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.
We present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed by fingerprinting a single bacterium. A wide-field fluorescence-detected mid-infrared photothermal microscope developed allows video-rate bond-selective imaging of biological specimens.
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) imaging is an emerging technique where a visible beam is deployed to sense the photothermal lensing effect induced by infrared absorption of molecules. This technology provides sub-micron spatial resolution defined by the visible probe beam. Yet, the photothermal lensing effect is a weak effect for most materials. For instance, the diffraction coefficient of poly (methyl methacrylate) changes around 0.1% per Kelvin. Thus, highly sensitive probes are desired to improve the detection sensitivity. Here, we report a fluorescence-enhanced mid-infrared photothermal (FE-MIP) microscope with high sensitivity. MIP microscopy uses a pump-probe strategy in which a mid-infrared light vibrationally excites a sample and a visible light probes the thermal effect. Instead of measuring the scattering modulated by mid-infrared absorption, we deploy thermal sensitive fluorescent dyes as the probe, and measure the modulated fluorescence intensity in both confocal mode and wide field mode. We demonstrate high imaging sensitivity and component specificity through fluorescence labeling.
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.
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.
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