Expansion microscopy of apicomplexan parasites

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting‐edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure‐expansion microscopy (U‐ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U‐ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

parasites were chosen as the focus of this review as they comprise much of apicomplexan diversity, they all represent significant causes of human disease, and they are the apicomplexan parasites whose cell biology is the most well studied.
Investigation of the cell biology of apicomplexan parasites has been vital for developing our understanding of the most fundamental processes in these parasites, along with identifying novel targets for drugs and defining resistance mechanisms for existing drugs.
Despite this, many aspects of the cell biology of apicomplexan parasites are extremely difficult to interrogate because of the small size of these parasites.For example, the nucleus of a Plasmodium merozoite can be <1 μm in diameter (Rudlaff et al., 2020), which makes resolving sub-nuclear biology difficult or impossible using conventional light microscopy techniques.Further, the rhoptries, secretory organelles that coordinate host cell invasion, are as narrow as ~30 nm in Toxoplasma (Mageswaran et al., 2021), making it impossible to individually distinguish each of the 8-12 rhoptries in a tachyzoite without using electron microscopy.Historically, the small size of apicomplexan parasites has been a driver for innovation in the biological imaging of parasites, with an impressive array of super-resolution light microscopy techniques including single-molecule localization microscopy (SMLM) (Sanchez et al., 2019), stimulated emission depletion microscopy (STED) (Mehnert et al., 2019;Schloetel et al., 2019), Airyscan (Liffner et al., 2022;Rudlaff et al., 2019) and structural illumination microscopy (SIM) (Gras et al., 2019;Riglar et al., 2011Riglar et al., , 2013) ) used to interrogate parasite biology.Further, electron microscopy or tomography (Cyrklaff et al., 2017;Ferreira et al., 2023;Hanssen et al., 2013;Mageswaran et al., 2021), X-ray microscopy (Hanssen et al., 2011) and atomic force microscopy (Considine et al., 2000;Millholland et al., 2011) have been widely applied to Apicomplexa.The downside of these techniques, however, is that they often involve expensive infrastructure, non-commercial reagents, specialized expertise to perform or some combination of these three.

| E XPAN S I ON MI CROSCOPY
While tremendous advancements have been made across multiple imaging modalities in recent decades, this has largely involved advancements in technology or microscopy infrastructure.In 2015, however, a sample preparation method was developed called expansion microscopy (ExM), which involves physically enlarging the sample rather than employing new microscopy technologies (Chen et al., 2015).Since the first ExM protocol was published, dozens of derivatives have been developed, but all are similar in at least four particular ways (Wassie et al., 2019).Firstly, a biological specimen is covalently anchored to a polymer solution (Wassie et al., 2019).This polymer solution is then turned into a hydrogel that will expand when deionized water is added or shrink when a solution of high osmolarity is added (Wassie et al., 2019).The sample is processed in a way that physically allows it to expand, such as denaturation or proteinase K digestion (Wassie et al., 2019).Finally, the sample will be stained with some kind of fluorescent markers, such as fluorophoreconjugated antibodies.Overall, these steps combine to physically expand your biological specimen between 4-fold and >20-fold depending on the protocol used (Gambarotto et al., 2019;Klimas et al., 2023;Louvel et al., 2022;Truckenbrodt et al., 2018;Wassie et al., 2019).While ExM represented a tremendous technical and conceptual breakthrough in the way we think about and approach microscopy experiments, early ExM protocols were not widely adopted by the parasitology community.

| Ultrastructure expansion microscopy (U-ExM)
Expansion microscopy became widely adopted within the parasitology community following the first published examples of ExM on apicomplexan parasites, with T. gondii being expanded using a protocol named ultrastructure-expansion microscopy (U-ExM) (Tosetti et al., 2020).U-ExM results in ~4.5-fold expansion of the sample and was specifically developed with isotropic ultrastructural preservation of both cells and organelles in mind (Gambarotto et al., 2019).
While not very technically different from other ExM protocols, U-ExM exclusively uses commonly commercially available reagents and is compatible with almost all reagents regularly used in fluorescence microscopy, making it easily deployable for parasite labs around the world.Since the first publication of U-ExM on Toxoplasma, a further 28 studies have been published on apicomplexan parasites using this technique in less than 3 years (see Tables 1-3 for summary of all U-ExM studies in Apicomplexa).These studies have spanned five species (Toxoplasma gondii, Cryptosporidium parvum, P. falciparum, P. yoelii and P. berghei), eight lifecycle stages and localized over 80 proteins using either protein-specific antibodies or tagged proteins (Tables 1-3 and Figure 1).A single study using an ExM technique other than U-ExM has been published on apicomplexan parasites (Pavlou et al., 2020), but to date, U-ExM has been the favoured technique for groups studying Apicomplexa.This review will briefly introduce the lifecycles of Plasmodium, Toxoplasma and Cryptosporidium, and summarize how U-ExM has changed our understanding of the cell biology of these parasites.

| Plasmodium
While there are many species of Plasmodium, this review will predominantly focus on those that infect humans (especially P. falciparum), with some information inferred from studies in rodent-infecting Plasmodium spp.(P.berghei and P. yoelii).Plasmodium parasites in the sporozoite stage of the lifecycle are transmitted from their mosquito hosts to their mammalian hosts during a blood meal (Frischknecht & Matuschewski, 2017;Shute, 1943).Sporozoites migrate from the skin to the liver, where they will invade a hepatocyte and replicate asexually to form a hepatic schizont that contains thousands of merozoites (Amino et al., 2006;Matsuoka et al., 2002;Prudêncio et al., 2006).Merozoites will egress from the hepatic schizont into the TA B L E 1 Summary of all proteins localized by U-ExM using antibodies or tagged cell lines in Plasmodium.

Organism
Lifecycle stage
Male and female gametocytes are transferred to a mosquito through a subsequent blood meal, where they will egress from the red blood cell and undergo gametogenesis (Dash et al., 2022).Each male gametocyte undergoes three rounds of mitosis to form eight gametes, which will fertilize a female gamete to form a diploid zygote (Guttery et al., 2015).The zygote will soon undergo meiosis and form a tetraploid ookinete (Guttery et al., 2015), which will invade the mosquito midgut wall and form an oocyst (Maccallum, 1897;Vinetz, 2005).
Inside this oocyst, the parasite replicates asexually to form tens of thousands of sporozoites (Frischknecht & Matuschewski, 2017;Ross, 1898).Once fully mature, sporozoites will rupture from the oocyst into the haemocoel of the mosquito before migrating to and invading the mosquito salivary gland (Grassi, 1901; Mueller  , 2010).Sporozoites will then reside in the salivary gland until the mosquito takes another blood meal.
Across their lifecycle, malaria parasites perform at least four distinct host cell invasion and egress events, transmission between hosts twice, and undergo asexual replication four times and sexual replication once.These major events are hubs for discovery and investigation of Plasmodium-specific, apicomplexan-specific and broad eukaryote cell biology.

| U-ExM in Plasmodium
The first study using U-ExM in Plasmodium expanded gametocytes, ookinetes and schizonts of P. berghei (Bertiaux et al., 2021).While the focus of this study was on the microtubules of the parasites, the authors importantly stained parasites with a fluorophore-conjugated N-hydroxysuccinimide (NHS) ester (Bertiaux et al., 2021).NHS ester binds free amine groups and so when parasites stained with NHS ester conjugated to a fluorophore (hereafter simply referred to as NHS ester) are visualized (Nanda & Lorsch, 2014), the outcome is essentially a protein density map of the parasite.The use of NHS ester, therefore, allows the visualization of a number of protein-dense structures within the parasite (similar to electron microscopy), without the need for protein-specific antibodies.
Across various stages of the Plasmodium lifecycle, NHS ester has been used to visualize the rhoptries, some cytoskeletal elements, basal complex, cytostomes, apical polar ring (APR) and nuclear microtubule organizing centre (MTOC) (Bertiaux et al., 2021;Liffner & Absalon, 2021;Liffner et al., 2023;Rashpa & Brochet, 2022;Rashpa et al., 2023;Simon et al., 2021;Yang, Cai, et al., 2023); highlighting it is a powerful tool for generally observing changes within the parasite.Following the publication of the first study using U-ExM in Plasmodium, further significant developments were made when the protocol was optimized for the most common Plasmodium fixative, paraformaldehyde (Simon et al., 2021), followed by the first U-ExM experiments using P. falciparum (Simon et al., 2021).Subsequently, modifications to the U-ExM protocol were made to streamline the process for harvesting malaria parasites at multiple timepoints/ lifecycle stages (Liffner & Absalon, 2021), along with the testing of a wide number of marker antibodies and chemical dyes (Liffner  et al., 2023).To date, nearly all lifecycle Plasmodium lifecycle stages have been expanded using U-ExM (Figure 2).These experiments have spanned P. falciparum, P. yoelii and P. berghei and localized dozens of proteins (Table 1).Below, we provide a brief summary of some of the cell biology findings in Plasmodium that have been enabled by U-ExM.

| Plasmodium microtubules redefined
Since the first application of U-ExM to malaria parasites (Bertiaux et al., 2021), parasite microtubules have been overwhelmingly the most studied aspect of parasite cell biology.The most significant finding of the first U-ExM study in Plasmodium was the conservation of a conoid during the ookinete stage of the parasite lifecycle (Bertiaux et al., 2021).The conoid is a tubulin-rich structure at the apical end of the motile/invasive stages of many apicomplexans (Dos Santos Pacheco et al., 2020).The presence or absence of the conoid in apicomplexan parasites was long thought to be a defining feature of two classes of parasites, the Conoidasida (conoid present) and Aconoidasida (conoid lacking) (Dos Santos Pacheco et al., 2020).
Plasmodium was considered to be part of the Aconoidasida, but using U-ExM, the authors showed not only that P. berghei ookinetes possess a conoid-like structure at their apical end, but also that Plasmodium homologues of canonical conoid markers localize to this structure (Bertiaux et al., 2021).In addition to this foundational observation of conoid conservation, the authors also visualized the microtubules of activated male gametocytes and gametes and merozoites (Bertiaux et al., 2021).They showed that in each of these stages, along with ookinetes, the subpellicular microtubules (SPMTs), cytosolic microtubules thought to define parasite shape, are stabilized by polyglutamylation and that this polyglutamylation can be recognized using anti-PolyE antibodies (Bertiaux et al., 2021).
The ability to specifically detect SPMTs has subsequently been utilized in other studies to distinguish intranuclear microtubules from SPMTs (Liffner et al., 2023).
Once the microtubules of gametocytes and ookinetes had been defined using U-ExM, subsequent studies have built on these observations by characterizing microtubules using U-ExM.A recent study using U-ExM in P. yoelii showed that Apical Polar Ring protein 2 (APR2) is involved with stabilizing ookinete SPMTs and anchoring them to the APR, with APR2 knockout parasites showing microtubule accumulations at their basal end (Qian, Wang, Guan, et al., 2022).
Other studies have shown that F-box protein 1 (FBXO1) (Rashpa et al., 2023) along with kinesins 13 and 20 (Zeeshan et al., 2022)  it is now being leveraged as a platform to identify and investigate cytoskeletal proteins and phenotypes.

| Description of a bipartite nuclear microtubule organizing centre (MTOC)
Apicomplexa undergo closed mitosis (Gerald et al., 2011;Voß, Klaus, Guizetti, et al., 2023), with the nuclear envelope remaining intact during mitosis.As a consequence of this, the microtubules that coordinate mitosis are formed from a microtubule organizing centre (MTOC) embedded within the nuclear envelope.Similar to the use of NHS ester to identify the conoid of ookinetes, the coupling of U-ExM and NHS ester was used to visualize the bipartite nuclear MTOC of Plasmodium (Liffner & Absalon, 2021;Simon et al., 2021).Using U-ExM along with other advanced imaging modalities, it has recently been shown in asexual blood-stage parasites (Liffner & Absalon, 2021;Liffner et al., 2023;Simon et al., 2021) and gametocytes (Li, Shami, et al., 2022;Rashpa & Brochet, 2022) that the nuclear MTOC comprised an intranuclear region and a cytoplasmic extension(s).Until the advent of U-ExM, the only marker of the nuclear MTOC for light microscopy was anti-centrin antibodies.
Anti-centrin antibodies, and likely all four centrin proteins encoded in Plasmodium, however, have recently been shown to localize exclusively to the cytoplasmic extension of the bipartite nuclear MTOC (Liffner et al., 2023;Simon et al., 2021;Voß, Klaus, Lichti, et al., 2023).
One finding of the study that first characterized the nuclear MTOC of Plasmodium by U-ExM was that microtubules could be observed before centrin (Simon et al., 2021).This suggested that centrin was not required for nuclear microtubule polymerization, but raised the question of when the nuclear MTOC was formed.A study that tracked a number of P. falciparum markers across the asexual blood stage of the lifecycle, which used NHS ester and centrin as markers for the MTOC, subsequently showed that the intranuclear portion of the MTOC was built before the cytoplasmic extensions (Liffner et al., 2023), and that only the intranuclear portion is necessary for the nucleation of intranuclear microtubules.Additionally, it was shown that the MTOC is not observable in fully formed merozoites or newly invaded ring-stage parasites (Liffner et al., 2023), suggesting that the MTOC disassembles prior to egress and forms de novo following reinvasion.
While the cytoplasmic portion of the nuclear MTOC is not required for making the microtubules that coordinate mitosis, it has subsequently been shown that they nucleate cytoplasmic microtubules (Li, Shami, et al., 2022;Liffner et al., 2023;Rashpa & Brochet, 2022).
In asexual blood stages, the SPMTs that define merozoites seem to emerge from the cytoplasmic extensions of the MTOC (Liffner et al., 2023).Further, it appears that the SPMTs of gametocytes and the axonemes of gametes emerge from the MTOC cytosolic extensions (Li, Shami, et al., 2022, Rashpa & Brochet, 2022).Collectively, this highlights the nuclear MTOC as both a bipartite and bifunctional structure, with the intranuclear portion nucleating the microtubules that coordinate mitosis and the cytoplasmic extensions nucleating SPMTs.

| The nuclear MTOC as a hub for cellular organization
In addition to its potential role in nucleating SPMTs, U-ExM studies have suggested that the cytoplasmic extensions of the nuclear MTOC may act as a global hub for cellular organization (Liffner et al., 2023;Rashpa et al., 2023).Using U-ExM, the cytoplasmic extensions of the MTOC were found in a small region between the nuclear envelope and parasite plasma membrane (PPM) (Liffner et al., 2023).This led to the suggestion that the nuclei of asexual blood-stage parasites are anchored in place to the PPM by the MTOC (Liffner et al., 2023).Temporally, this seems to occur when parasites start to undergo mitosis, with nuclei appearing anchored to the PPM from the onset of mitosis until partway through segmentation (Liffner et al., 2023).
When the MTOC first anchors to the PPM, there do not appear to be any other recognizable structures or organelles in the space between the nucleus and PPM.Eventually, however, this small area will go on to define the apical end of the forming merozoites (Liffner et al., 2023, Rashpa et al., 2023).Not long after nucleus anchoring, each MTOC is accompanied by a Golgi.Subsequently, the apical polar ring and rhoptries undergo their biogenesis in this small space between the PPM and nuclear envelope (Liffner et al., 2023).Marking the start of segmentation, the basal complex and inner-membrane complex (IMC) also undergo their biogenesis in this confined space (Liffner et al., 2023), turning an area of the cell that previously lacked any recognizable structures into the highly organized forming end of a merozoite.Once the parasite has begun segmentation, a branch of both the apicoplast and mitochondrion will closely associate with the cytoplasmic extension of the MTOC and subsequently these organelles will undergo fission (Liffner et al., 2023).It is tempting to speculate that these interactions anchor the apicoplast and mitochondrion within the forming merozoite, subsequently triggering their fission.Collectively, these events establish the nuclear MTOC as hub, around which merozoites are built.Additionally, as the schizont lacks obvious polarity prior to the anchoring of the nucleus, it is possible that the attachment of the MTOC to the PPM establishes regions of polarity within the schizont that will go on to define the apical end of its merozoites.

| Mitosis and meiosis at subnuclear resolution
Plasmodium parasites undergo closed mitosis (Gerald et al., 2011), where the nuclear envelope does not break down during mitosis, followed by nuclear division, and so with uncondensed chromosomes, so mitotic progress cannot be assessed using DNA dyes.
During the replicative stages of the Plasmodium lifecycle, the parasite undergoes mitosis or nuclear division in at least three distinct ways.Asexual blood-stage parasites undergo schizogony, where nuclei undergo rounds of mitosis followed by nuclear division in a shared cytoplasm (Voß, Klaus, Guizetti, et al., 2023).
During male gametogenesis, the parasite undergoes three rounds of mitosis without nuclear division, creating an 8n nucleus that undergoes combined nuclear and cell division to form eight gametes (Matthews et al., 2018).During liver-stage replication, merozoites are formed via schizogony (Roques et al., 2023), but the details of mitosis and nuclear division in liver-stage parasites are not well understood.It has recently been shown that during sporogony in oocysts, large nuclei likely contain multiple genome copies (Araki et al., 2020), but the dynamics of mitosis and nuclear division in this stage of the lifecycle are yet to be elucidated.

One of the major challenges of investigating mitosis and meiosis
in Plasmodium is a lack of available tools to study these processes.As Plasmodium undergoes closed mitosis, and some stages of the lifecycle contain multiple genome copies in a single nucleus, a marker of the nuclear envelope is of clear importance.Prior to U-ExM, however, no markers or stains discernibly and uniformly marked the nuclear envelope.To address this, a lipid stain BODIPY ceramide was applied to parasites following U-ExM (Liffner & Absalon, 2021).Using BODIPY ceramide, the authors were able to observe many membranous structures within the parasite but most notably the nuclear envelope (Liffner & Absalon, 2021).Leveraging this development, they studied the function of minichromosome maintenance binding protein (MCMBP), where they showed that knockdown leads to aneuploidy and the formation of anaphase chromatin bridges (Liffner & Absalon, 2021).
As DNA staining does not indicate mitotic/meiotic stage due to the uncondensed chromosomes of Plasmodium, this must be inferred from protein markers.Using U-ExM, a recent study validated a number of kinetochore markers that can be used to infer the position of during mitosis and meiosis (Brusini et al., 2022).
These markers allowed the authors to visualize either kinetochores or centromeres lining up on the metaphase plate, along with their separation during meiosis and mitosis of both gametocytes and asexual blood stages (Brusini et al., 2022).The ability to infer the position of individual chromosomes and kinetochores will have a significant impact on our understanding of the dynamics of mitosis and meiosis in malaria parasites in future.Further, these observations provide a platform for the characterization of proteins involved in mitosis and meiosis, with a recent study using U-ExM to show that end-binding protein 1 (EB1) mediates microtubulekinetochore attachment during mitosis of gametocytes (Yang, Cai, et al., 2023).
Of the organisms discussed in this review, U-ExM has been applied to by far the greatest diversity of lifecycle stages in Plasmodium.
At present, nearly the entire Plasmodium lifecycle has been visualized using U-ExM (Figure 2).This exemplifies the adaptability of U-ExM to different sample types, and the transferability of observations between lifecycle stages.A comprehensive list of all proteins localized in Plasmodium to date can be found in Table 1.

| Toxoplasma
Toxoplasma gondii can infect almost any nucleated cell of any warmblooded animal but it undergoes sexual replication in the intestine of felines, its definitive host (Tenter et al., 2000).Animals can be infected by either the cyst or oocyst stage of the lifecycle, cysts contain invasive parasite stages known as bradyzoites, while oocysts contain invasive parasite stages known as sporozoites (Hill & Dubey, 2002).When a non-felid animal is infected by an oocyst, the oocyst releases sporozoites, which differentiate into tachyzoites and invade host cells (Carruthers & Boothroyd, 2007).Following host cell invasion, tachyzoites can either replicate asexually to form daughter tachyzoites that will egress from the host cell and invade a new host cell, or differentiate into bradyzoites (Lyons et al., 2002).
Bradyzoites form tissue cysts, and when these cysts are ingested by another non-felid animal the cysts release the bradyzoites to invade host cells, differentiate into tachyzoites and repeat this cycle (Hill & Dubey, 2002).When a felid is infected by an oocyst, the sporozoites will differentiate first into tachyzoites and subsequently bradyzoites.Differentiated bradyzoites from the oocyst, or ones that come directly from tissue cysts, can then differentiate into either tachyzoites or sexual-stage gametocytes in the intestinal epithelium of the felid (Lourido, 2019).Male and female gametocytes can fuse to form a diploid zygote, which will subsequently undergo meiosis to form a haploid oocyst (Martorelli Di Genova & Knoll, 2020).This oocyst will then be shed from the felid to reinitiate the infectious cycle in another animal.

| U-ExM in Toxoplasma
Of the three genera of parasites discussed in this review, expansion microscopy has been the most widely applied in Toxoplasma, with seven different research groups having published using the technique.Overall, U-ExM experiments on Toxoplasma, to date, have heavily focussed on parasite microtubules, but more recent studies have started exploring other aspects of parasite cell biology.Importantly for users of U-ExM and other derivative expansion techniques, the tachyzoite conoid is used as a 'molecular ruler' to assess expansion factor because of its highly defined dimensions (Louvel et al., 2022).

| Defining the components and function of the Toxoplasma conoid
The past few years have seen tremendous efforts to define the apical end of Toxoplasma tachyzoites in incredible detail, using a variety of imaging modalities (Li et al., 2023;Mageswaran et al., 2021;Segev-Zarko et al., 2022;Sun et al., 2022).At the apical end of a tachyzoite is the conoid, a barrel-shaped structure comprised angled tubulin fibres and containing a further two intraconoidal microtubules (Dos Santos Pacheco et al., 2020).On its apex, the conoid is flanked by the preconoidal rings (PCRs), while on its basal end, the conoid is flanked by the APRs (Dos Santos Pacheco et al., 2020).The APRs form the apex of the parasite's inner-membrane complex, and are where the subpellicular microtubules (SPMTs) that define its shape are nucleated from.When tachyzoites are intracellular the conoid is retracted, but when parasites are extracellular the conoid is extruded, allowing parasite motility and invasion.
From the very first study using U-ExM on Toxoplasma, it was clear that it was going to serve as a powerful tool for defining the proteins that comprise the apical end of the parasite, and importantly characterizing their mutants.In this study, the authors utilized U-ExM to refine the localization of a number of apical cap (AC) proteins (Tosetti et al., 2020).In doing so, they immediately recognized the tremendous ability of U-ExM to visualize parasite microtubules and also to unequivocally define whether the conoid was retracted or extruded.Uniquely, AC9 and AC10 were localized specifically to the space between the SPMTs and their knockdown led to significant cytoskeletal defects (Tosetti et al., 2020).Since this study, 23 proteins have been localized to either the PCRs, conoid or APRs of Toxoplasma, highlighting the utility of this technique for precise protein localization.The flexibility of visualizing a range of conoid-localizing proteins using antibodies or tags, along with the ability to assess changes to the conoid by U-ExM, demonstrates the usefulness of U-ExM for investigating the role of proteins that define parasite ultrastructure.

| A closer view of the centrosome and kinetochore of Toxoplasma
Investigating mitosis in Toxoplasma is challenging for many of the same reasons as described previously for Plasmodium.During the tachyzoite stage of the lifecycle, Toxoplasma divides through a process called endodyogeny.In brief, the genome of a mother parasite will duplicate and undergo closed mitosis, with nuclear fission partitioning sister nuclei into two daughter cells that form within the mother (Gubbels et al., 2020).During mitosis, and the early stages of daughter cell formation, the two forming daughter buds can be difficult to distinguish from both each other and the mother parasite; complicating study of Toxoplasma mitosis by light microscopy.
Two separate studies have shown that using U-ExM and a combination of antibodies against tubulin and markers of either the centrosome or kinetochore, the stages of mitosis in tachyzoites can be precisely defined (Brusini et al., 2022;Tomasina et al., 2022).By observing these stages of mitosis, the authors of each study were then able to characterize the localization and function of proteins during mitosis.
The first of these studies focussed on describing new markers of the kinetochore and their relative positions during mitosis (Brusini et al., 2022).By complementing biochemical methods with U-ExM, the authors were not only able to distinguish between markers of the kinetochore and centromeric proteins but also able to define the position of proteins in sub-kinetochore compartments (Brusini et al., 2022).As the alignment of chromosomes along the metaphase plate, and their subsequent separation, could now be visualized, the authors subsequently characterized knockdown mutants of apicomplexan kinetochore protein 1 (AKiT1) and centromere protein C (CENP-C).Following either AKiT1 or CENP-C knockdown, it could be seen that chromosomes fail to properly align along the metaphase plate and then fail to separate appropriately during anaphase (Brusini et al., 2022).The ability to characterize the function of proteins involved in mitosis at this specificity represents a tremendous advance in parasite cell biology and will undoubtedly lead to a significant increase in our understanding of parasite mitosis in the future.
The tachyzoite centrosome comprises an outer core that contains two centrioles and a centriole-free inner core, which opposes a nuclear envelope elaboration called the centrocone.The second of these two studies assessed the role of the centrosome inner core on mitosis (Tomasina et al., 2022).Using U-ExM, the authors showed that the only known marker of the inner core Cep250L1 gets duplicated and separated during mitosis.Subsequently, they generated Cep250L1 knockdown parasites and showed that upon Cep250L1 depletion, parasites have a reduced ability to form their mitotic spindle (Tomasina et al., 2022).This suggests that the inner core of the tachyzoite centrosome, which previously had no known function, is involved in regulating mitotic spindle assembly.Collectively, these two studies highlight the utility of U-ExM to study parasite mitosis, and functionally characterize proteins involved in the process.

| Visualizing interactions between organelles
The organelles within a parasite are not static or standalone structures, instead, they are constantly morphing and interacting with each other.Investigating inter-organelle interactions, principally mediated by membrane contact sites, is a relatively but rapidly growing new area of parasite cell biology.A recent study using U-ExM investigated the interaction between the inner-membrane complex and mitochondrion of tachyzoites (Oliveira Souza et al., 2022).In intracellular parasites, the mitochondrion typically adopts a 'lasso-like' shape around the nucleus and knockout of an outer mitochondrial membrane protein known as lasso maintenance factor 1 (LMF1) has been shown to disrupt this shape (Jacobs et al., 2020).Surprisingly, when the authors looked for LMF1 interactors, their top hits were proteins of the inner-membrane complex including IMC10 (Oliveira Souza et al., 2022).The authors showed that LMF1 localizes in close proximity to the IMC, appearing to form a bridge between the mitochondrion and IMC (Oliveira Souza et al., 2022).Subsequently, the authors showed that IMC10 knockdown also results in defects in mitochondrial morphology (Oliveira Souza et al., 2022).Assessing the interaction between LMF1 and the IMC during daughter cell formation, the authors suggested that LMF1 and the IMC interact cytokinesis to uniformly distribute mitochondria around the nuclei of daughter cells and form the characteristic lasso shape (Oliveira Souza et al., 2022).
Toxoplasma holds a unique position as the first expanded apicomplexan parasite, with the researchers who pioneered this technique in Toxoplasma tremendously demonstrating how U-ExM can unlock new avenues to explore in parasite cell biology.A comprehensive list of all proteins localized in Toxoplasma to date can be found in Table 2.

| Cryptosporidium
Unlike both Toxoplasma and Plasmodium, Cryptosporidium can undergo its whole life cycle in the same host.Cryptosporidium spp.infect a wide range of animals, but this review will focus on those that infect mammals, which include the two major human pathogens C. parvum and C. hominis.Cryptosporidium is transmitted when a host ingests oocysts (Guérin & Striepen, 2020).Oocysts contain sporozoites, which will be released from the oocyst and invade the intestinal epithelium (Guérin & Striepen, 2020).Once the sporozoite has invaded an enterocyte it will form a trophozoite, which will replicate asexually to form a meront containing eight merozoites (Guérin & Striepen, 2020).Merozoites will egress from the infected cell, reinvade a new cell and repeat this cycle two times (English et al., 2022;Guérin & Striepen, 2020).During the third round of asexual replication, merozoites commit to sexual differentiation and following meront egress, the merozoites will invade a new host cell to form either a male or female gamont (English et al., 2022;Tandel et al., 2019).Male gametes undergo four rounds of mitosis to produce 16 gametes, while female gamonts differentiate into a single gamete (Guérin & Striepen, 2020).Subsequently, these gametes fuse to form a diploid zygote that will undergo meiosis and form an oocyst (Guérin & Striepen, 2020).Oocysts can either be shed to infect a new host, or undergo auto-infection within the same host, repeating this cycle.

Relative to our understanding of cell biology in Toxoplasma and
Plasmodium, the investigation of Cryptosporidium cell biology is still in its infancy.In large part, this is due to the fact that until relatively recently culture, cryopreservation and genetic manipulation of Cryptosporidium were either difficult or not possible (Guérin & Striepen, 2020).In recent years, however, genetically tractable and culturable systems for Cryptosporidium have been developed (Heo et al., 2018;Jaskiewicz et al., 2018;Sateriale et al., 2019;Vinayak et al., 2015;Wilke et al., 2019).Consequently, we have seen a long-overdue explosion in our understanding of Cryptosporidium biology.In only the last few years, an impressive array of microscopy techniques including live cell microscopy (English et al., 2022;Guérin et al., 2021), structured illumination microscopy (SIM) (Choudhary et al., 2020;Guérin et al., 2021), stimulated emission depletion (STED) microscopy (Guérin et al., 2021), cryo-electron tomography (Mageswaran et al., 2021) and most recently U-ExM (Guérin et al., 2023), have been employed to better understand Cryptosporidium cell biology.

| Visualizing the spatial proteome of Cryptosporidium
A significant advance in our understanding of Cryptosporidium biology was achieved with the recent publication of the spatial proteome of C. parvum (Guérin et al., 2023).Using a technique known as hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT) (Christoforou et al., 2016), the authors localized 1107 proteins to different organelles or structures within Cryptosporidium sporozoites.The output of hyperLOPIT clusters proteins based on fraction extraction profiles, as proteins in the same organelles will have similar extraction profiles.Proteins in the dataset with known localizations can then be used to identify which organelles or structures the observed clusters represent.To validate these data, the authors picked proteins predicted to localize to the micronemes or rhoptry neck and performed colocalization with known markers for these organelles (Guérin et al., 2023).In addition, the authors performed U-ExM on sporozoites coupled with NHS ester staining, which clearly revealed the rhoptry, conoid, crystalloid body and dense granules (Figure 1).The use of U-ExM coupled with NHS ester proved invaluable, as previously there were no antibodies that could be used as markers for either crystalloid body or dense granules.
This allowed the authors to validate four new dense granules proteins and one new crystalloid body protein (Guérin et al., 2023).Use of NHS ester with U-ExM in Cryptosporidium is likely to continue to be extremely useful in future, due to the relative dearth of markerspecific antibodies that are commonly or commercially available.
2.8.2 | Identification of two secretory organelles in Cryptosporidium, dense granules and small granules Based on the hyperLOPIT data, the authors observed two clusters of proteins distinct from dense granule clusters predicted to be secreted that contained no marker proteins, and tagged a protein from each of these clusters to investigate their localization (Guérin et al., 2023).
Using U-ExM coupled with NHS ester, the authors showed that both proteins were found in several small vesicles near the nucleus that were distinct from the dense granules.This organelle was termed the small granules, and was subsequently shown to be secreted following host cell invasion (Guérin et al., 2023).Curiously, some proteins predicted to localize to the small granules proteins are conserved in other Apicomplexa, but whether other Apicomplexa have these secretory organelles is not yet known.Collectively, this highlights the utility of U-ExM not only for performing detailed colocalization studies but also highlights its ability to reveal structures previously not visible by conventional light microscopy.
Due to the power of U-ExM for identifying organelles without the need for specific antibodies to visualize them, it is likely that of the three genera of parasites discussed in this review, U-ExM has the greatest ability to lead to significant advances in our understanding of Cryptosporidium biology.A comprehensive list of all proteins localized in Cryptosporidium to date can be found in Table 3.

| ADVANTAG E S , DR AWBACK S AND LIMITATI ON S OF U -E XM IN API COMPLE X A
As described above, U-ExM has already made a clear impact in the field of parasite cell biology.In the coming paragraphs, we will summarize some of the specific advantages, drawbacks and limitations that either have been observed with U-ExM, or maybe in future.It is difficult to infer the increase in "resolution" caused by expanding the sample, as the increase in sample size has no influence on the resolution of an image.Because of this complication, we will refer to U-ExM as providing an approximately 4.5-fold increase in image detail, rather than resolution.By contrast to some super-resolution methods, U-ExM increases image detail equally for all lasers/colours and does so equally in three dimensions.Another obvious advantage of U-ExM is that it is compatible with most super-resolution imaging modalities, increasing the potential usefulness of the technique.To date, Airyscan (Calla et al., 2023;Liffner & Absalon, 2021;Liffner et al., 2023;Morano et al., 2023) is the only super-resolution technique that has been used on expanded parasites to the best of our knowledge, but stimulated emission depletion (STED) (Gambarotto et al., 2019), 3D structured illumination microscopy (SIM) (Woglar et al., 2022) and single molecule localization microscopy (SMLM) (Chang et al., 2023) have been applied to U-ExM on other sample types.Additionally, U-ExM may increase antibody accessibility for certain proteins, with reduced antibody competition having been previously observed for expanded microtubules (Gambarotto et al., 2019).A final advantage of U-ExM, which is not the case for all expansion microscopy protocols, is that the linkage error (distance between fluorophore and biological target) is reduced proportionally to the expansion factor (Zwettler et al., 2020).
Like any imaging modality, U-ExM is accompanied by some limitations and artefacts.It is important, however, for early adopters of U-ExM to recognize these limitations and artefacts and share these findings with the community.Some broad potential limitations of U-ExM include target dilution, artefacts of denaturation, a lack of sample isotropicity and incompatibility with some fluorescent molecules.An expansion factor of 4.5× corresponds to an approximately 90-fold increase in volume, potentially meaning a 90-fold reduction in the concentration of a protein or molecule of interest.
This may cause difficulties in detecting proteins or molecules that are either particularly lowly expressed or lowly abundant within the cell.During U-ExM the sample is denatured using a combination of heat and detergents.As the sample is denatured, proteins will be linear and therefore antibody-antigen recognition may differ from conventional immunofluorescence.Additionally, denaturation and expansion could introduce some artefacts, but other than some parasite-specific artefacts detailed below, it is not easily predictable what these artefacts would be.One general concern of expansion microscopy overall is whether the sample is preserved isotropically (equally in all dimensions).This particular concern led directly to the development of U-ExM, where the authors observed that other expansion microscopy methods did not isotropically preserve centrioles (Gambarotto et al., 2019).To the best of our knowledge, no significant distortions of isotropicity have been reported in samples prepared by U-ExM, but users should be aware of this possibility.
Finally, U-ExM is incompatible with some fluorescent molecules.
Most notably, conventional fluorescent protein tags (such as GFP or mCherry) will lose their fluorescence during expansion and would therefore need to be detected using antibodies instead.Recently, however, fluorescent tags that are highly resistant to both thermal and chemical denaturation have been developed (Campbell et al., 2022).These tags have been shown to be compatible with some expansion microscopy protocols (Campbell et al., 2022), and may be compatible with U-ExM.
To date, the most notable parasite-specific limitation occurs in blood-stage malaria parasites that biomineralize haem into a crystal called haemozoin (Matz, 2022).The large haemozoin crystal in the centre of the parasite either does not expand, or does not get anchored to the gel, leaving a large empty space in the food vacuole where it previously resided (Liffner et al., 2023).Considering this, any observations about the parasite food vacuole should be made with caution as we know it has been altered in a non-physiologically relevant way.Curiously, it has also been observed that non-specific antibody fluorescence accumulates in the region of the cell that used to contain the haemozoin crystal (Liffner et al., 2023).
To date, U-ExM in apicomplexan parasites has only been used to study cultured cells, but U-ExM is applicable to tissues or whole organs.Considering this, we will likely in future see publications that visualize parasites in situ, performing U-ExM on intestinal sections containing Toxoplasma or Cryptosporidium, or whole expanded salivary glands or midguts from Plasmodium-infected mosquitoes.
One significant challenge this is likely to impose is that it will significantly increase the depth of the gel that you need to image to capture your sample.In our experience, the sample is always brightest at the surface of the gel closest to the objective lens and becomes progressively dimmer the deeper into the gel you image; which is to be expected as working distance increases.While imaging of parasites in situ will likely be possible irrespective of this limitation, it will prevent imaging of entire organs or tissues using conventional confocal or widefield microscopy.Imaging modalities such as light sheet or lattice light sheet fluorescence microscopy (Stelzer et al., 2021), where the sample is more evenly illuminated could likely overcome this hurdle and provide high-resolution expanded images of tissues or organs infected with parasites.
While U-ExM is extremely useful for revealing previously indistinguishable structures, it is likely not compatible with absolute fluorescence quantification.Absolute fluorescence quantification (where two different samples are compared to each other), like in colocalization studies, for example, is complicated because of the variables that need to be controlled for.In U-ExM, there is a small difference between the expansion factors of gels, with reported ranges between 3.9-and 4.3-fold expansion and currently (Bertiaux et al., 2021;Gambarotto et al., 2019;Liffner & Absalon, 2021;Liffner et al., 2023), there is no way to control for this variance.
Compounding this problem is the previously mentioned issue about cells deeper into the gel not being as bright as those at the imaging surface.Further, it remains currently unclear how much gel-to-gel variation there is in fluorescence intensity, or whether fluorescence intensity is influenced by position within the gel.
While >80 proteins have been localized in Apicomplexa by U-ExM using either protein-specific antibodies or tagged proteins (Tables 1-3), authors have described not being able to localize a small number of proteins using U-ExM.For U-ExM, the sample is completely denatured and so antibodies will be encountering linear epitopes, while in conventional immunofluorescence assays the antibodies would typically encounter conformational epitopes.Considering this, it is unsurprising that some proteins that are readily localized using conventional immunofluorescence assays can sometimes not be localized using U-ExM.To the best of our knowledge, only two apicomplexan proteins have been published as not being localizable by U-ExM; MCMBP in P. falciparum (Liffner & Absalon, 2021) and CCP1 in Cryptosporidium (Guérin et al., 2023).

| P OTENTIAL FUTURE APPLI C ATI ON S AND EMERG ING E XPAN S I ON MI CROSCOPY TECHNI Q U E S
U-ExM has already had a drastic influence on our understanding of cell biology in apicomplexan parasites, but expansion microscopy is a rapidly evolving technique.Additionally, due to its relatively recent application to apicomplexan parasites, there are some well-studied aspects of parasite cell biology that have not yet been interrogated using U-ExM.In the final few paragraphs of this review, we will summarize emerging expansion microscopy techniques and speculate on areas of apicomplexan cell biology where U-ExM could provide breakthroughs.

| An expanded repertoire of fluorescent dyes
A common feature of many of the studies using U-ExM on apicomplexan parasites has been the application of fluorescent chemical dyes in addition to antibodies.
This >4-fold expansion has been achieved in a number of ways including using more swellable hydrogels (Damstra et al., 2022), and performing iterative expansion where an already expanded hydrogel is expanded again (Louvel et al., 2022;M'Saad & Bewersdorf, 2020).
An iterative expansion microscopy technique known as iterative U-ExM (iU-ExM) has already been applied to T. gondii, where the individual microtubules that comprise the conoid were observed; something previously only seen using electron microscopy (Louvel et al., 2022).These next-generation expansion microscopy techniques will likely be extremely useful for answering questions about highly protein-dense structures not readily resolved by U-ExM, such as the conoid.25-fold linear expansion, however, corresponds to >10,000-fold volumetric expansion, which would decrease protein concentration so much that many proteins/stains may become impossible to visualize.We think that iU-ExM, and other expansion techniques that expand beyond 4-fold will be extremely useful for determining whether there is subcompartmentalization of proteins within protein-dense structures.Considering this, some logical candidates to investigate using these techniques are the basal complex, apical polar rings, nuclear MTOC and nuclear pore complexes.

| Nature versus nurture
The last few years have seen the publication of groundbreaking studies that allow for the in vitro production of infectious Plasmodium sporozoites (Eappen et al., 2022) and Toxoplasma cat-restricted sexual stages (Antunes et al., 2023).U-ExM could be used to compare the ultrastructure of these in vitro generated parasites against their in vivo generated counterparts to define the ways in which they are both similar and different.The advantage of U-ExM over electron microscopy in this context would be the ability to sample hundreds of cells in three dimensions, at different stages of their development/differentiation, and do so with antibodies against proteins of interest.

F
Comparison of unexpanded and U-ExM parasites across Apicomplexa.Example images of Plasmodium falciparum schizonts, Cryptosporidium parvum sporozoites and Toxoplasma gondii tachyzoites that are either unexpanded, or prepared using U-ExM.For all images, DNA stains are represented in cyan.For all U-ExM images, greyscale represents NHS ester (protein density).For Plasmodium, magenta represents anti-tubulin (microtubule) staining and zoom region of interest shows a single merozoite from a schizont.For Cryptosporidium, magenta represents the dense granule protein DG3, white (unexpanded only) represents the sporozoite antigen Cp23 and zoom region of interest shows individual dense granules.For Toxoplasma, magenta represents anti-acetylated tubulin (microtubule) staining and zoom region of interest shows a forming daughter cell.White scale bars = 2 μm, yellow scale bars = 500 nm.Plasmodium images are derived from the dataset in Liffner et al. (2023), Cryptosporidium images are unpublished images derived from the dataset in Guérin et al. (2023) and Toxoplasma images are from an unpublished dataset.
play a role in microtubule integrity and organization in both ookinetes and male gametocytes.Collectively, this highlights the importance of the initial description of Plasmodium microtubules by U-ExM as F I G U R E 2 An expanded view of the Plasmodium lifecycle.Generalized Plasmodium lifecycle with all represented stages depicted as expanded parasites.For all images, greyscale represents protein density (NHS ester).Cyan represents either DNA staining (SYTOX deep red) or GEX1 in the macrogamete.For liver stages, magenta represents UIS4 (parasitophorous vacuole).For ookinete, gametes and the gametocyte, magenta represents microtubules.Oocyst and sporozoite images are P. yoelii (currently unpublished dataset).Ookinete (replicated with permission from Bertiaux et al., 2021), gametes (replicated with permission from Rashpa & Brochet, 2022) and liver stages are P. berghei (currently unpublished dataset).Asexual blood-stage (from dataset in Liffner et al., 2023) and gametocyte (currently unpublished dataset) are P. falciparum.Images are not depicted to scale.
Importantly, the ability to visualize all the different regions at the apical end of tachyzoites gave the ability to characterize mutants that deform these structures.A recent study localized a number of proteins to the different subregions of the tachyzoite apical end, including five new PCR proteins, and functionally characterized 10 of them using knockdown parasites (Dos SantosPacheco et al., 2022).Specifically, the authors assessed conoid extrusion using U-ExM and found seven proteins whose knockdown inhibited parasite motility and conoid extrusion (Dos SantosPacheco et al., 2022).The authors also characterized homologues of two of these proteins in P. berghei ookinetes, and showed that both localize at the conoid, with knockdown inhibiting ookinete motility and decreasing the distance between the conoid and APR; a likely equivalent of inhibiting conoid extrusion (Dos SantosPacheco et al., 2022).Collectively, using U-ExM, the authors defined a new hypothetical model for the process and function of conoid extrusion.When the conoid is extruded, actin flux occurs from the apical end of the conoid and accumulates between the parasite plasma membrane (PPM) and innermembrane complex (IMC) before moving to the basal end of the parasite to generate its actin-based motility.When the conoid is retracted, the PCRs prevent the flux of actin through the space between the PPM and IMC, therefore inhibiting actin-based motility and making conoid extrusion essential for motility (Dos SantosPacheco et al., 2022).
Perhaps the most exciting avenue for the future development of expansion microscopy-based techniques is the ability to combine ExM with other kinds of experiments.Fluorescence in situ hybridization (FISH) experiments have been a mainstay of nucleic acid biology for many years.Protocols have been developed that enable both DNA(Klimas et al., 2023) and RNA(Chen et al., 2016) FISH on expanded samples, opening the possibility of studying individual genomic loci or RNAs on expanded parasites.Omics technologies have also recently been combined with expansion microscopy, with protocols recently published to perform in situ spatial transcriptomics(Alon et al., 2021) and proteomics(Li, Sun, et al., 2022) on expanded samples.These techniques are not yet at the level of single-cell transcriptomics or proteomics, but we can image this kind of technique being applied to decipher a single-cell transcriptome or proteome of something like the P. vivax hypnozoite.
complexan cell biology but to the best of our knowledge, there are no published examples of expanded parasites in the process of invading.It is not hard to imagine taking U-ExM and revisiting one of the seminal works on host cell invasion using light microscopy or electron microscopy (such asRiglar et al. (2011) for Plasmodium or DelRosario et al. (2019) for Toxoplasma).To date, U-ExM has not significantly altered our view of host cell invasion by apicomplexan parasites, but we think this is very likely to change in the near future.4.6 | Expanding the worldThis review has focussed primarily on studies in P. falciparum, P. berghei, P. yoelii, T. gondii and C. parvum largely because there are well-established culture systems for these organisms.Perhaps the biggest strength of U-ExM, however, is that when it is coupled with general stains such as NHS ester or BODIPY ceramide, we can learn a great deal about the cell biology of organisms we cannot genetically manipulate and for which we lack protein-specific antibody.In 2023, a groundbreaking and ambitious study known as Traversing European Coastlines (TREC), led by EMBL and the Fondation Tara yond the walls of a laboratory.Applying a similar thought process to apicomplexan parasites, one could imagine using U-ExM to look at the ultrastructure of malaria parasites inside field-caught mosquitoes, haemosporidian parasites in blood samples from wildlife or Toxoplasma cysts from livestock animals.The future of expansion microscopy and what that means for that will mean for the study of parasite cell biology is truly exciting.AUTH O R CO NTR I B UTI O N S Benjamin Liffner: Conceptualization; writing -original draft; writing -review and editing; investigation.Sabrina Absalon: Conceptualization; investigation; writing -original draft; writingreview and editing; supervision.

TA B L E 2
Summary of all proteins localized by U-ExM using antibodies or tagged cell lines in Toxoplasma.