Analogous on-axis interference topographic phase microscopy (AOITPM)

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Abstract

Summary:

The refractive index (RI) of a sample as an endogenous contrast agent plays an important role in transparent live cell imaging. In tomographic phase microscopy (TPM), 3D quantitative RI maps can be reconstructed based on the measured projections of the RI in multiple directions. The resolution of the RI maps not only depends on the numerical aperture of the employed objective lens, but also is determined by the accuracy of the quantitative phase of the sample measured at multiple scanning illumination angles. This paper reports an analogous on-axis interference TPM, where the interference angle between the sample and reference beams is kept constant for projections in multiple directions to improve the accuracy of the phase maps and the resolution of RI tomograms. The system has been validated with both silica beads and red blood cells. Compared with conventional TPM, the proposed system acquires quantitative RI maps with higher resolution (420 nm @λ = 633 nm) and signal-to-noise ratio that can be beneficial for live cell imaging in biomedical applications.

Lay description:

The refractive index (RI) of a sample as an endogenous contrast agent plays an important role in transparent cell imaging, since it enables to reveal the unique aspects of the cellular structure naturally without damaging the cells. In tomographic phase microscopy (TPM), 3D quantitative RI maps can be reconstructed based on the measured projections of the RI in multiple directions, which is similar to the computed tomography (CT) that measures the projection of the X-ray absorption in different orientations. The resolution of the RI maps not only depends on the numerical aperture of the employed objective lens, but is also determined by the accuracy of the quantitative phase of the sample measured at multiple scanning illumination angles. In this paper, we have proposed an implementation of an improved version of conventional TPM, which is called analogous on-axis interference TPM (AOITMP), where the interference angle between the sample and reference beams is kept constant by rotating the reference arm with the sample arm. Because of the zero interference angle characteristics, interference strip noise can be eliminated, which on one hand is capable of acquiring phase images with a high accuracy even at large oblique illumination angles. On the other hand, removing the interference strip enables to obtain the quantitative phase of complex samples with larger phase gradient. Furthermore, we also adopt two methods to improve the phase image accuracy and signal-to-noise ratio. These techniques contribute to higher accuracy quantitative phase measurement, thus leading to a higher resolution RI maps. We also improve the cyclical scanning control method to reduce the vibration frequency limit of the piezoelectric ceramics mounted mirror, allowing the TPM to capture interference images at a more rapid speed and enhance the system's stability. In order to demonstrate the performance of our setup, we image both silica beads and blood cells and obtain satisfactory results. The tomographic RI maps are proved to have a higher lateral resolution (420 nm @λ = 633 nm) and signal-to-noise ratio than different interference angle TPM (conventional TPM). These quantitative RI maps can provide an important intrinsic contrast agent for live cell imaging in biological applications without the need of fluorescent labelling.

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