing. Besides, the whole measurement process is fairly high efficiency, because only three steps: placing object, exposure, and reconstruction. Large area and high resolution recovery image is our target, and also the characteristic of this microscopy system. It can be used in detection of micro optical element, biological recognition, path tracking of plankton etc. Especially in biology and medicine research, high efficient, flexible working distance and field of view characters are much suitable.

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