Journal: Nature Methods

Article Title: Differentiation of sphingomyelin and cholesterol by hyperspectral mid-infrared detection of single-bond vibrational modes in the fingerprint region

doi: 10.1038/s41592-026-03025-w

Figure Lengend Snippet: a , Differential spectra between 50 Chol-enriched and unenriched HEK cells. Data are presented as mean values ± s.d. For comparison, MβCD–Chol complex spectrum acquired with ATR–FTIR spectroscopy is shown in black. Chol-enriched HEK cells showed a peak at 1,048 cm −1 , corresponding to the absorption peak of the MβCD–Chol complex. b , Violin plots showing the kernel density of the AUC of the peak at 1,048 cm −1 before (T0) and 16 h (T16) after exposure to the MβCD–Chol complex. Box plot minima and maxima inside the violin plots indicate the interquartile range, the white circles indicate the mean values and the whiskers indicate the s.d. (coefficient of 1). Outliers are shown as individual points. *** P = 1.46 × 10 −28 from a two-sided paired t -test. N = 3. c , HyFOPM micrographs of HEK cells before (T0) and after treatment (T16) with MβCD–Chol complexes. Overlay maps are obtained from Chol (1,048 cm −1 ) and SM contrast (1,464 cm −1 ). d , e , Box plots representing the OptA contrast of the micrographs in c : the Chol contrast (1,048 cm −1 ) ( d ) and the SM contrast (1,464 cm −1 ) ( e ) for MβCD-treated HEK cells at time 0 h (T0) and 16 h (T16). Box plots indicate the s.e.m. (coefficient of 1; box limits), the white circles indicate the mean values, while the centered lines the median values, and the whiskers indicate the s.d. (coefficient of 1). Outliers are shown as individual points. *** P = 6.19 × 10 −12 ( d ) and P = 5.48 × 10 −8 ( e ) from a two-sided paired t -test. Scale bar, 300 μm, N = 3. OptA, optoacoustic; NOptA, normalized optoacoustic.

Article Snippet: Accumulation of SM and Chol was performed respectively on a human adenocarcinoma alveolar basal epithelial cell line (A549, American Type Culture Collection (ATCC): CCL-185) and a HEK cell line (HEK293, ATCC: CRL-1573).

Techniques: Comparison, Spectroscopy

Journal: Nature Methods

Article Title: Differentiation of sphingomyelin and cholesterol by hyperspectral mid-infrared detection of single-bond vibrational modes in the fingerprint region

doi: 10.1038/s41592-026-03025-w

Figure Lengend Snippet: a) Comparison between the hyperspectral fingerprint optoacoustic microscopy (hyFOPM)’s spectra of a Chol liquid solution (green line) and of MβCD - Chol complex (purple line). The Chol absorption peak shifts from 1056 cm −1 to 1048 cm −1 when Chol is complexed with cyclodextrins. b) Comparison between the Fourier transform infrared (FTIR) spectra of MβCD complex with Chol (in red) and MβCD without Chol (in blue). For comparison, the FTIR spectrum of a Chol liquid solution is plotted in black. c) HyFOPM images monitoring Chol loading in HEK cells at 1375 cm −1 (Chol contrast), at 1540 cm −1 (protein contrast), and at 2852 cm −1 (lipid contrast) before (T0) and after 16 hours (T16) of treatment with MβCD-Chol. Boxplots representing the optoacoustic contrast of HEK cells in (c) at (d) 1375 cm −1 (Chol contrast), at (e) 2852 cm −1 (lipid contrast) and at (f) 1540 cm −1 (protein contrast). Box plots indicate the standard error (SE, coefficient =1; box limits), the white circles indicate the mean values, while the centered lines the median values, and the whiskers indicate the standard deviation (SD, coefficient =1). Outliers are shown as individual points. ***p = 2.24 × 10 −28 ( d ), p = 3.16 × 10 −8 ( e ) and p = 0.0018 ( f ) from a two-sided paired t-test. N = 3. OptA: Optoacoustic; NOptA: Normalized Optoacoustic.

Article Snippet: Accumulation of SM and Chol was performed respectively on a human adenocarcinoma alveolar basal epithelial cell line (A549, American Type Culture Collection (ATCC): CCL-185) and a HEK cell line (HEK293, ATCC: CRL-1573).

Techniques: Comparison, Microscopy, Fourier Transform Infrared Spectroscopy, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Optimized ND4 allotopic expression for gene therapy of Leber’s hereditary optic neuropathy

doi: 10.3389/fbioe.2026.1765995

Figure Lengend Snippet: (A) Schematic representation of the generation of the HEK-293 LHON cell line with the MT-ND4 mutation (mt.11778 G>A); (B) TMRE fluorescence (accumulates in mitochondria) in native HEK-293 and HEK-293 Rho 0 cells; (C) sequence of the MT-ND4 region.

Article Snippet: The HEK-293 cell line (ATCC cat. CRL-1573) was transferred to DMEM medium containing 4.5 g/L glucose (Biosera cat. PM-D1115), 5% v/v FBS (Biowest, cat. S1600), 2.5 mM pyruvate (PanEco, cat. F023), 1× NEAA (PanEco, cat. F115), 100 μg/mL uridine (Sigma-Aldrich, cat. U3750), and 25 ng/mL EtBr (Sigma-Aldrich, cat. 46067) with a monthly increase in concentration to 50, 100, 200, 300, and finally 400 ng/mL ( ).

Techniques: Mutagenesis, Fluorescence, Sequencing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Optimized ND4 allotopic expression for gene therapy of Leber’s hereditary optic neuropathy

doi: 10.3389/fbioe.2026.1765995

Figure Lengend Snippet: Values of ROS, hydrogen peroxide, calcium ions, and ΔΨm levels in HEK-293 (HEK) and HEK-293 LHON (HEK(LHON)) cell lines. (A) DCF (ROS) fluorescence level in cells; (B) HyPer7 fluorescence level (hydrogen peroxide) in mitochondria; (C) Case12 fluorescence level (calcium ions) in mitochondria; (D) TMRE fluorescence level (ΔΨm) in cells; n = 4 biological replicates. Data are shown as means ± SD. In each panel, statistics were evaluated using the Mann–Whitney U test; * p < 0.05.

Article Snippet: The HEK-293 cell line (ATCC cat. CRL-1573) was transferred to DMEM medium containing 4.5 g/L glucose (Biosera cat. PM-D1115), 5% v/v FBS (Biowest, cat. S1600), 2.5 mM pyruvate (PanEco, cat. F023), 1× NEAA (PanEco, cat. F115), 100 μg/mL uridine (Sigma-Aldrich, cat. U3750), and 25 ng/mL EtBr (Sigma-Aldrich, cat. 46067) with a monthly increase in concentration to 50, 100, 200, 300, and finally 400 ng/mL ( ).

Techniques: Fluorescence, MANN-WHITNEY

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Optimized ND4 allotopic expression for gene therapy of Leber’s hereditary optic neuropathy

doi: 10.3389/fbioe.2026.1765995

Figure Lengend Snippet: Effects of delivery of the functionally active ND4opt gene with various MTS into HEK-293 (LHON) cells on the level of ROS, hydrogen peroxide, calcium ions, and ΔΨm; (A) DCF (ROS) fluorescence levels in cells after delivery of the ND4opt gene with different MTS using plasmid vectors; (B) DCF fluorescence level (ROS) in cells after delivery of the ND4opt gene with various MTS using AAV vectors; (C) HyPer7 fluorescence level (hydrogen peroxide) in mitochondria after delivery of the ND4opt gene with various MTS using plasmid vectors; (D) Case12 (calcium ion) fluorescence level in mitochondria after delivery of the ND4opt gene with various MTS using plasmid vectors; (E) TMRE (ΔΨm) fluorescence level in cells after delivery of the ND4opt gene with different MTS using plasmid vectors; n = 4 biological replicates. Data are shown as means ± SD. In each panel, statistics were evaluated using the Mann–Whitney U test, where each variant “+MTSx_ND4” was compared with “+MTS8k_ND4mut” or AAV_empty (for B ), and the effect of the variant on the figure (shown by *) is indicated only for the variant “+MTS8k_ND4”; * p < 0.05.

Article Snippet: The HEK-293 cell line (ATCC cat. CRL-1573) was transferred to DMEM medium containing 4.5 g/L glucose (Biosera cat. PM-D1115), 5% v/v FBS (Biowest, cat. S1600), 2.5 mM pyruvate (PanEco, cat. F023), 1× NEAA (PanEco, cat. F115), 100 μg/mL uridine (Sigma-Aldrich, cat. U3750), and 25 ng/mL EtBr (Sigma-Aldrich, cat. 46067) with a monthly increase in concentration to 50, 100, 200, 300, and finally 400 ng/mL ( ).

Techniques: Fluorescence, Plasmid Preparation, MANN-WHITNEY, Variant Assay