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.
Caldini et al. (Sat,) studied this question.