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).