Multi-moded high-index contrast optical waveguide for super-contrast high-resolution label-free microscopy

Abstract

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2–10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells.

Label-free incoherent super-resolution optical microscopy

“…Our work helps synthesize a label-free incoherent imaging system and is termed Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy (EPSLON), which builds and extends the concepts outlined by Goodman [39], Ruh et.al. [25], Wicker and Heinztmann [13] and previous work based on photonic-chip microscopy [29]. These concepts of EPSLON which enable circumventing the label-free far-field diffraction-limit are explained and experimentally demonstrated in Fig.…”

“…This gives rise to similar images for different illumination angles of the incident plane wave, i.e., the particle concentration and image plane intensity obey a linear relationship as the intensity registered by the camera becomes independent of 𝛥𝜑(𝑡). It is this property that allows the usage of structured light in SIM [13,42] or the intrinsic photo-kinetics of the molecules in IFON algorithms to enhance the resolution [29],…”

“…In EPSLON, this problem is resolved by resorting to Si3N4 waveguides of the following types: (1) Straight waveguides with strip geometry and large widths that supports a large number of the guided modes, generating MMI patterns (Fig. 2a) [29], (2) Four-arm junction multi-moded strip waveguides for speckle illumination from different azimuthal angles (Fig. 2b) [29] and, (3) a single moded SIM chip with rib geometry and phase modulation for one-dimensional structured illumination (Fig.…”

“…2a) [29], (2) Four-arm junction multi-moded strip waveguides for speckle illumination from different azimuthal angles (Fig. 2b) [29] and, (3) a single moded SIM chip with rib geometry and phase modulation for one-dimensional structured illumination (Fig. 2c) [50].…”

“…Poor-contrast and diffraction-limited resolution are major impediments to the development of label-free optical microscopy. To mitigate the issue of poor contrast, various approaches have emerged: phase contrast microscopy [19], differential interference contrast [20], interferometric scattering microscopy [21], interferometric techniques [22], holographic non-interferometric techniques [23], Fourier Ptychography [24], rotating coherent scattering microscopy [25], manipulating the coherence of light sources used for illumination [26], ultraviolet microscopy [27], optical waveguides [28,29] etc. However, circumventing the diffraction-limit in label-free regime is still at a nascent stage in life sciences, as opposed to fluorescence microscopy [30].…”