SM-SPAD · Single-molecule imaging with SPAD array detection

Single-molecule imaging with SPAD array detection

Microscopes have become a fundamental tool ubiquitous with high-quality biological research, and have enabled the visualization of cells, cell organelles, and even single molecules. However, the resolving power of conventional widefield microscopes is limited to about 250 nm due to the diffraction of light. Consequently, two structures closer than this limit are unresolvable from one another. This limit is explicitly detrimental to structural biology studies, where the species of interest, typically proteins, nucleic acids, or other macromolecules, is much smaller than 100 nm. Our understanding of the organization of these structures and the way they generate responses in cells is therefore currently restricted. Several imaging techniques that are able to circumvent the diffraction limit have recently been introduced in fluorescence microscopy. Single-molecule (SM) localization microscopy was demonstrated to yield a lateral resolution of 20 nm by sequentially and stochastically switching on single fluorescent molecules and determining their position in the image plane by analyzing the observed fluorescence. However, these techniques require a lot of emission photons to get a reasonable localization precision. Unfortunately, the fluorescence emission is limited by photobleaching of the fluorophores. To overcome this problem, the concept of MINFLUX was recently introduced. MINFLUX is a technique in which a doughnut-shaped spot of light is used to find the position of a single fluorescence-tagged molecule. By moving the spot so that the center of the doughnut is in close proximity to the position of the molecule, only a minimum dose of fluorescence is generated. A triangulation procedure that is realized by slightly moving the excitation beam allows estimation of the molecule’s location with very high accuracy and minimum photon flux. Although MINFLUX provides a significantly better resolution compared to standard localization microscopy, it still neglects crucial information needed to improve the resolution further and speed up the imaging process, namely: (i) the point-detection system does not allow directly recording the image of the fluorescent molecule in the image plane, thus a rather complex beam-scanning approach is required; (ii) it does not leverage the fluorescence lifetime information, which can provide nanometer-scale information on the structure of interest. Recording this information is crucial for understanding the organization of molecular complexes. In this project, both of these limitations were tackled. The main goal of the SM-SPAD project is to develop a 3D single-molecule fluorescence lifetime imaging technique for structural biology. The goal will be reached by combining the concept of MINFLUX with three new ideas: (i) 3D motionless structured illumination and structured detection for improved spatial resolution; (ii) fluorescence antibunching analysis to speed up the data acquisition by enabling simultaneous localization of several active molecules; (iii) SM level fluorescence lifetime analysis to extract the maximum amount of information from the sample. The proposed molecular-scale imaging technique is perfectly suited for the study of complex biological samples. SM-SPAD is particularly promising in the field of neuroscience research in which the organization of large protein complexes are of high interest, as they may play a crucial role in the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s.

Data: CORDIS, © European Union