Following immunofluorescent staining, cells were imaged using a Nikon A1R Laser Scanning Confocal microscope equipped with an Andor iXon 897 Ultra EMCCD and NIS Elements software (v. 5.30). Cells with detectable HLB puncta were selected using a 10× NA 0.45 air objective and their XY coordinates were saved. Individual Z-stacks of selected cells were then captured sequentially using a Plan Apo λ 100×1.45 NA oil objective with 0.1 μm step size and 1.0 AU pinhole size per channel. Chromatic aberration between the TRITC and Cy5 channels was quantified using 0.5 μm fluorescent microspheres on a FocalCheck test slide (Invitrogen, F36909). The Cy5 channel was computationally shifted by +0.06 μm in the X plane for all nuclear Z-stacks to compensate for the measured aberration.
A1r laser scanning confocal microscope
Manufactured by Oxford Instruments
The A1R Laser Scanning Confocal Microscope is a high-performance imaging system designed for fluorescence microscopy applications. It utilizes a laser-based scanning mechanism to capture high-resolution, optical sectioning images of samples. The A1R provides precise control over the excitation light and collection of emitted fluorescence, enabling the visualization of fine details within complex biological specimens.
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3 protocols using a1r laser scanning confocal microscope
1
Confocal Imaging of HLB Puncta
2
Confocal Imaging of HLB Puncta
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Following immunofluorescent staining, cells were imaged using a Nikon A1R Laser Scanning Confocal microscope equipped with an Andor iXon 897 Ultra EMCCD and NIS Elements software (v. 5.30). Cells with detectable HLB puncta were selected using a 10× NA 0.45 air objective and their XY coordinates were saved. Individual Z-stacks of selected cells were then captured sequentially using a Plan Apo λ 100×1.45 NA oil objective with 0.1 μm step size and 1.0 AU pinhole size per channel. Chromatic aberration between the TRITC and Cy5 channels was quantified using 0.5 μm fluorescent microspheres on a FocalCheck test slide (Invitrogen, F36909). The Cy5 channel was computationally shifted by +0.06 μm in the X plane for all nuclear Z-stacks to compensate for the measured aberration.
3
Quantifying Cytosolic and Nuclear RNA
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Fixed cell imaging was performed using a 100× oil objective on a Nikon A1R Laser Scanning Confocal microscope with an Andor iXon 897 Ultra detector or a Nikon SoRa Spinning Disc Confocal microscope with an Andor iXon Life 897 EMCCD Camera. We imaged 10–20 z-slices at a distance of 0.2 μM/slice. All images shown are a max projection in Z of the z-stack.
We used ImageJ (version 2.9.0/1.53t) to quantify GAPDH smFISH spots. After creating a max intensity projection in Z, we set an intensity threshold for the experiment and analysed the number of pixels above that threshold within manually defined regions of interest (ROIs). Each ROI represents a single cell. To quantify AHNAK, NORAD and GAS5 smFISH spots, we first made a mask of the nuclei using CellProfiler (version 4.2.5) to specifically analyse the cytosol (or nuclei), set an intensity threshold as described above, then used the ImageJ feature Analyse Particles to count the number of smFISH spots above the intensity threshold in each ROI.
AHA intensity was quantified using ROIs manually defined in ImageJ. We then measured the average intensity of each ROI.
We used ImageJ (version 2.9.0/1.53t) to quantify GAPDH smFISH spots. After creating a max intensity projection in Z, we set an intensity threshold for the experiment and analysed the number of pixels above that threshold within manually defined regions of interest (ROIs). Each ROI represents a single cell. To quantify AHNAK, NORAD and GAS5 smFISH spots, we first made a mask of the nuclei using CellProfiler (version 4.2.5) to specifically analyse the cytosol (or nuclei), set an intensity threshold as described above, then used the ImageJ feature Analyse Particles to count the number of smFISH spots above the intensity threshold in each ROI.
AHA intensity was quantified using ROIs manually defined in ImageJ. We then measured the average intensity of each ROI.
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