nikon ti2 e tirf microscopy  (Nikon)

Journal: Redox Biology

Article Title: Redox compartmentalization drives functional heterogeneity of mature insulin secretory vesicles in pancreatic β-cells

doi: 10.1016/j.redox.2025.103847

Figure Lengend Snippet: Oxidative level is strongly correlated to the fate of individual mature ISV within the population. (A) Representative TIRF images of NPY-Timer labeled INS-1E cell during glucose-stimulated insulin secretion (GSIS) process. Starvation (2.8 mM glucose for 30 min), Phase1(16.7 mM glucose for 5 min), Phase2(16.7 mM glucose for 30 min). Scale bar = 3 μm. (B) Histogram of the total ISV number change from the TIRF images of NPY-Timer labeled INS-1E cells. p < 0.0001/p = 0.0477(Paired, Dunnett's multiple comparisons test), n = 20/20/20, respectively. (C) Histogram of the Low-oxidative ISV number change from the TIRF images of NPY-Timer labeled INS-1E cells. p < 0.0001/p = 0.0027(Paired, Dunnett's multiple comparisons test), n = 20/20/20, respectively. (D) Histogram of the High-oxidative ISV number change from the TIRF images of NPY-Timer labeled INS-1E cells. p = 0.07819/p = 0.5567(Paired, Dunnett's multiple comparisons test), n = 20/20/20, respectively. (E) Representative TIRF images for the fusion event of one Low-oxidative ISV and Non-fusion property of High-oxidative ISV in NPY-Timer labeled INS-1E cells. 1.3s per frame. Scale bar = 100 nm. (F) Fluorescent intensity curve of each ISV subtype along time from E. (G) Representative volumetric images of raw data (left panel) of NPY-Timer labeled Endoc-βH5 cell during GSIS, Scale bar = 2 μm. 3D cartoon model (right panel) shows how to measure “Distance to PM” for Low-oxidative ISVs and High-oxidative ISVs. (H) Histogram of the relative number change of Low-oxidative ISVs and High-oxidative ISVs near PM (Distance ≤ 1 μm) normalized to the Starvation group. Starvation (2.8 mM glucose for 30 min), Phase1(16.7 mM glucose for 5 min), Phase2(16.7 mM glucose for 30 min). n = 13/13/13, respectively. (I) Frequency distribution of the number change of Low-oxidative ISVs during GSIS in NPY-Timer labeled Endoc-βH5 cell during GSIS. Over 50 % of Low-oxidative ISVs are locate near PM (Distance ≤ 1 μm). (J) Frequency distribution of the number change of High-oxidative ISVs during GSIS in NPY-Timer labeled Endoc-βH5 cell during GSIS. Less than 30 % of High-oxidative ISVs are locate near PM (Distance ≤ 1 μm). (K) Representative images of the Low/High-oxidative ISVs distribution under Glutamine starvation for 1h/2h/3h within NPY-Timer labeled INS-1E cell, Scale bar, 5 μm. (L) Curve of the Distance to PM of Low-oxidative ISVs and High-oxidative ISVs under Glutamine starvation for 1h/2h/3h within NPY-Timer labeled INS-1E cell. p = 0.0383(Paired t -test), n = 15/15/15 cells, respectively. (M) Representative colocalization images of the Low/High-oxidative ISVs to lysosomes under Glutamine starvation for 1h/3h within NPY-Timer and Lysotracker co-labeled INS-1E cell. 1h group at the top row, and 3h group at the bottom row. Scale bar, 5 μm. (N) Histogram of the Pearson correlation coefficient for quantifying the colocalization level of the Low-oxidative ISVs and High-oxidative ISVs to lysosomes under Glutamine starvation for 1h/3h within NPY-Timer and Lysotracker co-labeled INS-1E cell. For Low-oxidative ISV's 1h vs 3h: p = 0.1975 (Šídák's multiple comparisons test), n = 22/70 cells, respectively. For High-oxidative ISV's 1h vs 3h: p < 0.0001 (Šídák's multiple comparisons test), n = 22/70 cells, respectively. Data are presented as mean ± SEM.

Article Snippet: Then the images were captured as Starvation (2.8 mM glucose for 30 min), Phase1(16.7 mM glucose for 5 min), Phase2(16.7 mM glucose for 30 min) using Nikon Ti2-E TIRF microscopy with a 1.518 oil-immersion 60x objective lens in PSF mode.

Techniques: Labeling