inverted confocal microscope zeiss axioobserver, z.1  (Carl Zeiss)

Journal: Nature Communications

Article Title: Single-cell quantification and dose-response of cytosolic siRNA delivery

doi: 10.1038/s41467-023-36752-1

Figure Lengend Snippet: Airyscan confocal imaging of HeLa cells during treatment with lipoplexed AF647-siRNA. a HeLa cells stably expressing d1-eGFP exhibiting the cytosolic distribution of AF647-siRNA after 4 h siRNA treatment. The scale bar is 50 µm. Representative images of eight independent experiments. b HeLa cells expressing YFP-galectin-9 were imaged every 5 s during siRNA transfection, to visualize de novo galectin-9 recruitment to lipoplex-containing vesicles, followed by endosomal release and gradual cytosolic dispersion of AF647-siRNA. Outlines indicate cell boundaries and arrows indicate the releasing particle. The scale bar is 20 µm. Representative images of three independent experiments. c Representative images showing redistribution of AF647-siRNA to cytoplasmic foci after endosomal release and initial homogenous cytosolic dispersion. The scale bar is 20 µm. Images are representative of eight independent experiments. d The redistribution of siRNA-AF647(as) into cytoplasmic foci after release to the cytosol was monitored in cells expressing GFP-AGO2. The image detail shows a single AGO2 + structure. Scale bars, 20 µm; details, 2 µm. Representative images of two experiments. Cells were incubated with 0.4 nM ( a , c ) or 0.67 nM ( b , d ) siRNA.

Article Snippet: An inverted AxioObserver Z.1 LSM 710 epifluorescence confocal microscope with an Airyscan array detector unit (Carl Zeiss AG, Oberkochen, Germany) and equipped with a 40× Plan-Neofluar 1.3 numerical aperture (NA) oil-immersion objective was used for live-cell imaging acquisition.

Techniques: Imaging, Stable Transfection, Expressing, Transfection, Dispersion, Incubation

Journal: Nature Communications

Article Title: Single-cell quantification and dose-response of cytosolic siRNA delivery

doi: 10.1038/s41467-023-36752-1

Figure Lengend Snippet: Airyscan confocal imaging of HeLa cells expressing YFP-galectin-9 every 5 min during treatment with lipoplexed AF647-siRNA. a Endosomal release and cytosolic dispersion of AF647-siRNA, coinciding with the recruitment of YFP-galectin-9 to the lipoplex-containing vesicle (indicated by arrowheads). The scale bar is 20 µm. b Measurements of cytosolic AF647-siRNA in single cells, aligned in time so that t = 0 is the first imaging timepoint with detectable de novo YFP-galectin-9 recruitment to lipoplex-containing vesicles. c Traces of individual cells (columns) with the recruitment of YFP-galectin-9 to vesicles containing siRNA-lipoplex, indicating the performance of the automated event detection in combination with manual quality control. Cell traces are aligned in time with t = 0 being the first frame with detectable galectin-9 recruitment. For a – c , N = 187 cells from two independent experiments. d Performance of manual and automated detection of endosomal siRNA release. Detection sensitivity and specificity were determined by comparing events indicated by galectin-9 recruitment and the manual or automated detection of AF647-siRNA release to the cytosol. Manual quality control (MQC) was performed after automated event detection, to exclude false positive events. Mean ± s.d. is shown. N = two independent experiments. e – g Continuous monitoring of cytosolic AF647-siRNA fluorescence intensity was translated into absolute concentrations using condition-matched reference measurements. siRNA release magnitude estimations were made by fitting a mathematical model (lines) to the cytosolic siRNA concentration from single-cell measurements (circles). Examples are shown for the three different modeling approaches used, depending on the magnitude and kinetics of siRNA release: e a typical high-magnitude release event with exponential decay, f a high-noise low-magnitude release event (step-function), and g two separate events occurring in quick succession. Source data for b – g are presented in the Source Data file.

Article Snippet: An inverted AxioObserver Z.1 LSM 710 epifluorescence confocal microscope with an Airyscan array detector unit (Carl Zeiss AG, Oberkochen, Germany) and equipped with a 40× Plan-Neofluar 1.3 numerical aperture (NA) oil-immersion objective was used for live-cell imaging acquisition.

Techniques: Imaging, Expressing, Dispersion, Control, Fluorescence, Concentration Assay

Journal: Nature Communications

Article Title: Single-cell quantification and dose-response of cytosolic siRNA delivery

doi: 10.1038/s41467-023-36752-1

Figure Lengend Snippet: HeLa cells stably expressing d1-eGFP were treated with 40–2000 pM lipoplexed AF647-siRNA targeting eGFP. A confocal microscope with an Airyscan detector was used for live-cell imaging, followed by single-cell analysis. a , b Cells were ordered and divided into equal groups based on the model-estimated magnitude of siRNA release events. Traces are aligned with t = 0 at the time of cytosolic siRNA detection. Lines are mean eGFP expression per quantile, shaded areas are 80% confidence intervals. Median cytosolic siRNA concentration (nM) and mean R 2 -value per quantile are indicated. Model-fit ( R 2 ) thresholds of >0.3 ( a ) and >0.75 ( b ) were used. N ≥ 92, 58 ( R 2 > 0.3) 90, and 59 ( R 2 > 0.75) cells per quantile for siGFP-1 and siGFP-2, respectively. c , d Single-cell knockdown kinetics between 2 and 10 h after siRNA release. Model-estimated peak cytosolic siRNA concentration and eGFP expression relative to t = 0 is shown per cell, for c siGFP-1 and d siGFP-2. Release events with model R 2 > 0.3 are shown. Red line is the sigmoidal curve fitted by least squares regression. Absolute knockdown IC50 (nM) is indicated. Between 2 and 10 h, N ranges from 419 to 119 and 264 to 117 cells, for siGFP-1 and siGFP-2, respectively. e Relative knockdown IC50 for each time point based on data shown in ( c , d ). Time from the detected release event is indicated. Cells with release events with model R 2 > 0.3 are shown. The line is mean IC50 and the shaded area is 80% confidence intervals. f A model was used to estimate eGFP knockdown mediated by siGFP-1 or siGFP-2, predicted from the median peak cytosolic siRNA concentration after release in each quantile (indicated in graphs). All data shown are from 13 and 6 independent experiments, for siGFP-1 and siGFP-2, respectively. Source data are presented in the Source Data file.

Article Snippet: An inverted AxioObserver Z.1 LSM 710 epifluorescence confocal microscope with an Airyscan array detector unit (Carl Zeiss AG, Oberkochen, Germany) and equipped with a 40× Plan-Neofluar 1.3 numerical aperture (NA) oil-immersion objective was used for live-cell imaging acquisition.

Techniques: Stable Transfection, Expressing, Microscopy, Live Cell Imaging, Single-cell Analysis, Concentration Assay, Knockdown