Super‐resolution fluorescence microscopy for investigating bacterial cell biology

Super‐resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single‐molecule resolution on a millisecond time scale. Here we review recent applications of super‐resolution fluorescence microscopy 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 molecular machines in action.


| INTRODUC TI ON
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 (Figure 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, for example, 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 with an emphasis on secretion systems.Special attention will also 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 applicable in other cell types.A widely applicable workflow guiding researchers toward in cellulo MINFLUX imaging at molecular scale has been described previously (Carsten et al., 2022).

| FLUORE SCEN CE SUPER-RE SOLUTION MI CROSCOPY TECHNOLOG IE S
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 system components (Figure 2a; Lin et al., 2022;Nauth et al., 2018).
Two fluorescence microscopy technologies achieving resolutions down to about 20-30 nm have been particularly successful in imaging biological systems.These are, on the one hand, laser scanning-based super-resolution approaches like stimulated emission depletion (STED) nanoscopy (Hell & Wichmann, 1994;Klar & 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).In these cases, fluorescence emissions of single fluorophores are localized below the diffraction limit of light over time (Betzig et al., 2006;Rust et al., 2006;Schnitzbauer et al., 2017).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 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 (Chen et al., 2015;Truckenbrodt et al., 2018).Presently there are only a few published studies in which Expansion Microscopy has been used in bacteria (i.e., Gotz et al., 2020;Kunz et al., 2021).It needs to be carefully evaluated whether individual components of structures of interest, for example, secretion systems, are expanded with the same factor in all dimensions (Buttner et al., 2021).
Detailed overviews about different SRM technologies have been published elsewhere (Bond et al., 2022;Jing et al., 2021;Valli et al., 2021).Advantages and limitations of SIM, STED, SMLM, and MINFLUX are also described in Table 2.
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 & Lehner, 2018).Another aspect to consider, that is particularly relevant when using SRM, 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 this label error, fluorescent proteins, self-labeling enzyme (SLE) tags (SNAP, Halo, CLIP) and nanobodies of around 3 nm in size have successfully been employed (Banaz et al., 2019;Carsten et al., 2022;Liss et al., 2015;Ries et al., 2012; Figure 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).

| APPLI C ATI ON S OF FLUORE SCEN CE S RM IN BAC TERIAL CELL B I OLOGY
The selected papers on SRM and single-molecule tracking (SMT) and their applications in bacteria described in the following sections are also listed and summarized in Table 1.

| CELL WALL AND MEMB R ANE PRO CE SS E S
Deciphering the precise timing and localization of peptidoglycan synthesis in Gram-negative and Gram-positive bacteria is a longstanding 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 Streptococcus pneumoniae was successfully combined with dSTORM (Trouve et al., 2021).
Further, cephalosporin-, metabolic-and hydroxylamine-based fluorescent probes in combination with d-STORM revealed the molecular details of how peptidoglycan dynamics in Staphylococcus aureus are controlled during growth and division (Lund et al., 2022).
CcmA aids coordinate cell shape-determining proteins and peptidoglycan synthesis machinery to organize cell wall synthesis and curvature (Sichel et al., 2022).3D-SIM demonstrated that SepF transiently co-localizes with FtsZ at the septum of the archaeon Methanobrevibacter smithii.It was found that SepF is the relevant FtsZ anchor and possibly primes the future division plane (Pende et al., 2021).The production of extracellular vesicles (EVs) by Grampositive 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).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 (Figure 2g; Morris et al., 2022).

| ADHE S IN S AND B I OFILM
PALM and dSTORM were employed to study the subcellular localization of proteins on the surface and in the cytoplasm of Mycoplasma spp., which are the smallest known bacteria with sizes of 300-800 nm.Because of their tiny genomes and sizes, these human and animal pathogens also represent important model organisms for synthetic biology, and super-resolution microscopy techniques will greatly improve the understanding of their biology (Rideau et al., 2022).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 S. aureus, fibronectin patches of 300 nm or larger and the involvement of two or more bacterial receptors are required (Khateb 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).

| DNA AND RNA ME TABOLIS M
Different SRM and SMT techniques have been employed to visualize activities of bacterial RNA-polymerases (Figure 2f), DNAbinding proteins, the exact spatial organization of DNA replication and transcription, and DNA repair processes in real time (Cassaro & Uphoff, 2022;Stracy et al., 2015;Uphoff et al., 2013).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).Singlemolecule localization microscopy in 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 DNAbinding proteins at the replication fork likely contribute to the Pol IV enrichment (Thrall et al., 2022).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).

| FLUORE SCEN CE S RM IN S ECRE TI ON SYS TEMS
Bacteria have developed different types of systems to secrete molecules into the extracellular space or to translocate them into host cells (Filloux, 2022).In pathogens, 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 (Jenkins et al., 2022;Wagner et al., 2018).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 Yersinia, Pseudomonas, Shigella, and 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) (Figure 1a).The needle complex is a multi-ringed cylindrical structure embedded in the bacterial cell envelope and connected to a 30-to 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 (Deane et al., 2006;Veenendaal et al., 2007).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 ask for an investigation with TA B L E 1 Selected super-resolution microscopy studies in bacteria.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 (Figure 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 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, 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, T3SS and bacterial species (Singh & Kenney, 2021).
Using PALM, the distribution of the ATPase SecA, which is the driving force for protein secretion by the SecYEG translocon, was evaluated in 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 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 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 Acinetobacter baylyi, Acinetobacter baumanii and 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 (Figure 2a; Lin et al., 2022).
SIM was also used to show distributions of the type 9 secretion system components GldL, GldM, GldK, and GldN in 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 SRM study of a T3SS, various T3SS components in Salmonella Typhimurium were labeled with fluorescent antibodies or the photoswitchable fluorophore mEos 3.2 and visualized with 2D and 3D SMLM (Figure 2b; 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 (Figure 2e; Diepold et al., 2017;Prindle et al., 2022).
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(Diepold et al., , 2017;;Zilkenat et al., 2016).Further, due to the estimated resolution of ∼35 nm of the microscopic technique, the needle complex protein PrgH (unified nomenclature: SctD; Wagner & Diepold, 2020) and the tip complex protein SipD (unified nomenclature: SctA) were visualized at a distance of ∼100 nm in individual injectisomes (Figures 1a and 2b).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 Yersinia enterocolitica T3SS pore complex proteins YopB and YopD (uni- With an isotropic localization precision of ~5 nm, these experiments could reproduce the size of the YscL structure determined by Cryo ET to be ~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 ~10 nm apart; Figure 2d; Carsten et al., 2022).
Recently, Halo-tagged S. enterica effectors PipB2, SseF, SseJ, and SifA were visualized using SMT and SMLM.A bidirectional motility of SseF, SifA, and PipB2 along tubular membrane structures was revealed providing novel and comprehensive information about the mobility of Salmonella SPI-2 effectors.Co-motion tracking analysis showed identical movement patterns of PipB2 together with the GFP-labeled host protein LAMP1 (Goser et al., 2023).

| PER S PEC TIVE S
SRM and SMT are rapidly developing technologies that are expected to allow major advances in understanding bacterial cell biology.( 1) Live-STED microscopy has the potential to image fluorescently labeled molecules during complex bacterial processes with up to five 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, for example, 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.For example, the internal ALFA-tag in 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).
Correlative approaches combining the strengths of fluorescence SRM and (cryo) electron microscopy (EM), that is, CLEM, also hold great potential to provide high-resolution data on molecular processes in bacteria and their interaction with host cells.While EM can provide label-free and high-resolution data of essentially all cellular structures, fluorescence SRM adds localization and dynamics of specifically labeled proteins.As this review focuses on fluorescence SRM, we will not discuss the multitude of excellent EM studies, some of which focusing on secretion systems have recently been published (Bergeron & Marlovits, 2022;Kooger et al., 2018;Rapisarda et al., 2018).CLEM just begins to emerge in the context of bacterial infection biology.For example, EM combined with SIM (Figure 2h), super-resolution confocal microscopy or STORM, produced data on Brucella-containing vacuole extensions that colocalize with the endoplasmic reticulum, the cell envelope architecture of Deinococcus radiodurans and Salmonella typhimurium-host interactions, respectively (Sedzicki et al., 2018;Sexton et al., 2022;van Elsland et al., 2018).
It is expected that, for example, the possibilities of the tracking mode in MINFLUX nanoscopy and potentially other upcoming technologies such as MINSTED nanoscopy, which enable the direct observation of movements of individual molecules, will provide completely new insights into complex molecular activities.The spatial and temporal resolutions associated with this technology, for example, visualization of 8-nm-long steps with a millisecond temporal resolution in case of the kinesin motor protein, could not have been dreamed of a while ago (Deguchi et al., 2023;Wolff et al., 2023).In bacterial cells, 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 center of a donut-shaped excitation beam that is scanned through the sample.Moving the excitation beam with an F I G U R E 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 μm width, 2.5 μm length) carrying a T3SS (~40 nm width, ~150 nm length) and a ribosome (~20 nm diameter).The ellipsoids represent the approximate resolution in x, y, and 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, a bacterial ribosome, and the 3D-MINFLUX sphere are shown enlarged in the insets.IM, inner membrane; PG, peptidoglycan layer; OM, outer membrane; HCM, host cell membrane.(b) True-scale representation of common molecules used for fluorescent labeling 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).
fied 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 (Figure 2c; 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 Y. enterocolitica T3SS (Rudolph et al., 2022).MINFLUX nanoscopy allowed to visualize single YopD-ALFA molecules bound by fluorescent nanobodies in Yersinia translocation pores.The localization precision was ~5 nm and thus the size of the pore could be determined to be ~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.
recordings of single-molecule movements may help to eventually produce molecular movies of processes such as protein secretion across membranes, cell wall assembly and/or DNA/RNA synthesis.AUTH O R CO NTR I B UTI O N S Alexander Carsten: Conceptualization; visualization; writingoriginal draft; methodology; formal analysis.Manuel Wolters: Conceptualization; visualization; writing -original draft.Martin Aepfelbacher: Conceptualization; writing -original draft; funding acquisition; visualization; project administration; resources; supervision.
Advantages and limitations of the described SRM approaches.