Quantitative phase imaging of cells by Digital Holographic Microscopy

G, Hanu Phani Ram (2014) Quantitative phase imaging of cells by Digital Holographic Microscopy. Masters thesis, Indian Institute of Technology, Hyderabad.

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Abstract

We constructed digital holographic microscopy (DHM) setup for extracting the quantitative phase information of biological cells. Here we record the digital hologram of the object and perform computational reconstruction. The hologram recording is carried out on a CCD camera. CCD camera will digitize the information hence the method is known as ‘Digital Holographic Microscopy’. From the quantitative phase information, we can calculate the specimen (cell) thickness and volume. This method is advantageous compared to the existing techniques like bright field microscopy, phase contrast microscopy, differential interference contrast and other qualitative phase imaging techniques since they cannot give us exact phase information. In addition, this method is very attractive for live cell imaging as it does not require any contrast agents. In order to improve the resolution and field of view, the principle of Synthetic Apertures (SA) has been applied by moving the CCD camera to 9 positions and the acquired digital holograms were stitched together to increase the field of view with 22 Kilo pixels. We performed 3-D image reconstructions of a transparent ITO electrode. DHM being a quantitative phase imaging technique, could estimate the height and thickness of the ITO electrode. In order to show the improvement in resolution using synthetic apertures, we have imaged the USAF resolution chart. We have shown that the amplitude reconstruction of the USAF resolution chart has given better resolution in synthetic aperture digital holographic microscopy (SA-DHM) compared to DHM. We reconstructed 3 dimensional structure of an E.coli bacteria using SA-DHM and quantified its length and thickness.

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