microscope platform  (Genoscope)

Journal: iScience

Article Title: Genetically encoded and modular subcellular organelle probes reveal dysfunction in lysosomes and mitochondria driven by PRKN knockout

doi: 10.1016/j.isci.2025.112816

Figure Lengend Snippet: Timer fluorophore measures changes in subcellular mitochondrial turnover in PRKN −/− (A) Schematic demonstrating how the fluorescent protein timer works. Timer undergoes a fluorescent shift from a green emission to a red emission overtime; analysis of timer relies on examining the ratio between the red and green emission. (B) Schematic of the lentiviral construct used in the following panels localizing timer to the mitochondria by using the targeting sequence of COX8A. (C) Representative images of astrocytes transduced with COX8A-Timer and treated with a vehicle (DMSO; top) or 100 nM bafilomycin A1 (bottom) for 24 h. Images were acquired with a 488-excitation line and a 594-excitation line. Far right: ratiometric representation of the red channel divided by the green channel and pseudo colored so that orange, yellow and white indicate more relative red emission and black and purple indicate more relative green emission. Nuclei were stained with DRAQ5. Scale bars, 50 μm. (D) Quantification of the ratio of mean intensity of emission from excitation with a 594 nm laser to the mean intensity of emission from excitation with a 488 nm laser in astrocytes expressing COX8A-timer and treated with a vehicle (DMSO) or 100 nM bafilomycin A1 for 24 h. Central bars represent mean and error bars represent standard deviation (unpaired t test; n = 6; each replicate is an average over 25 images). (E) Representative images of PRKN +/+ and PRKN −/− astrocytes transduced with COX8A-timer. Images were acquired with a 488-excitation line and a 594-excitation line. Far right: ratiometric representation of the red channel divided by the green channel and pseudo colored so that orange, yellow and white indicate more relative red emission and black and purple indicate more relative green emission. Nuclei were stained with DRAQ5. Scale bars, 50 μm. (F) Quantification of the ratio of mean intensity of emission from excitation with a 594 nm laser to the mean intensity of emission from excitation with a 488 nm laser in PRKN +/+ and PRKN −/− astrocytes expressing COX8A-Timer. Central bars represent mean and error bars represent standard deviation ( n = 3; each replicate is an average over 25 images). (G) Quantification of mean intensity normalized by nuclear area of green and red fluorescence in PRKN +/+ and PRKN −/− astrocytes expressing COX8A-timer. Central bars represent mean and error bars represent standard deviation (two-way ANOVA with Sidak correction; n = 3; each replicate is an average over 25 images). All images were acquired on a CX7 HCS platform with a 20x objective lens. For all graphs, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ <0.0001.

Article Snippet: We thank the Microscopy CoRE at Icahn School of Medicine for providing access to the Leica DMi8 and Thermo Scientific CX7 High Content Screening Platform, as well as training and technical expertise for using these microscopes.

Techniques: Construct, Sequencing, Transduction, Staining, Expressing, Standard Deviation, Fluorescence

Journal: iScience

Article Title: Genetically encoded and modular subcellular organelle probes reveal dysfunction in lysosomes and mitochondria driven by PRKN knockout

doi: 10.1016/j.isci.2025.112816

Figure Lengend Snippet: Nucleus-localized fluorophores offer improvements over available stains for multiday imaging (A) Schematic of the lentiviral construct localizing mTagBFP2 to the nucleus with an H2B fusion protein and antibiotic resistance to puromycin used in the following panels. (B) Representative images of H2B-mTagBFP2 and DRAQ5 nuclear stain 1 h (top) and 24 h (bottom) after adding DRAQ5 to the cell cultures. (C) Quantification of the percent of stain localized to the nuclear area versus the cytoplasm 1 h and 24 h after adding DRAQ5 to cell cultures. The central bar represents the mean and the error bars represent the standard deviation (two-way ANOVA with Tukey’s HSD; n = 3 per time point; each replicate is an average over 36 images). (D) Schematic of the lentiviral construct localizing Emerald or mCherry to the nucleus with an H2B fusion protein and antibiotic resistance to puromycin used in the following panels. (E) Representative images of nuclei from PRKN +/+ astrocytes, labeled with H2B-Emerald, and PRKN −/− astrocytes, labeled with H2B-mCherry, cultured together during a proliferation assay. Images were taken 24 h (top) after seeding and 72 h (bottom) after seeding. (F) Quantification of number of PRKN +/+ and PRKN −/− nuclei per image field 24 h and 72 h after seeding. Dots represent mean values and error bars represent standard deviation (two-way ANOVA with Tukey’s HSD; n = 3 per time point; each replicate is an average over 25 images). Scale bars, 50 μm. All images were acquired on the CX7 HCS platform with a 20x objective lens. For all graphs, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ <0.0001. See also and .

Article Snippet: We thank the Microscopy CoRE at Icahn School of Medicine for providing access to the Leica DMi8 and Thermo Scientific CX7 High Content Screening Platform, as well as training and technical expertise for using these microscopes.

Techniques: Imaging, Construct, Staining, Standard Deviation, Labeling, Cell Culture, Proliferation Assay