Total internal reflection-based nearfield illumination for label-free high-contrast imaging of biological samples

Label-free, high-contrast imaging of biological specimens and microstructures is crucial due to the translucent nature of the sample; the incident light hardly affects the light passing through the specimen, resulting in a low-contrast image. Labeled imaging techniques are extensively used in cell biology to visualize the cellular and subcellular structures of biological specimens with high contrast. Also, photobleaching, quenching, and phototoxicity are the phenomena that limit the labeled imaging. Here, we propose a label-free high-contrast imaging technique utilizing total internal reflection (TIR) based incoherent near-field illumination on a coverglass. Light is coupled into coverglass and guided through it by TIR. Glass coverslip's guided light of wavelength 488nm generates auto-fluorescence (AF) of glass which illuminates the sample in the near field. The scattered light from the specimen's structure gives structural information about the sample with a dark background, similar to darkfield microscopy. The potential of the current system has been first tested on polystyrene spheres of size ~ 5.2 ± 0.07μm and compared with bright field (BF) microscopy and performed size measurement accuracy. Thereafter, we demonstrated label-free high-contrast imaging of bull sperm cells and also compared with BF. The current coverslip-guided TIR mode near-field illumination system supports high contrast scattering and scalable field-of-view (FOV) imaging, which is useful for biological applications. Furthermore, our proposed technique is single-shot, easy to use, cost-effective and avoids specific sample preparation, unlike other specifically designed waveguide mode coherent imaging systems, which use spatial scanning to record several frames and then average them into one image.