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Updated: Jul 26, 2025
Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
Published on: February 3, 2013
Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network
Elina Mäntylä1, Toni Montonen1, Lucio Azzari2
1BioMediTech, Faculty of Medicine and Health Technology, Tampere University, 33100 Tampere, Finland.
Insights
We developed an iterative indirect immunofluorescence (IT-IF) staining method combined with expansion microscopy (ExM) for enhanced superresolution imaging of nuclear lamina organization. This approach improves signal quality and reveals nanoscopic details of the lamin network.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biophysics
Background:
- Nuclear lamina architecture is crucial for cellular function and is studied using superresolution microscopy.
- Challenges in superresolution microscopy include epitope accessibility, labeling density, and detection precision in crowded nuclear environments.
Purpose of the Study:
- To develop and validate an improved superresolution microscopy method for visualizing subnuclear nanostructures, specifically the nuclear lamina.
- To enhance signal-to-background ratio and labeling density for more precise imaging of nuclear components.
Main Methods:
- Iterative indirect immunofluorescence (IT-IF) staining combined with expansion microscopy (ExM).
- Structured illumination microscopy (SIM) for superresolution imaging.
- Development of 3D-printed gel casting equipment for ExM.
- A signal-processing pipeline for image denoising and deblurring.
Main Results:
- IT-IF significantly improves signal-to-background ratio and mean fluorescence intensity compared to conventional immunostaining.
- Expansion microscopy (ExM) is validated for analyzing compacted nuclear multiprotein complexes, including viral capsids.
- The developed signal-processing pipeline aids quantitative image analysis.
- Nanoscopic details of the lamin network organization were revealed using signal-resolved IT-IF and superresolution ExM.
Conclusions:
- The combined IT-IF and ExM approach offers enhanced superresolution imaging of the nuclear lamina.
- This method provides a valuable platform for quantitative imaging and studying nuclear structure-function relationships.
- The findings are crucial for understanding the intranuclear structural coregulation of cell function and fate.
Abstract:
Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional-printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization-a prerequisite for studying intranuclear structural coregulation of cell function and fate.
