Abstract
Single particle level visualization of biological nanoparticles such as viruses and exosomes is challenging due to their small size and low dielectric contrast. Fluorescence based methods are highly preferred, however they require labelling which may perturb the functionality of the particle of interest. On the other hand, wide-field interferometric microscopy can be used to detect sub-diffraction limited nanoparticles without using any labels. Here we demonstrate that utilization of defocused images enhances the visibility of nanoparticles in interferometric microscopy and thus improves the detectable size limit. With the proposed method termed as Depth Scanning Correlation (DSC) Interferometric Microscopy, we experimentally demonstrate the detection of sub-35nm dielectric particles without using any labels. Furthermore, we demonstrate direct detection of single exosomes. This label-free and high throughput nanoparticle detection technique can be used to sense and characterize biological particles over a range between a few tens to a few hundred nanometers, where conventional methods are insufficient.
Highlights
Single particle level visualization of biological nanoparticles such as viruses and exosomes is challenging due to their small size and low dielectric contrast
One of the most successful implementation of this method is interferometric scattering microscopy[23,24], in which, detection of single virus[25], single proteins[26,27], and visualization of lipid membrane formation[28] is demonstrated. iSCAT uses standard cover glass as a sample substrate and scattered light from the nanoparticles is interfered with a reference beam which is reflected by the interface of medium and the cover glass
We propose a method termed Depth Scanning Correlation (DSC) Interferometric Microscopy, where depth scan images of the immobilized nanoparticles on top of a sample substrate are acquired, and a correlation analysis is performed to enhance the visibility of the nanoparticles while diminishing the noise in the background
Summary
Single particle level visualization of biological nanoparticles such as viruses and exosomes is challenging due to their small size and low dielectric contrast. A popular contrast mechanism is fluorescence-based detection that most commonly provide ensemble measurements Advanced microscopy techniques such as STORM5,6, Stimulated Depletion Emission (STED) Microscopy[7,8,9], and Photoactivated Localization Microscopy (PALM)[10] generate high resolution images of fluorophore tagged nano-structures[11,12] with single particle resolution, they require labelling of nanoparticles that may alter their properties and the outcome of the assay of interest[13,14]. Noise due to impurities in sample substrate and light source is eliminated by subtracting the background and frame averaging[26] using high frame rate cameras with a trade-off in the field of view Another interferometric detection method called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) uses a special sample surface, which is a layered substrate where the thickness of the layer is optimized for optimum interferometric signal[21,29]. By implementing aperture-shaping filters, detection of 50 nm polystyrene particles have been demonstrated[33] in air
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