Fluorescence super-resolution microscopy techniques
Fluorescence SRM techniques can achieve resolutions and localization precisions far below the diffraction limit of light of about 200-300 nm. Among them, structured illumination microscopy (SIM) is a wide field approach that illuminates the sample with a periodical (most of the time sinusoidal patterned) excitation light. While the excitation pattern is shifted and turned with respect to the sample, multiple pictures need to be acquired. Fourier transformation-based algorithms are applied on the acquired frames to produce the final image (Gustafsson, 2000). Linear SIM can improve the lateral resolution to about 120 nm and the axial resolution to about 300 nm (Gustafsson et al., 2008). Linear SIM has been used widely in bacterial cell biology and e.g., allowed an improved visualization of different secretion systems (Nauth et al., 2018, Lin et al., 2022).
Two fluorescence microscopy technologies achieving resolutions down to about 20-30 nm have been particularly successful in imaging diverse biological systems. These are on the one hand laser-scanning based super-resolution approaches like stimulated emission depletion (STED) nanoscopy, which directly records super-resolved images (Hell and Wichmann, 1994, Klar and Hell, 1999) and, on the other hand, wide field based single molecule localization microscopy (SMLM) approaches including (direct) stochastic optical reconstruction microscopy ((d)STORM), photoactivated localization microscopy (PALM) and points accumulation for imaging in nanoscale topography (PAINT). Here, fluorescent emissions of single fluorophores are localized below the diffraction limit over time (Betzig et al., 2006, Rust et al., 2006, Schnitzbauer et al., 2017).
Recently, minimal photon flux (MINFLUX) nanoscopy has been shown to reach resolutions and localization precisions down to 1 nm. MINFLUX nanoscopy is a laser scanning SMLM technique that combines features of STED nanoscopy and SMLM. The precise localization of single fluorophores is achieved by determining their position with respect to the centre of a donut-shaped excitation beam that is scanned through the sample. Moving the excitation beam with an excitation minimum at its center around the target molecule in order to find the minimum excitation point, MINFLUX nanoscopy enables localization of fluorophores using a minimal number of photons (Balzarotti et al., 2017, Schmidt et al., 2021).
An entirely different but still worth mentioning approach to visualize sub-diffraction limited details in biological samples is Expansion Microscopy. Here, the biological samples (e.g., tissues, cells, bacterial infection models) are expanded isotropically with help of a swellable polymer matrix thus physically enlarging the biological structures by a factor of 4.5 to 10, which results in an increased (pseudo-) resolution independent of the microscopy technique used (Truckenbrodt et al., 2018, Chen et al., 2015). Presently there are only a few published studies in which Expansion Microscopy has been used in bacteria (i.e. (Kunz et al., 2021, Gotz et al., 2020). It needs to be carefully evaluated whether individual components of structures of interest, e.g., secretion systems, are expanded with the same factor in all dimensions (Buttner et al., 2021).
A key role for performing successful super-resolution microscopy in microbiology is played by the sample preparation with respect to the fluorescent labels that are available. For fluorescence microscopy in biological specimens, the molecules of interest must be marked with a fluorescent probe. If a tag is introduced into the endogenously- or heterologously expressed molecule of interest, care must be taken to ensure that the tag does not interfere with the function of the molecule. Also, overexpression of heterologous molecules can produce artifacts in biological samples (Bolognesi and Lehner, 2018). Another aspect to consider, especially when using super-resolution microscopy techniques, is that the fluorescent label must be positioned as close as possible to the molecule of interest to take full advantage of the achievable single-digit nanometer resolution. For example, a combination of primary and fluorescently labeled secondary antibodies can already offset the fluorescent label by up to 20 nm relative to the molecule of interest (Fruh et al., 2021). To minimize the label error, fluorescent proteins, self-labeling enzyme (SLE) tags (SNAP, Halo, CLIP) and nanobodies of around 3 nm in size have successfully been employed (Liss et al., 2015, Ries et al., 2012, Carsten et al., 2022, Banaz et al., 2019) (Fig. 1B). The label error in the visualization of proteins can also be reduced to the subnanometer scale by introducing noncanonical amino acids with bioorthogonal (“clickable”) side chains (Mihaila et al., 2022). An overview and more detailed information on labeling approaches and fluorescent probes suitable for super-resolution fluorescence microscopy have been published elsewhere (Liu et al., 2022).