3D Expansion-PALM (PhotoActivated Localization Microscopy) Dissects Protein-Protein Interactions Down to the Molecular Scale in Bacteria
Year: 2026
Authors: Caldini C., Del Duca S., Vassallo A., Semenzato G., Fani R., Pavone F.S., Gardini L.
Autors Affiliation: European Lab Nonlinear Spect Lens LENS, Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, Via Giovanni Sansone 1, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Biol, Via Madonna Piano 6, I-50019 Sesto Fiorentino, Italy; Council Agr Res & Econ CREA, Res Ctr Genom & Bioinformat, Via S Protaso 69, I-29017 Fiorenzuola Darda, Italy; Natl Res Council Italy, Inst Sustainable Plant Protect, I-50019 Sesto Fiorentino, Italy; Natl Res Council CNR INO, Natl Inst Opt, Largo Enr Fermi 6, I-50125 Florence, Italy; CNR, Inst Complex Syst, Via Madonna Piano 10, I-50019 Sesto Fiorentino, Italy.
Abstract: Super-resolution microscopy has transformed biological imaging by enabling nanoscale visualization of cellular structures beyond the diffraction limit. However, its effective application in highly dense molecular environments still poses challenges. This is the case for 3D PhotoActivated Localization Microscopy (PALM) achieved through astigmatism in bacterial cells. The limited volume of a single bacterium highly increases the probability of the intensity profiles emitted by single chromophores to overlap, thus strongly decreasing the number of localizations, leading to dramatic undersampling. Dual-color 3D super-resolution in Escherichia coli is achieved through a combination of PALM with Expansion Microscopy (Ex-PALM). PALM provides high specificity through photoactivable (PA) fusion proteins and high localization precision, while ExM physically expands the specimen and separate densely packed molecules. This hybrid approach enables dual-color 3D single-molecule localization with about 3 nm spatial resolution, thus allowing one to measure distances down to the molecular scale. This is achieved by optimizing ExM protocols in bacteria to achieve a 4-fold isotropic expansion, by minimizing both chromatic aberrations and signal crosstalk, and by improving single-molecule sensitivity through highly selective inclined illumination. The method is applied to measure the spatial distribution of HisF and HisH proteins, involved in E. coli histidine biosynthesis. By tagging each protein with a photoactivable fluorescent protein, Ex-PALM reveals that after being synthetized, they co-localize in the bacterial volume with an average 3D distance of 19 nm. By combining labeling specificity with Ex-PALM, an effective method is developed for studying molecular organization in prokaryotes and in high-density samples in general, such as cell organelles or molecular condensates, with broad applications in microbiology, synthetic biology, and cellular biophysics.
Journal/Review: MICROORGANISMS
Volume: 14 (4) Pages from: 772-1 to: 772-20
More Information: This research was co-funded by the European Union under HEU-GA 101131771 Lasers4EU. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. The European Union cannot be held responsible for them. This research was supported by the European Union’s Horizon2020 research and innovation program under grant Agreement No. 871124 Laserlab-Europe and by the project CNR-FOE-LENS.KeyWords: E. coli; super-resolution microscopy; expansion microscopy; expansion-PALM; dual color; molecular distanceDOI: 10.3390/microorganisms14040772
