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\begin{document}

\title{Super-resolution fluorescence microscopy for investigating bacterial
macromolecular complexes}



\author[1]{Alexander Carsten}%
\author[1]{Manuel Wolters}%
\author[1]{Martin Aepfelbacher}%
\affil[1]{Universitatsklinikum Hamburg-Eppendorf Institut fur Medizinische Mikrobiologie Virologie und Hygiene}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Super-resolution fluorescence microscopy techniques developed over the
past two decades have pushed the resolution limit for fluorescently
labeled molecules into the nanometer range. These techniques have the
potential to study bacterial macromolecular complexes such as secretion
systems with single-molecule resolution on a millisecond time scale.
Here we review recent applications of super-resolution fluorescence
microscopy in molecular bacteriology with a focus on bacterial secretion
systems. We also describe MINFLUX fluorescence nanoscopy, a relatively
new technique that promises to one day produce molecular movies of
bacterial molecular machines in action.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The dimensions of most bacterial cells are in the micrometer range and
thus come close to the diffraction limit of visible light of about
200-300 nm (Fig. 1A). Therefore, to resolve the details and dynamics of
crucial bacterial activities, novel techniques are necessary. Recent
developments in fluorescence super-resolution microscopy (SRM) are
promising to move closer to the goal of observing the localization and
motion of single proteins in living bacterial cells. SRM techniques have
the potential to revolutionize the understanding of central processes in
bacteria e.g., peptidoglycan assembly, the mode of action of DNA-binding
proteins, the function of macromolecular machines involved in protein
secretion, DNA replication or antibiotic resistance. In this review, we
describe recent work showing the importance of fluorescence SRM for
understanding complex molecular structures and functions in bacteria.
Special attention will be paid to the MINFLUX nanoscopy technique, which
is a promising approach to visualize the molecular motions and dynamic
interactions of single molecules with a spatiotemporal resolution in the
single-digit nanometer and low millisecond range. Applications of SRM
methods including MINFLUX nanoscopy to observe individual components of
a molecular machine, the bacterial type 3 secretion system (T3SS), will
be described in more detail. While the focus of this review is on the
visualization of molecular processes in bacteria, all of the described
microscopy approaches are also applied in other cell types. A widely
applicable workflow guiding researchers towards \emph{in cellulo}MINFLUX
imaging at molecular scale has been described previously (Carsten et
al., 2022).

\textbf{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).

\textbf{Super-resolution fluorescence microscopy in bacterial cell
biology}

Deciphering the precise timing and localization of peptidoglycan
synthesis in Gram-negative and Gram-positive bacteria is a long-standing
goal in molecular bacteriology and may be of medical importance, for
example, in the development of new antibiotics. In one study,
fluorescent D-amino acids were incorporated into bacterial
peptidoglycans and imaged by STED nanoscopy at below 100 nm resolution
(Soderstrom et al., 2020). In another study, bioorthogonal metabolic
labeling of peptidoglycan in \emph{Streptococcus pneumoniae} was
successfully combined with dSTORM (Trouve et al., 2021). Cephalosporin-,
metabolic- and hydroxylamine-based fluorescent probes in combination
with d-STORM revealed the molecular details of how peptidoglycan
dynamics in \emph{Staphylococcus aureus} are controlled during growth
and division (Lund et al., 2022). 3D-SIM demonstrated that SepF
transiently co-localizes with FtsZ at the septum of the
archaeon\emph{Methanobrevibacter smithii} . It was found that SepF is
the relevant FtsZ anchor and possibly primes the future division plane
(Pende et al., 2021). Combinations of STORM or SIM super-resolution
fluorescence microscopy with atomic force microscopy (AFM), named
STORMForce or SIMForce respectively, revealed the intricate
spatiotemporal 3D dynamics of peptidoglycan synthesis during growth and
division in \emph{Bacillus subtilis} (Tank et al., 2021). 3D-SIM helped
identify the molecular function and interaction partners of
a\emph{Helicobacter pylori} bactofilin (CcmA), that plays a crucial role
in generating the distinctive helical shape of the bacteria. CcmA aids
coordinate cell shape-determining proteins and peptidoglycan synthesis
machinery to organize cell wall synthesis and curvature (Sichel et al.,
2022). SIM of fluorescently labeled teixobactin, a recently introduced
antibiotic, allowed visualization of teixobactin interactions and
structural organization in the Gram-positive cell wall. This provided
insights into the mechanism of action of teixobactin and could
contribute to the development of antibiotics with similar properties
(Morris et al., 2022). PALM and dSTORM were employed to study the
subcellular localization of proteins at the surface and in the cytoplasm
of \emph{Mycoplasma} spp., which are the smallest known bacteria with
sizes of 300 to 800 nm. Because of their tiny genomes and sizes, these
human and animal pathogens represent important model organisms for
synthetic biology, and super-resolution microscopy techniques will
greatly improve the understanding of their biology (Rideau et al.,
2022). \emph{S. aureus} fibronectin binding receptor organization and
adhesion to patches of fibronectin of systematically varied size
(100-1000 nm) was investigated using DNA-PAINT. The results suggest that
for strong adhesion of \emph{S. aureus} fibronectin patches of 300 nm or
larger and the involvement of two or more receptors are required (Khateb
et al., 2022). Different SRM and single-molecule tracking (SMT)
techniques have been employed to visualize activities of bacterial
RNA-polymerases, DNA-binding proteins, the exact spatial organization of
DNA replication and transcription, and DNA repair processes in real time
(Cassaro and Uphoff, 2022, Uphoff et al., 2013, Stracy et al., 2015).
DNA methyltransferases (MTases) have central functions in restriction
modification systems, cell cycle regulation, and the control of gene
expression. SMLM and SMT of the MTase DnmA revealed its preferential
localization to the nucleoid and the replisome region as well as its
intracellular dynamics (Fernandez et al., 2023). Single-molecule
localization microscopy in \emph{E. coli} cells was employed to
investigate enrichment of the translesion synthesis polymerase Pol IV at
stalled replication forks in the presence of DNA damage. It turned out
that alterations in the dynamics of single-stranded DNA-binding proteins
at the replication fork likely contribute to the Pol IV enrichment
(Thrall et al., 2022). Microbial biofilms play an important role in
both, human infections and biotechnological processes. SR-SIM revealed
the distribution of extracellular polysaccharides and DNA in resident
biofilms (Wang et al., 2022). The production of extracellular vesicles
(EVs) by Gram-positive bacteria was investigated by STORM. The results
showed that EV can be formed by membrane blebbing and explosive cell
lysis, suggesting that cell wall degradation plays a significant role in
their biogenesis (Jeong et al., 2022). Listeriolysin S (LLS) a
thiazole/oxazole-modified microcin from Listeria monocytogenes was
localized to the bacterial cell membrane and cytoplasm using
PAINT/dSTORM. This contributed to the understanding of LLS as a
contact-dependent bacteriocin (Meza-Torres et al., 2021).

\textbf{Super-resolution in secretion systems:}

Pathogenic bacteria have developed different types of systems to secrete
molecules into the extracellular space or to translocate them into host
cells (Filloux, 2022). The secreted or translocated molecules often
serve as virulence factors, for example, to compete with other bacterial
species, to invade mammalian host cells or to evade the host immune
system (Galan, 2009, Le et al., 2021). Bacterial secretion systems can
consist of a single protein or more than 20 different proteins that
combine to form complex macromolecular machines, as in the case of the
T3SS (Wagner et al., 2018, Jenkins et al., 2022). Most of our knowledge
about the structure and function of secretion systems comes from
genetic, biochemical and structural biological as well as electron
microscopic studies (Berger et al., 2021, Hu et al., 2018, Lunelli et
al., 2020, Marlovits et al., 2004, Worrall et al., 2016).

T3SSs, also called injectisomes, are found in numerous pathogens
including \emph{Yersinia, Pseudomonas, Shigella} and
\emph{Salmonella,}and translocate effector proteins into eukaryotic host
cells (Wagner et al., 2018). Although the T3SSs of the various pathogens
are highly conserved, the effectors injected by them differ
significantly in structure and function and can manipulate a variety of
cellular processes. This ultimately determines the interaction of each
pathogen with the host and the outcome of the infection (Galan, 2009).
T3SSs have a width of ~40 nm and a length of ~150 nm and consist of
both, stable components (needle complex, export apparatus) and
transiently associated components (sorting platform, tip complex, pore
complex) (Fig. 1A). The needle complex is a multi-ringed cylindrical
structure embedded in the bacterial cell envelope and connected to a
30-70 nm long needle filament that points into the extracellular space.
Together with the export apparatus, it forms a channel through which the
structural and effector proteins of the system are transported (Miletic
et al., 2021). At the distal end of the needle is the tip complex, which
is involved in host cell recognition, activation of secretion, and
regulation of the assembly of the pore complex upon host cell contact
(Veenendaal et al., 2007, Deane et al., 2006). Lastly, several
cytoplasmic proteins form a heteromultimeric complex known as the
sorting platform, which is involved in the selection and sorting of
proteins destined for secretion and translocation (Lara-Tejero et al.,
2011).

Even though the complexity and presumed molecular dynamics of bacterial
secretion systems asks for an investigation with fluorescence
microscopic methods, the resolution of these methods has long been
insufficient for this purpose. However, novel super-resolution
microscopy techniques developed in the last two decades pushed the
resolution limit for fluorescently labeled molecules into the nanometer
range (Fig. 1A). Such techniques now also allow the study of bacterial
secretion systems at much higher resolution (Sahl et al., 2017). Some
relevant reports on this subject are presented below.

In an early systematic analysis, the suitability of the SLEs HaloTag and
SNAP-tag for super resolution microscopy of different \emph{Salmonella
enterica} secretion system subunits was tested. The tags were
genetically linked to subunits of a type I secretion system (T1SS) and
T3SS (and to the flagellar rotor and a transcription factor), the tagged
proteins were labeled by cell-permeable dyes and analyzed by dSTORM and
SMT. This allowed determination of the number, subcellular localization
and dynamics of protein complexes in living bacteria (Barlag et al.,
2016). In a follow-up study, \emph{S. enterica} T3SS effectors fused to
SLEs were found to be translocated into host cells where they remained
functional and were properly located (Goser et al., 2019). However, it
is important to consider that the SLEs may be secreted with greatly
varying efficiency depending on the type of tagged protein and T3SS
involved species (Singh and Kenney, 2021).

Using PALM, the intrabacterial distribution of the ATPase SecA, which is
the driving force for protein secretion by the SecYEG translocon, was
evaluated in \emph{E. coli} . SecA was mostly localized as a homodimer
along the cytoplasmic membrane and diffused along it in three different
diffusion rate populations as found by SMT (Seinen et al., 2021).

The type I secretion system substrate hemolysin A (HlyA) was imaged on
the surface of \emph{E. coli} using SIM. In contrast to other bacterial
secretion systems, HlyA showed no polarization on the cell surface and
its distribution was not influenced by cell growth and division cycle
(Beer et al., 2022).

Using SIM, the sfGFP-labeled inner membrane component VirB6 of
the\emph{Agrobacterium tumefaciens} type 4 secretion system was found to
preferentially localize to the cell poles (Mary et al., 2018). SIM was
also employed to subcellularly localize type 6 secretion system (T6SS)
assembly in response to cell-cell contact in \emph{Acinetobacter baylyi}
, \emph{Acinetobacter baumanii} and \emph{Burkholderia thailandensis} .
Employing sfGFP-tagged sheath protein TssB, the polymerization rate and
time as well as the disassembly of the contractile sheaths could be
visualized. The individual T6SSs were mainly assembled at the site of
contact with neighboring bacterial cells, whereby periplasmic proteins
as well as the outer membrane protein OmpA mediated this localization
(Lin et al., 2022).

SIM was also used to show distributions of the type 9 secretion system
components GldL, GldM, GldK and GldN in \emph{Flavobacterium johnsoniae}
. All of these proteins seem to be distributed in foci along the
bacterial circumference. GldK and GldN, which are part of the GldKN
complex, showed in average less foci per cell than GldL and GldM,
suggesting two subpopulations of GldLM complexes, one free and one
associated with GldKN rings (Vincent et al., 2022).

In a comprehensive super-resolution microscopy study of a T3SS, various
T3SS components in \emph{Salmonella Typhimurium} were labeled with
fluorescent antibodies or the photoswitchable fluorophore mEos 3.2 and
visualized with 2D and 3D SMLM (Zhang et al., 2017). Thereby,
subcellular distributions and rough numbers of needle complexes, sorting
platform components, tip complex and an effector could be determined.
Needle complexes including export apparatus were almost exclusively
located at the bacterial plasma membrane, whereas a considerable
fraction of sorting platform components was also in the cytoplasm,
suggesting that sorting platforms are transiently and dynamically
associated with the needle complexes (Prindle et al., 2022) (Diepold et
al., 2017). The relative stochiometries of components of the sorting
platform and export apparatus could be determined, confirming previous
observations using other techniques (Diepold et al., 2015, Zilkenat et
al., 2016, Diepold et al., 2017). Further, due to the estimated
resolution of ~ 35nm of the microscopic technique, the needle complex
protein PrgH (unified nomenclature: SctD) and the tip complex protein
SipD (unified nomenclature: SctA) could be visualized at a distance of ~
100 nm in individual injectisomes (Fig. 1A). It was also found that
needle complexes are essential for the assembly of sorting platforms and
that the effector SopB is mainly found in clusters in the cytoplasm and
this does not depend on the parallel presence of needle complexes or
sorting platforms (Zhang et al., 2017).

STED microscopy and SIM were used to visualize the \emph{Yersinia
enterocolitica} T3SS pore complex proteins YopB and YopD (unified
nomenclature: SctE and SctB, respectively) in infected host cells. Per
bacterium ~ 30 what appeared to be single translocation pores at the tip
of injectisome needles formed upon host cell contact. The two pore
proteins YopB and YopD on one side and the needle complex/basal body
protein component YscD (unified nomenclature: SctD) on the other side of
single injectisomes could be resolved at a mean distance of ~ 109 nm.
Further, 3D-STED microscopy allowed to localize YopB in translocation
pores which formed in a peculiar pre-vacuolar compartment in the
infected cells (Nauth et al., 2018). To minimize the label error for
MINFLUX nanoscopy, an ALFA-tag was introduced into YopD's extracellular
domain (giving rise to YopD-ALFA). It was demonstrated that the ALFA-tag
did not compromise the central functions of YopD during protein
translocation by the \emph{Y. enterocolitica} T3SS (Rudolph et al.,
2022). MINFLUX nanoscopy allowed to visualize single YopD-ALFA molecules
bound by fluorescent nanobodies in \emph{Yersinia} translocation pores.
The localization precision was \textasciitilde{} 5 nm and thus the size
of the pore could be determined to be \textasciitilde{} 18 nm. Further,
clusters consisting of 12 molecules of sorting platform protein YscL
(unified nomenclature: SctL) fused with a HaloTag were recorded by 2D
and 3D MINFLUX microscopy. With an isotropic localization precision of
\textasciitilde{} 5 nm, these experiments could reproduce the size of
the YscL structure determined by Cryo ET to be \textasciitilde{}16 nm in
diameter (Carsten et al., 2022) (Berger et al., 2021). 3D MINFLUX
experiments performed in whole bacteria showed that the YscL complexes
localized almost exclusively at the plasma membrane and at very low
distances to each other (down to \textasciitilde{}10 nm apart) (Carsten
et al., 2022).

SMT and SMLM in live \emph{Yersinia enterocolitica} revealed distinct
diffusive states of the eYFP, eGFP and PAmCherry labelled sorting
platform components YscQ, YscL and YscN (unified nomenclature: SctQ,
SctL and SctN) and suggested that they form distinct cytosolic complexes
before binding to the needle complex (Rocha et al., 2018, Prindle et
al., 2022, Diepold et al., 2015). SMT of eGFP labelled YscD (unified
nomenclature: SctD) showed partial disassembly of the T3SS basal body
component at low external pH (Wimmi et al., 2021).

PALM was used to show that the Salmonella pathogenicity island-2 (SPI-2)
signaling proteins SsrA/B labeled with PAmCherry were induced under low
pH conditions. Furthermore, SMT identified pH-dependent DNA binding of
SsrB (Liew et al., 2019).

Recently Halo-tagged S. \emph{enterica} effectors PipB2, SseF, SseJ and
SifA were visualized using SMT and SMLM. A bidirectional motility along
tubular membrane structures of SseF, SifA and PipB2 was revealed
providing novel and comprehensive information about the mobility
of\emph{Salmonella} SPI-2 effectors. Co-motion tracking analysis showed
identical movement patterns of PipB2 together with the GFP labelled host
protein LAMP1 (Goser et al., 2023).

\textbf{Perspectives}

SRM and SMT are rapidly developing technologies that are expected to
allow major advances in understanding bacterial cell biology in the
future. 1) Live-STED microscopy has the potential to image fluorescently
labeled molecules during complex bacterial processes with up to 5-times
better resolution than conventional live cell microscopy (Stockhammer,
2020). 2) New developments and optimizations of fluorescent probes
(e.g., concerning on-/off-switching properties, brightness), especially
for MINFLUX nanoscopy, will increase the versatility and flexibility of
the method, e.g., in multicolor and live imaging as well as SMT (Remmel
et al., 2023). 3) Combining MINFLUX nanoscopy with a PAINT-labeling
approach may enable parallel imaging of three or more molecules of
interest (Ostersehlt et al., 2022). 4) Advanced labeling approaches of
molecules may also open up unforeseen methodological options. E.g., the
internal ALFA tag in \emph{Y. enterocolitica} YopD (see above) can be
bound by fluorescent nanobodies added extracellularly during a bacterial
cell infection, allowing the kinetics of T3SS pore assembly and
disassembly to be visualized in living bacteria and host cells (Rudolph
et al., 2022).

Finally, the ability of MINFLUX tracking to directly observe the
movements of single molecules promises new insights into the
structure-function relationship of complex molecular processes in living
bacterial and host cells. Particularly the spatiotemporal resolution
that could be achieved with this technology was something that could not
have been dreamed of not so long ago (Deguchi et al., 2023, Wolff et
al., 2023).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1: Approximate resolution of different fluorescence
microscopy methods using the example of a Gram-negative bacterial cell
with a ribosome and a type 3 secretion system (T3SS).} (A) Schematic
true-scale representation of a Gram-negative rod-shaped bacterium (1 \selectlanguage{greek}µ\selectlanguage{english}m
width, 2.5 \selectlanguage{greek}µ\selectlanguage{english}m length) carrying a T3SS (\textasciitilde{}40 nm width,
\textasciitilde{}150 nm length) and a ribosome (\textasciitilde{}20 nm
diameter). The ellipsoids represent the approximate resolution
in\emph{x} , \emph{y} and \emph{z} for confocal microscopy, structured
illumination microscopy (SIM), stimulated emission depletion (STED)
nanoscopy, single molecule localization microscopy (SMLM) and minimal
photon flux (MINFLUX) nanoscopy. Approximate resolution values in 2D
(blue) and 3D (green) mode of the indicated microscopy methods are
shown. The T3SS, its translocon and YscL complex and the 3D-MINFLUX
ellipsoid are shown enlarged in the insets. IM, inner membrane; PG,
peptidoglycan layer; OM, outer membrane; HCM, host cell membrane. (B)
True-scale representation of various molecules used for fluorescent
labelling of target structures in relation to the 3D-MINFLUX nanoscopy
resolution. Structures were obtained from the Protein Data Bank (PDB):
1igt (IgG), 5dty (green fluorescent protein), 6i2g (NbALFA bound to
ALFA-tag), 6y7a (Halo-tag) and 7k00 (bacterial ribosome).

Table 1: Selected super resolution microscopy studies in bacteria\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Technique(s)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Biological target}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Labelling method}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Live/fixed}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Organism}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Reference}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{cell wall}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3D STORM (dual-color imaging)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Membrane N-acetylglucosamineprotein A enterotoxin B\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
nile red WGA (AF647) Indirect immunofluorescence staining (AF647)
Indirect immunofluorescence staining (AF647)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Staphylococcus aureus}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1186/s12915-022-01472-3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
dSTORM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
peptidoglycan\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
azido-D-Ala-D-Ala probe linked to AF647\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Streptococcus pneumoniae}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1016/j.xpro.2021.101006\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
dSTORM/ 3D-SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
penicillin-binding proteins glycan strands\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
CephCy5 azide N-acetylmuramic acid (AzNAM)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Staphylococcus aureus}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1021/acschembio.2c00741\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
STED/SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
peptidoglycan\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fluorescein/OregonGreen488/ TAMARA-labelled d-amino acids\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Staphylococcus aureus} \emph{Streptococcus mutans} \emph{Bacillus
subtilis} \emph{Escherichia coli} \emph{Zymomonas mobilis}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1099/mic.0.000996\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3D-SIM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Z-ring (SepF and FtsZ)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
indirect immunofluorescence staining (AF555 and AF488)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Methanobrevibacter smithii}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1038/s41467-021-23099-8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3D-SIM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
bactofilin (CcmA)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
HaloTag (JF549)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Helicobacter pylori}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.7554/eLife.80111\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
SIM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
teixobactin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fluorescent teixobactin analogues (fluorescein, Cy3 and Cy5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
B\emph{acillus subtilis}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1039/d2sc01388f\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{DNA}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
DNA methyltransferase (DnmA)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PAmCherry\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Bacillus subtilis}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1128/mbio.03185-22\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
translesion synthesis polymerases (Pol IV) single-stranded DNA-binding
protein (SSB)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PAmCherry, mYPet\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Escherichia coli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1073/pnas.2208875119\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (imaging/ SMT) PAINT\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
two-component system SsrB, SsrA membranes\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PAmCherry Nile red\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Salmonella enterica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.7554/eLife.45311\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (imaging/ SMT) 3D-SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
RNA polymerase Nucleoid\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PAmCherry HU-mCherry\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Escherichia coli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1073/pnas.1507592112\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (imaging/ SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
DNA polymerase I ligase\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PamCherry\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Escherichia coli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1073/pnas.1301804110\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
extracellular polysaccharides eDNA\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fluorescein isothiocyanate-labeled concanavalin A propidium iodide\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Bacillus subtilis} \emph{Brevundimonas diminuta}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1007/s11356-022-21645-5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{secretion systems}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2D/3D-MINFLUX/ STORM/ STED\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T3SS translocon (YopD) T3SS basal body (YscD)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
ALFA-Tag/NbALFA based labelling (AbberiorSTAR635P, AF647) Halo-Tag
(AF647)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Yersinia enterocolitica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1088/2050-6120/aca880\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PALM (SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
T3SS basal body (YscD)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PAmCherry\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
live\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Yersinia enerocolitica}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1038/s41467-021-21863-4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM (imaging/ SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
SecYEG translocon ATPase (SecA)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Ypet/mEos3.2\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/ live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Escherichia coli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1038/s41598-021-81081-2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2D/3D-PALM (dual-color imaging)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T3SS PrgH, SpaO, OrgA, OrgB, InvC, InvI, InvA SipD\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
mEos3.2 indirect immunofluorescence staining (flag-tag, AF647)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Salmonella enterica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1073/pnas.1705823114\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
dSTORM (dual-color imaging/ SMT)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
flagellar basal body complex (FliN) T1SS ATPase subunit (SiiF) T1SS
Secretin (SiiC) T3SS export aparatus (SpaS) T3SS ATPase (InvC)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Halo (TMR, Atto6) SNAP (TMR-Star)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/ live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Salmonella enterica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1038/srep31601\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2D/3D-STED (dual-color imaging)/ SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T3SS translocon (YopB, YopD, LcrV) T3SS basal body YscD\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
indirect immunofluoescence staining (AbberiorSTARRed and AF594)
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Yersinia enterocolitica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1371/journal.ppat.1007527\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2D/3D-STED\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T3SS translocon (YopD)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
ALFA-Tag/NbALFA based labelling (AF647, AbberiorSTAR635P)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Yersinia enterocolitica}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1371/journal.ppat.1010251\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2D/3D-SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T6SS proteins (TssB,TssA) \emph{T}ype \emph{s}ix secretion dynamic
\emph{l}ocalization protein A (TslA)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
sfGFP/\emph{mCherry2/mNeonGreen} indirect immunofluorescence staining
(AF594)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed/live\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Acinetobacter spp.} \emph{Burkholderia thailandensis}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{10.15252/embj.2021108595}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
SIM\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
T9SS components (GldL and GldM) Adhesin (SprB)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
indirect immunofluoescence staining (AF488, AF561)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Flavobacterium johnsoniae}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1371/journal.pbio.3001443\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
SIM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
T4SS protein (VirB6a)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
sfGFP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Agrobacterium tumefaciens}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1074/jbc.RA118.002751\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{adhesins, virulence factors}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DNA-PAINT\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fibronectin binding proteins (FnBPA and FnBPB)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Fn conjugated to a DNA-PAINT docking sequence, Cy3B-labeled
complementary imager strands\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Staphylococcus aureus}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1021/acsnano.2c00630\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PAINT/ dSTORM (dual-color imaging)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
listeriolysin S membrane\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Direct immunofluoescence staining (HA-tag, AF647) Nile red\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Listeria monocytogenes}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1073/pnas.2108155118\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PALM, dSTORM; dSTORM/PALM (dual-color imaging)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
F-type ATPase (F\textsubscript{1-like}-X\textsubscript{0}) protease
(MIP\textsubscript{0582})\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
mEos3.2 indirect immunofluoescence staining (HA-tag, AF 647)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Mycoplasma spp.}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
10.1128/spectrum.00645-22\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
SIM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hemolysin A (HlyA)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
eGFP/ Indirect immunofluoescence staining (Cy3 or
AbberiorStar635P)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
fixed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Escherichia coli}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10.1128/AEM.01896-21\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

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Acknowledgements:

We thank Jost Enninga for helpful discussions and Antonio Virgilio
Failla for critical reading of the manuscript. The selected papers on
SRM/SMT and their applications in bacteria that we mention and cite in
this article do not claim to be complete. We apologize to all colleagues
whose papers we have not cited here due to space limitations or because
we simply did not discover them during our extensive literature
searches.

Funding:

This study was supported by a grant from the Joachim Herz Foundation to
Alexander Carsten and by the DFG-funded RTG 2771 Humans and Microbes.
Acquisition of the MINFLUX microscope was made possible through funding
from the European Regional Development Fund (ERDF) under the Operational
Programme Hamburg ERDF 2014-2020, REACT-EU, awarded by the Hamburgische
Investitions- und F\selectlanguage{ngerman}örderbank (grant number 51164232).

Conflict of interest:

The authors declare no conflict of interest.

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