Deep-tissue chemical imaging is essential for many biomedical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with micron lateral resolution. By pumping the overtone transition of carbon-hydrogen bonds and probing the subsequent photothermal lens with shortwave infrared light, SWIP can obtain chemical contrast from one-micron polymer particles located at 800-μm depth in a highly scattering phantom. We demonstrated that SWIP can resolve intracellular lipids across an intact tumor spheroid and the layered structure in thick liver, skin, brain, and breast tissues. SWIP microscopy fills a gap in vibrational imaging with sub-cellular resolution and millimeter-level penetration, which heralds broad potential for life science and clinical applications.
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.
The detection sensitivity of SRS microscopy is fundamentally limited by shot noise. To address this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index, as suggested by simulation and fluorescence thermometer measurement. By introducing a CW laser beam to probe the refractive index change, we demonstrate stimulated Raman photothermal (SRPT) imaging of particles, cells, and tissues with high signal to noise ratio at the fingerprint region. SRPT microscopy opens a new way to vibrational spectroscopic imaging with ultrahigh sensitivity.
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.
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