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“OPTIR brings significant benefits to biophysics and biology. As a vibrational method, OPTIR allows for detailed spectral information on samples, including label-free identification of biopolymers and assignments of protein structures. Low laser powers are nonperturbative to the system.”
Reporting in Analytical Chemistry, researchers at Yale University tackled fundamental limitations that have hampered infrared imaging applications in biological systems. The promise of applying infrared imaging to biological systems has been hampered by low spatial resolution and the overwhelming water background arising from the aqueous nature of in-cell and in vivo work. The team systematically evaluated optimal data collection modes on a commercial OPTIR microscope across three cell lines and three organisms, while testing the compatibility of various IR active probes for metabolic tracking.
The researchers demonstrated that multispectral imaging provides comparable information to hyperspectral imaging while dramatically reducing acquisition times. Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times 20-fold. The authors contrast different modes of operation assessing sensitivity and spatial resolution and make valuable workflow recommendations for both fixed and live cell imaging.
The study validated multiple IR probes for OPTIR compatibility, successfully demonstrating global protein labeling with azidohomoalanine and site-specific labeling with 4-azidomethyl-L-phenylalanine in both E. coli and mammalian cells. Live cell metabolic tracking experiments showed 13C glucose metabolism monitored in live fat cells and E. coli highlights that the same probe may be used in different pathways. The researchers also demonstrated drug visualization capabilities by imaging neratinib uptake in live cell lysosomes using the compound’s intrinsic nitrile moiety.
OPTIR emerges as a versatile platform enabling dynamic biological imaging across multiple scales, from single cells to whole organisms including tardigrades and zebrafish embryos. OPTIR has a spatial resolution of under 500 nm and is less impacted by water absorption as water’s high heat capacity reduces its photothermal signal. Because of this, OPTIR can be used to collect vibrational images in living cells and tissues in water. The technique’s combination of high spatial resolution, reduced water interference, and compatibility with live samples positions it as a powerful tool for spatially resolved metabolic studies in living systems.
Authors:
Sydney O. Shuster, Anna E. Curtis, and Caitlin M. Davis First Author: Sydney O. Shuster, Yale University
