How can light-based methods provide maximal information on biological processes? What are the limits to the information we can extract? How deep and fast can we look? How can we use extreme resolution to answer concrete questions? What answers can multimodal approaches deliver?

The development of novel optical methods like MINFLUX (see below) shows that the answers to these questions remain unexplored or incomplete, and that there is much to gain from synergistically combining expertise from diverse areas like optics, electronics, statistics, chemistry and biology. By solving transverse methodological challenges, we strive to push forward the maturation of light microscopy and profoundly influence life sciences along the way.

Super Resolution Microscopy

Fluorescence microscopy is an invaluable tool for exploring the structure and function of biological processes. It provides high specificity and contrast for the observation of cellular components (DNA, RNA, proteins, lipids, etc.) tagged with fluorescent molecules in a minimally invasive fashion, even allowing the study of live specimens.

The spatial resolution of classical fluorescence microscopy is limited to hundreds of nanometers due to the diffraction of light; however, higher resolutions were unlocked with the development of the so-called super-resolution methodologies (stimulated emission depletion (STED) microscopy, photo-activated localization microscopy/stochastic optical reconstruction microscopy (PALM/STORM), among others).

In the last decade, achieving resolutions in the order of 10 nm to 100 nm became routine and has revealed details of subcellular organelles and new structures as well as ultrastructural anatomy in tissue, granting the Nobel Prize in Chemistry in 2014 to the developers of super resolution. Despite this revolution, the development of an ultimate microscope – revealing the precise nanometric location of all molecules of interest at all times without affecting the sample under study – remains elusive. Among several adversities, the photon budget of fluorescent probes is a fundamental bound for the tradeoff between spatial and time resolution.

Figure 1. (A) Principle of PALM/STORM, where the resolution depends on the wavelength, numerical aperture and number of collected photons. (B) MINFLUX depicted as a ruler, where emitters are located from a sequence of exposures to tailored light patterns. The resolution now depends on the size of the ruler (L, separation between sequential excitations), instead of the wavelength. (C) A MINFLUX scheme in 2D with multiple excitations with doughnut-shaped beams, each coloured dot is the center of an exposure. (D) A MINFLUX scheme in 3D, with a beam created by top-hat wavefront shaping. (E) Iterative MINFLUX, where successive approximations to the location of the molecule produce a localization that surpasses the typical N-1/2 dependence.

MINFLUX Nanoscopy

To overcome this, maximally informative luminescence excitation or MINFLUX, merges elements of information theory with (i) the single-emitter nature of PALM/STORM (fig. 1A) and (ii) the beam geometries typically used in STED (e.g the so-called “doughnut” beam). This technique (fig. 1 B–E) has shown that the information that each photon contains on the location of its emitter is a flexible quantity and that it can be dramatically increased in imaging and tracking applications (fig. 2B–D). Therefore, a given localization precision can be obtained by using much fewer photons (e.g. 20 times) than in conventional centroid-localization techniques, such as PALM and STORM.

The concept was demonstrated in two and three dimensions by imaging DNA origami constructs and fixed and live cells, reaching 1– 3 nm isotropic resolutions (fig. 2A,D). Tracking of the movement of DNA origami constructs (fig. 2B) resulted in ~2 nm precision and sub‑­millisecond time resolution. Maximal photon economy was sought by tracking of 30S ribosomal subunit proteins in living E. coli fused with the photoconvertible protein mEos2 (fig. 2C). Precisions of ~50 nm using 10 photons per localization were obtained, increasing the temporal resolution and the number of localizations per track hundredfold with respect to state-of-the-art tracking experiments under similar conditions.

Figure 2. (A) MINFLUX nanoscopy by sequentially localizing individually blinking fluorophores; a DNA-origami arrangement is imaged. (B) MINFLUX tracking of a DNA-origami flipping device, a fluorophore is followed with 2.5 nm precision every ~0.5 ms. (C) MINFLUX tracking of the small ribosomal subunit protein in living E. coli. (D) Iterative MINFLUX imaging of nuclear pore complex in 3D (fixed) and 2D (live, with comparison to classical SMLM), and of the PSD-95 protein at a neuronal synapse.