Mid-infrared photothermal (MIP) microscopy is an emerging tool for high-resolution vibrational imaging of biological and material samples. In a MIP microscope, a pulsed IR beam induces a thermal lens of a liquid specimen or thermal expansion of a particle. The photothermal effects are probed by a visible beam. MIP imaging can be implemented in scanning mode or wide-field mode. In wide-field mode, we present a wide-field interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope, that addresses the unmet need for identification of single virus, including detection of viral proteins, DNA and RNA. In scanning mode, we demonstrate click-free MIP imaging of azide-tagged trehalose in live mycobacteria and reveal trehalose pathway dynamics without photobleaching. MIP mapping of unreacted azide after click reaction further reveals click reaction heterogeneity within a bacterium. Broader applications of azido photothermal probes (Apps) in unraveling carbohydrate utilization inside in yeast and mammalian cells are demonstrated.
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.
Real-time tracking of intracellular carbohydrates remains a grand challenge. While click chemistry enables bio-orthogonal tagging of small molecules with azido groups, subsequent fluorescence visualization is hindered by larger fluorophores, potentially altering intrinsic metabolism. Here, we introduce a click-free, dye-free method for dynamic tracking of trehalose within single mycobacteria via mid-infrared photothermal (MIP) imaging of azido-trehalose. Through spectroscopic MIP imaging, the metabolic pathways of trehalose and its interactions with intracellular proteins is demonstrated without click reaction and photobleaching, offering valuable insights into mycobacterial physiology. Our approach paves the way for further advancements in studying metabolic dynamics within living cells.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.