Label-free spectroscopic detection of single viruses provides component analysis of virus strains. Current methods suffer from low throughput and weak signal contrast of individual virions. Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscopy for high-throughput fingerprinting of single viruses. WIDE-MIP not only reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses, but also uncovers an enriched β sheet components in DNA varicella-zoster virus proteins. Different nucleic acids signatures of thymine and uracil residue vibrations are also obtained to differentiate DNA and RNA viruses.
Label-free spectroscopic detection of single viruses provides component analysis of virus strains. Current methods suffer from low throughput and weak signal contrast of individual virions. Here, we report a wide-field interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscopy for high-throughput fingerprinting of single viruses. Comprehensive theoretical frameworks for WIDE-MIP signal is developed and experimentally validated by acquiring defocused interferometric and photothermal images. WIDE-MIP not only provides the amide I and amide II vibrations in viral proteins, but also reveals the unique IR signature of thymine and uracil residues vibration in DNA vaccinia viruses and RNA vesicular stomatitis viruses, respectively.
Optical coherence tomography (OCT) has been a powerful 3D optical imaging tool in the last decade while it lacks molecular information. In this work, we integrate the mid-infrared photothermal microscopy with the OCT approach to demonstrate a bond-selective full-field optical coherence tomography (BS-FF-OCT), in which a pulsed mid-infrared laser is used to modulate the full-field OCT signal through the photothermal effect. This method achieves label-free volumetric infrared spectroscopic imaging at 1-μm isotropic resolution, demonstrated by a variety of samples, including 1 μm PMMA beads embedded in agarose gel, polypropylene fiber mattress, myelinated nerve bundle in mouse brain tissue, Caenorhabditis elegans, and cancer cell spheroids.
Mid-infrared photothermal microscopy (MIP) has been a promising chemical imaging technique for functionality characterization of biological and pharmaceutical specimens owing to its enhanced resolution and high-specificity. Recently developed wide-field MIP modalities drastically improved the imaging speed and thus enabled high-throughput imaging. However, the sensitivity of the wide-field MIP technique has been limited by shot-noise of background photons. Here, we demonstrate a dark-field MIP modality to allow 4-fold signal-to-noise ratio improvement. Our technique is based on selectively blocking the reflected light. Simulation and experimental results are both provided, and they are consistent with each other.
Mid-infrared photothermal (MIP) microscopy overcomes the resolution and huge water background limits in conventional mid-infrared imaging by probing the mid-infrared absorption induced photothermal effect. However, to detect the subtle MIP signal, large probe power and lock-in detection are needed, which limit the imaging speed of current MIP systems. To overcome this limitation, we develop a single-pixel pump-probe camera that leverages the large well-depth capacity of photodiode to achieve high-speed wide-field MIP imaging. With compressive sensing applied, close to video-rate MIP imaging can be achieved, offering a powerful label-free chemical imaging tool to scrutinize the complex biological systems.
Morphological parameters of biological nanoparticles (BNPs) have strong implications on their fate and functionality in vivo e.g., circulation, biodistribution, and clearance. Although interferometric scattering microscopy modalities have demonstrated the label-free detection of sub-100 nm BNPs including viruses and exosomes; they have an insufficient spatial resolution roughly limited to the illumination wavelength. Here, we introduce computational imaging to interferometric scattering microscopy with asymmetric illumination and demonstrate a two-fold resolution enhancement. We demonstrate high-resolution imaging of nanoparticles across a large field-of-view of 100 µm × 100 µm. This novel imaging platform enables ultrasensitive and label-free morphological visualization of low-index sub-diffraction-limited BNPs in a high-throughput manner at subwavelength resolution.
Chemical characterization of biological specimens in the mid-infrared (IR) window plays a central role in the analysis of their functionalities. Although recent advances in mid-IR microscopy have demonstrated detection of the sample’s chemical contrast at a sub-micron resolution using a visible probe beam, they have limited sensitivity at high-throughput. To overcome this limit, we employ wide-field interferometric microscopy to detect the minute change in the optical path induced by mid-IR absorption. Our technique enables high-speed fingerprinting of more than thousands of sub-200 nm nanoparticles at once. This method paves the way for high-throughput, ultrasensitive, and label-free chemical imaging of individual bio-nanoparticles at sub-micron resolution.
Wide-field interferometric microscopy is a common-path interferometry technique that allows for label-free and high-throughput detection of weakly scattering sub-diffraction-limited biological nanoparticles. Such nanoparticles appear as diffraction-limited-spots in the image and optically resolving them beyond their ‘digital’ detection still remains a challenge owing to the diffraction barrier as well as the typical signal levels that fall below the noise floor. In this study, we demonstrate the utility of computational optics in the interference enhanced nanoparticle imaging to improve its resolving power to obtain structural information on clinically relevant and often complexed-shaped biological nanoparticles such as viruses and exosomes. We consider a spatially incoherent structured illumination based image reconstruction strategy in wide-field interferometric microscopy to achieve high contrast nanoparticle imaging with super-resolution. Our reconstruction technique makes use of the optical transfer function of the system derived via an analytical model based on angular spectrum representation. We provide experimental demonstrations using an artificial sample to quantify the resolution enhancement as well as a biological sample for concept demonstration. We also benchmark the results against gold standard images obtained using an electron microscope. Our highly-sensitive super-resolution imaging system constitutes a noncomplex optical design, which can be realized with simple modifications to a conventional epi-illumination microscope, offering a cost-effective alternative to the laborious and expensive standard high-resolution microscopy techniques. It has a broad spectrum of applications ranging from clinical diagnostics to biotechnological research.
Current state-of-the-art technology for in-vitro diagnostics employ laboratory tests such as ELISA that consists of a multi-step test procedure and give results in analog format. Results of these tests are interpreted by the color change in a set of diluted samples in a multi-well plate. However, detection of the minute changes in the color poses challenges and can lead to false interpretations. Instead, a technique that allows individual counting of specific binding events would be useful to overcome such challenges. Digital imaging has been applied recently for diagnostics applications. SPR is one of the techniques allowing quantitative measurements. However, the limit of detection in this technique is on the order of nM. The current required detection limit, which is already achieved with the analog techniques, is around pM. Optical techniques that are simple to implement and can offer better sensitivities have great potential to be used in medical diagnostics. Interference Microscopy is one of the tools that have been investigated over years in optics field. More of the studies have been performed in confocal geometry and each individual nanoparticle was observed separately. Here, we achieve wide-field imaging of individual nanoparticles in a large field-of-view (~166 μm × 250 μm) on a micro-array based sensor chip in fraction of a second. We tested the sensitivity of our technique on dielectric nanoparticles because they exhibit optical properties similar to viruses and cells. We can detect non-resonant dielectric polystyrene nanoparticles of 100 nm. Moreover, we perform post-processing applications to further enhance visibility.
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