Related Experiment Video
Updated: May 5, 2026
16:21
Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
Published on: March 10, 2014
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3D Expansion-PALM (PhotoActivated Localization Microscopy) Dissects Protein-Protein Interactions Down to the
Chiara Caldini1,2, Sara Del Duca3,4, Alberto Vassallo3
1European Laboratory for Non-Linear Spectroscopy (LENS), Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy.
Microorganisms
|May 4, 2026
Summary
This study introduces Expansion-Assisted PhotoActivated Localization Microscopy (Ex-PALM) for high-resolution imaging in dense bacterial cells. Ex-PALM achieves nanoscale visualization, revealing protein co-localization at 19 nm in Escherichia coli.
Area of Science:
- Microscopy
- Cellular Biophysics
- Molecular Biology
Background:
- Super-resolution microscopy enables nanoscale imaging but struggles with dense cellular environments like bacteria.
- 3D PhotoActivated Localization Microscopy (PALM) faces challenges with overlapping signals and undersampling in small bacterial volumes.
Purpose of the Study:
- To develop a dual-color 3D super-resolution imaging technique for dense bacterial cells.
- To overcome the limitations of conventional PALM in high-density molecular environments.
- To enable precise measurement of molecular distances within prokaryotes.
Main Methods:
- Combined PhotoActivated Localization Microscopy (PALM) with Expansion Microscopy (ExM) to create Ex-PALM.
- Optimized ExM for a 4-fold isotropic expansion in bacteria.
- Utilized photoactivable fusion proteins, inclined illumination, and minimized chromatic aberrations for high specificity and sensitivity.
Main Results:
- Achieved dual-color 3D single-molecule localization with ~3 nm spatial resolution.
- Successfully imaged the spatial distribution of HisF and HisH proteins in Escherichia coli.
- Revealed co-localization of newly synthesized HisF and HisH proteins at an average 3D distance of 19 nm.
Conclusions:
- Ex-PALM provides an effective method for studying molecular organization in prokaryotes and other high-density samples.
- This hybrid approach advances nanoscale imaging in challenging biological systems.
- Broad applications are foreseen in microbiology, synthetic biology, and cellular biophysics.
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