Super-Resolution Microscopy as a Versatile Tool in Probing Molecular Assembly

Abstract

Molecular assembly is promising in the construction of advanced materials, obtaining structures with specific functions. In-depth investigation of the relationships between the formation, dynamics, structure, and functionality of the specific molecular assemblies is one of the greatest challenges in nanotechnology and chemistry, which is essential in the rational design and development of functional materials for a variety of applications. Super-resolution microscopy (SRM) has been used as a versatile tool for investigating and elucidating the structures of individual molecular assemblies with its nanometric resolution, multicolor ability, and minimal invasiveness, which are also complementary to conventional optical or electronic techniques that provide the direct observation. In this review, we will provide an overview of the representative studies that utilize SRM to probe molecular assemblies, mainly focusing on the imaging of biomolecular assemblies (lipid-based, peptide-based, protein-based, and DNA-based), organic-inorganic hybrid assemblies, and polymer assemblies. This review will provide guidelines for the evaluation of the dynamics of molecular assemblies, assembly and disassembly processes with distinct dynamic behaviors, and multicomponent assembly through the application of these advanced imaging techniques. We believe that this review will inspire new ideas and propel the development of structural analyses of molecular assemblies to promote the exploitation of new-generation functional materials.

Keywords: dynamics; interactions; molecular assembly; super-resolution microscopy.

Figure 2

(A) Imaging of vesicles: images of a supported bilayer on glass and high-resolution synthetic image of the contour of a supported bilayer. Reproduced with permission from [53]. (B) Dynamic characteristics of OO4 lipid assembly. Reproduced with permission from [71].

Figure 4

(A) The red-green fibrils with a deblock structure investigated using representative STORM imaging revealed the unidirectional living growth of self-assembled protein nanofibrils. Reproduced with permission from [90]. (B) Angle-dependent heterogeneity of the distance to the protein maxima and mean protein intensity around a specific fat droplet. Reproduced with permission from [93].

Figure 1

Schematic illustration of SRM application in molecular assembly research.

Figure 3

(A) Fluorescent images of FF assemblies illustrating the assembly dynamics at different time points. Reproduced with permission from [86]. (B) 3D imaging of Fmoc-FF hydrogels and mesh size identification using the PAINT method. Reproduced with permission from [87]. (C) In situ and real-time STED imaging demonstrating the disintegration of peptide-based supramolecular nanofibers over time. Reproduced with permission from [88].

Figure 5

(A) BALM and reconstructed images of triangle DNA origami nanostructures. (B) Different position relations and interactions between the tube DNA origami and lysosomes in NIH 3T3 cells. Reproduced with permission from [106].

Figure 6

3D DNA-PAINT super-resolution fluorescence imaging of a polyhedral. Reproduced with permission from [107].

Figure 7

(A) Nanostructures of the aimed vaterite microspheres and the gelatin distribution. (B) Structures of calcite rhombohedra and the distribution of gelatin. (C) The distribution of gelatin in CaCO₃ over time investigated using dSTORM imaging in a switch buffer, Scale bar: 1 μm. Reproduced with permission from [121].

Figure 8

Zoomed dSTORM images of protein adsorption onto MSNs and MSNs-PEGx (x = 2k, 5 k, 10 k) particles after incubation with BSA solution for different timescales (Scale bars: 1 μm). Reproduced with permission from [147].

Figure 9

STORM imaging of Cy5- and Cy3-labeled BTA polymers at different mixing time points (Scale bars: 1 μm). Reproduced with permission from [153].