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.
Focalcheck test slide
Manufactured by Thermo Fisher Scientific
The FocalCheck test slide is a laboratory equipment product designed to aid in the assessment and verification of microscope focus and image quality. It provides a standardized test target for evaluating the performance of microscope systems.
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2 protocols using focalcheck test slide
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Confocal Imaging of HLB Puncta
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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.
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