Journal: Science Advances

Article Title: Optical photothermal infrared imaging of fatty acid esterification in the ER of living cells

doi: 10.1126/sciadv.aed6477

Figure Lengend Snippet: ( A ) Schematic of live-cell imaging using OPTIR imaging. CaF 2 , calcium fluoride; IR, infrared. ( B ) Bright-field image of a live control Huh-7 cell. ( C ) Single wave number image of the live control cell collected at the C═O lipid carbonyl band (1744 cm −1 ). ( D ) Single wave number image of the live control cell collected at the v as C─D 2 stretch (2198 cm −1 ). ( E ) Bright-field image of a live Huh-7 cell 24 hours after feeding 60 μM PA-d 31 conjugated to BSA at a 2:1 ratio. ( F ) Single wave number image of the live PA-d 31 –fed cell collected at the C═O lipid carbonyl band (1744 cm −1 ). ( G ) Single wave number image of the live PA-d 31 –fed cell collected at the v as C─D 2 stretching mode (2198 cm −1 ). ( H ) Average of 20 normalized OPTIR spectra collected across 20 LDs of four control Huh-7 cells (blue) and average of 20 normalized OPTIR spectra collected across 20 LDs of four Huh-7 cells 24 hours after feeding 60 μM PA-d 31 conjugated to BSA at a 2:1 ratio (pink). Figure displays four biological replicates and 20 technical replicates. Shading is one SD of the average normalized spectrum. The dominant C═O peak at 1744 cm −1 , attributed to the ester carbonyl of TAGs/CEs, confirms that both cells are imaged on LDs. Only the cell fed PA-d 31 exhibits C─D 2 asymmetric and symmetric stretches at 2198 and 2098 cm −1 , respectively. ( I ) A close-up of the C═O spectral region from 1700 to 1780 cm −1 . A shoulder at 1734 cm −1 is observed only in PA-d 31 –fed cells. ( J ) Second derivative of the C═O region resolves the shoulder at 1734 cm −1 .

Article Snippet: Cells were then fed 60 or 250 μM PA-d 31 and 30 μM DGAT1 inhibitor (T863, MedChemExpress, Princeton, NJ) and 30 μM DGAT2 inhibitor (PF06424439, MedChemExpress).

Techniques: Live Cell Imaging, Imaging, Control

Journal: Science Advances

Article Title: Optical photothermal infrared imaging of fatty acid esterification in the ER of living cells

doi: 10.1126/sciadv.aed6477

Figure Lengend Snippet: ( A to F ) Bright-field images of representative Huh-7 cells fed at 2, 12, 27, 39, 54, and 69 hours, respectively. Colored crosses indicate line scans where corresponding spectra were collected and are plotted in (G). Scale bars, 10 μm. ( G ) Average normalized IR spectra with SD of Huh-7 cells fed PA-d 31 at 2, 12, 27, 39, 54, and 69 hours. Each time point represents the average spectra from the points labeled in (A) to (F). Shading is one SD of the average normalized spectrum. Figure displays 1 biological replicate and an average of 25 technical replicates per time point. ( H ) Second derivative of the average spectra in (G) with corresponding SDs.

Article Snippet: Cells were then fed 60 or 250 μM PA-d 31 and 30 μM DGAT1 inhibitor (T863, MedChemExpress, Princeton, NJ) and 30 μM DGAT2 inhibitor (PF06424439, MedChemExpress).

Techniques: Labeling

Journal: Science Advances

Article Title: Optical photothermal infrared imaging of fatty acid esterification in the ER of living cells

doi: 10.1126/sciadv.aed6477

Figure Lengend Snippet: ( A ) Bright-field image of a cell 24 hours after feeding 120 μM PA-d 31 conjugated to BSA at a 2:1 ratio. Scale bar, 10 μm. ( B ) Fluorescence image of the cell in (A) stained for the ER (red), nucleus (blue), and LDs (green). A white box indicates the hyperspectral image bounds. Scale bar, 10 μm. ( C ) Fluorescence signal in the hyperspectral image area with the LD outlined in white. Scale bar, 1 μm. ( D ) Single wave number image of the second derivative of the 1734-cm −1 band in the hyperspectral image with the LD outlined in white. ( E ) Bright-field image of a cell 24 hours after feeding 120 μM PA-d 31 conjugated to BSA at a 2:1 ratio. ( F ) The green fluorescence channel of the cell in (E) stained for LD. Pink and blue boxes indicate the studied LDs. Scale bar, 10 μm. ( G ) The red fluorescence channel of the cell in (E) stained for ER. Pink and blue boxes indicate the studied LDs. Scale bar, 10 μm. ( H ) The cell in (E) stained for the ER (red), LD (green), and nucleus (blue). Pink and blue boxes indicate the studied LDs. Scale bar, 10 μm. ( I ) Spectra corresponding to the points where a line scan was collected across the LD in the blue box (inset). Lipid bands at 1744 and 1734 cm −1 are marked. Scale bar, 1 μm. ( J ) Spectra corresponding to the points where a line scan was collected across the LD in the pink box (inset). Lipid bands at 1744 and 1734 cm −1 are marked. Scale bar, 1 μm. All images collected on the OPTIR microscope.

Article Snippet: Cells were then fed 60 or 250 μM PA-d 31 and 30 μM DGAT1 inhibitor (T863, MedChemExpress, Princeton, NJ) and 30 μM DGAT2 inhibitor (PF06424439, MedChemExpress).

Techniques: Fluorescence, Staining, Microscopy

Journal: Science Advances

Article Title: Optical photothermal infrared imaging of fatty acid esterification in the ER of living cells

doi: 10.1126/sciadv.aed6477

Figure Lengend Snippet: ( A ) Bright-field image of a Huh-7 cell fed 60 μM PA-d 31 conjugated to BSA at a 2:1 ratio. The spectrum was collected as a single spectrum on the edge of a LD near the ER 24 hours after being fed PA-d 31 (indicated by the pink crosshairs). ( B ) Bright-field image of a Huh-7 cell fed 60 μM PA-d 31 conjugated to BSA at a 2:1 ratio. The spectrum was collected as a single spectrum on the center of a LD of a cell 69 hours after being fed PA-d 31 (blue crosshairs). ( C ) A redshift of the asymmetric and symmetric CD 2 stretches and the appearance of a Fermi resonance at 2158 cm −1 are correlated with the appearance of a 1734-cm −1 shoulder off the lipid carbonyl. Enlarged view of the C─D region and its corresponding second derivative spectra is shown in ( D ) and ( E ), respectively. Measurement of multiple cells and samples produced the same results (fig. S8). Spectra were normalized by prominent peak heights in the C─D region. Figure displays one biological and one technical replicate.

Article Snippet: Cells were then fed 60 or 250 μM PA-d 31 and 30 μM DGAT1 inhibitor (T863, MedChemExpress, Princeton, NJ) and 30 μM DGAT2 inhibitor (PF06424439, MedChemExpress).

Techniques: Produced