Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains

Area of Science:

• Cellular Biology
• Biochemistry
• Microscopy

Background:

• Understanding protein complex organization is crucial for cell biology.
• Current methods struggle with visualizing multiple proteins at physiological densities.
• Heterogeneous organization of protein complexes remains poorly understood.

Purpose of the Study:

• To develop a novel technique for in situ visualization of multiple protein species.
• To overcome limitations in studying protein complex organization at high densities.
• To enable detailed analysis of protein colocalization and interactions within cells.

Main Methods:

• Resin-embedded multicycle imaging (REMI) was developed.
• Proteins were stabilized in acrylic resins for repeated labeling and imaging.
• Computational techniques and super-resolution microscopy were used for image alignment and colocalization analysis.

Main Results:

• REMI allows for sequential imaging, removal, and replacement of affinity labels.
• Novel preparative methods were created for imaging proteins in plasma membranes and cytoplasm.
• An example demonstrated multiplexed imaging of eight proteins on a fibroblast membrane.

Conclusions:

• REMI significantly enhances the ability to study complex protein organization in situ.
• Multiplexed protein imaging provides deeper insights into cellular processes.
• This technique offers powerful analytical capabilities for membrane proteomic datasets.