Direct imaging of metabolism at sub-cellular resolution provides insights to understand single-cell physiology at spatially resolved manner. Here, we presented a metabolic profiling platform based on Optical Photothermal Infrared (OPTIR) spectroscopic imaging that enables in situ lipid metabolism characterization at sub-cellular resolution. We applied the developed metabolic imaging platform to study lipid synthesis from azide-labeled fatty acids across various systems including neuroglioma cells, human-induced pluripotent stem cells (iPSCs), iPSC-derived microglia cells, and iPSC-derived brain organoids. The specificity of the developed platform was further improved by coupling a fluorescence module that enables cell type specific-metabolic profiling. Collectivity, the proposed metabolic imaging platform opens a vast potential for in situ metabolism characterization at the single-cell level.
We developed an optical infrared photothermal (OPTIR) metabolic imaging platform that enables in situ lipid metabolism characterization at sub-cellular resolution. The cell-type specific lipid metabolic imaging was achieved through the fluorescence module integrated into the OPTIR system. We successfully imaged newly-synthesized lipids after azide-palmitic acid treatment in cells across different model systems including neuroglioma, human-induced pluripotent stem cells (iPSCs), iPSC-derived microglia cells, and iPSC-derived brain organoids. The metabolic heterogeneity in different cell types in brain organoids was observed. Collectivity, the proposed metabolic imaging platform opens a vast potential for in situ metabolism characterization at the single-cell level.
Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.
We developed a mid-infrared photothermal imaging (MIPI) - fluorescence in-situ hybridization (FISH) microscope that enables simultaneous identity probing and metabolic activity imaging for bacteria in a complex environment. We added the 13C-glucose in the bacteria culture medium and monitored the newly synthesized protein composition by MIPI at amide I band. Bacterial taxa were targeted with fluorescently-labelled FISH probes and imaged with the widefield epi-fluorescence imaging that built on to the MIP microscope. The sub-micrometer spatial resolution of MIPI enables the characterization at single bacterium level. The proposed platform provides the link between genotype and phenotype and would be a powerful tool to deepen our understanding of microbiome.
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.
We developed a versatile mid-infrared photothermal (MIP) modality that enabled study of metabolic activities in living cells coupled with stable isotope probing. Performance of laser and light emitting diode were compared and system detection limit was demonstrated for different stable isotope probes. We treated the cancer and bacteria cells with deuterium, 13C and 15N labelled nutrients, and acquired the multi-spectral images with the MIP microscope. We observed the red shift in the infrared spectrum, indicating the incorporation of heavy atoms into cell metabolism. Sub-cellular spatial distribution of metabolites including carbohydrates, nucleic acids, proteins and lipids were profiled with high chemical specificity, sub-micrometer spatial resolution and high throughput.
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.
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.