Fig. 1: Concept, instrumentation, and characterization of imaging lipid metabolism with OPTIR microscopy and IR probes.

A Azide tags were metabolically incorporated into newly-synthesized lipids and thus enabled the selective detection of these metabolites. B Schematic setup of a fluorescence-integrated OPTIR system. IR infrared, PBS polarizing beam splitter, PD photodiode. C OPTIR spectra of Azide-PA, PA, and a biological cell. Spectra were normalized (Norm.) to maximum intensity and offset for clarity. D Representative total lipids (1744 cm⁻¹), newly synthesized lipid (2096 cm⁻¹), and BODIPY imaging from a single cell. Scale bars, 10 μm. E Line profile along the horizontal and vertical direction of a small lipid droplet indicated with a white arrow in 2096 cm⁻¹ image in D. The raw data is shown in black dots and the Gaussian fitting is shown in the red curve. Full-width half maximum (FWHM) of the fitted curve is shown to demonstrate the spatial resolution. F Representative OPTIR images at 1744 cm⁻¹ and 2096 cm⁻¹ of human neuroglioma H4 cells after incubation in Azide-PA and PA-containing media for 24 h. Corresponding brightfield images are also shown. Scale bars, 20 μm G OPTIR spectra for azide-PA and PA incubated cells. Raw spectra were normalized to protein signal at 1654 cm⁻¹ and offset for clarity. Mean curve (solid) and standard deviation (shade) were generated from 22 azide-PA cultured cells and 14 PA cultured cells. H, I Statistical analysis of total lipid and newly synthesized lipid from spectral fitting results. Area under curve centered around 1654  cm⁻¹, 1740 cm⁻¹, and 2096 cm⁻¹ were used for quantification. A non-significant difference was observed for total lipids between azide-PA incubated (n = 22) and PA incubated cells (n = 14). Statistical test: two-sided two-sample t-test H. Central horizontal lines in the box plot indicate medians, box limits indicate first and third quartiles, vertical whisker lines indicate minimal and maximum values, the outliers were identified using a coefficient of 1.5 times the interquartile range (H-I). The spectral range of 1780 to 2030 cm⁻¹ was omitted since no observable peaks were presented. Source data are provided as a Source Data file.