Microsphere-assisted super-resolved Mirau digital holographic microscopy for cell identification

Abstract

In this paper, we use a glass microsphere incorporated into a digital holographic microscope to increase the effective resolution of the system, aiming at precise cell identification. A Mirau interferometric objective is employed in the experiments, which can be used for a common-path digital holographic microscopy (DHMicroscopy) arrangement. High-magnification Mirau objectives are expensive and suffer from low working distances, yet the commonly used low-magnification Mirau objectives do not have high lateral resolutions. We show that by placing a glass microsphere within the working distance of a low-magnification Mirau objective, its effective numerical aperture can be increased, leading to super-resolved three-dimensional images. The improvement in the lateral resolution depends on the size and vertical position of microsphere, and by varying these parameters, the lateral resolution and magnification may be adjusted. We used the information from the super-resolution DHMicroscopy to identify thalassemia minor red blood cells (tRBCs). Identification is done by comparing the volumetric measurements with those of healthy RBCs. Our results show that microsphere-assisted super-resolved Mirau DHMicroscopy, being common path and off-axis in nature, has the potential to serve as a benchtop device for cell identification and biomedical measurements.

Compensated Microsphere-Assisted Interference Microscopy

“…By compensating the optical path difference in both arms, the super-resolution phenomenon is therefore enhanced in comparison with the previous results reported in Refs. [24][25][26]. Nevertheless, such a high-quality measurement contributes to the clear visibility of the areas of artifact generation [green arrows in Fig.…”

“…The immersion of high-refractiveindex microspheres in a surrounding medium has also been suggested to improve the imaging quality [21,22]. More recently, microsphere-aided microscopy has been combined with dark-field illumination for the label-free visualization of translucent samples [23] and with interferometry for the high-resolution reconstruction of surface topography [24,25] or of cell volume [26]. These previously reported methods in microsphere-aided interference microscopy have been implemented by placing a glass microsphere in the object arm of a quantitative-phase interferometer, leading, first, to an unbalanced optical path difference between the arms and, second, to the generation of optical aberrations, as highlighted in Ref.…”

Super-Resolution of Nano-Materials and Quantum Effects Obtained by Microspheres

“…Digital holography for quantitative phase-contrast imaging was developed, in which numerical hologram reconstruction was described [104,105] and these methods were applied for phase-contrast imaging by microspheres [106,107]. Microsphereassisted super-resolved Mirau digital holographic-microscopy has also been attempted [108,109]. In the Mirau interferometry, the interferometric system consists of a single microscope objective, and the reference wave is obtained by reflection from a small mirror that is built inside the objective.…”

Illumination conditions in microsphere‐assisted microscopy

“…Although the phenomenon of microsphereassisted microscopy is still not fully explained and the claimed resolution values are debatable (Allen et al, 2015), several experimental results and computer simulations have not only determined the role of evanescent waves (Ben-Aryeh, 2016), the influence of the surrounding medium (Yao et al, 2015;Darafsheh, 2017) and the effect of the coherence (Maslov & Astratov, 2016;Perrin et al, 2017a), but also improved the field of view using stitching techniques (Wang et al, 2016a;Huszka et al, 2017). Moreover, microsphere-assisted microscopy has been successfully combined with interferometry for the three-dimensional (3D) topography reconstruction of nanostructures (Wang et al, 2016b;Perrin et al, 2017b;Kassamakov et al, 2017) or cell morphology (Aakhte et al, 2017), and with dark-field illumination for the imaging of translucent biological samples . Nevertheless, the illumination conditions of white-light microsphere-aided ultramicroscopy have not yet been considered in detail although they play a key role in the imaging process.…”