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CitationREVIEW ARTICLE| APRIL 25 2025
Nanoscale imaging of biological systems via expansion and
super-resolution microscopy
Daria Aristova  
 ; Dominik Kylies  
 ; Mario Del Rosario  
 ; Hannah S. Heil  
 ; Maria Schwerk  
 ;
Malte Kuehl  
 ; Milagros N. W ong 
 ; Ricardo Henriques  
 ; Victor G. Puelles 
Appl. Phys. Rev . 12, 02131 1 (2025)
https://doi.org/10.1063/5.0240464
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Nanoscale imaging of biological systems via
expansion and super-resolution microscopy
Cite as: Appl. Phys. Rev. 12, 021311 (2025); doi: 10.1063/5.0240464
Submitted: 25 September 2024 .Accepted: 1 April 2025 .
Published Online: 25 April 2025
Daria Aristova,1,2,3
Dominik Kylies,4,5
Mario Del Rosario,1,2,3
Hannah S. Heil,2,3,6
Maria Schwerk,4,5
Malte Kuehl,7,8
Milagros N. Wong,4,5,7,8
Ricardo Henriques,1,2,9,a)
and Victor G. Puelles4,5,7,8,a)
AFFILIATIONS
1Instituto de Tecnologia Química e Biol /C19ogica Ant /C19onio Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
2Instituto Gulbenkian de Ci ^encia, Oeiras, Portugal
3Gulbenkian Institute for Molecular Medicine, Oeiras, Portugal
4III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
5Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany
6Department of Clinical Sciences Lund, Division of Infection Medicine, Faculty of Medicine, Lund University, Lund, Sweden
7Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
8Department of Pathology, Aarhus University Hospital, Aarhus, Denmark
9UCL Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
a)Authors to whom correspondence should be addressed: r.henriques@itqb.unl.pt ;vgpuelles@clin.au.dk andv.puelles@uke.de
ABSTRACT
Super-resolution microscopy (SRM) has revolutionized life sciences by overcoming the diffraction limit, enabling the visualization of biologica l
structures at the nanoscale. Expansion Microscopy (ExM) has emerged as a powerful and accessible technique that enhances resolution by
physically enlarging the specimen. Importantly, the principles of ExM provide a unique foundation for combinations with SRM methods,
pushing the boundaries of achievable resolution. This review explores the fundamental principles of ExM and examines its successful integration
with various SRM techniques, including fluorescence fluctuation-based SRM, structured illumination microscopy, stimulated emission depletion
microscopy, and single-molecule localization microscopy. We discuss the applications, strengths, limitations, and resolutions achieved by thes e
combined approaches, providing a comprehensive guide for researchers to select the most suitable method for their specific scientific needs. Key
considerations when combining ExM with SRM include the impact on fluorophores, the requirement for specialized buffers, and the challenges
posed by the sensitivity of expanded hydrogels to temperature and hydration. Strategies to address these challenges, such as optimized labeling
techniques and gel re-embedding, are discussed in detail. This review aims to assist researchers in navigating the rapidly evolving landscape of
ExM and SRM, facilitating the development of tailored imaging pipelines to advance our understanding of biological systems at the nanoscale.
VC2025 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license ( https://
creativecommons.org/licenses/by/4.0/ ).https://doi.org/10.1063/5.0240464
TABLE OF CONTENTS
I. INTRODUCTION. ................................. 1
II. EXPANSION MICROSCOPY . . ..................... 2
A. Terminology ................................. 2
B. Principles. . . ................................. 2
C./C244-Fold expansion . . ......................... 3
D. Beyond fourfold expansion . . . ................. 4
E. Potential and challenges . . ..................... 4
III. COMBINING EXM WITH SRM. . . ................. 6
A. Fluorescence fluctuation-based super-resolution
microscopy . ................................. 6
B. Combining FF-SRM with ExM ................. 6C. Structured illumination microscopy . . ........... 8
D. Combining SIM with ExM . ................... 8
E. Stimulated emission depletion . . . ............... 1 0
F. Combining STED with ExM ................... 1 0
G. Single-molecule localization microscopy . . ....... 1 2
H. Combining SMLM with ExM . . . ............... 1 2
IV. DISCUSSION AND OUTLOOK . ................... 1 3
I. INTRODUCTION
Super-resolution microscopy (SRM) encompasses various meth-
ods that overcome the diffraction limit, a physical restriction that limits
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the optical resolution of fluorescent microscopes to around 250 nm.1,2
Subcellular structures such as cellular organelles, cytoskeletal filaments,
membrane receptors, and macromolecular complexes are often smaller
than the diffraction barrier. Notable examples in different organs include
the gap between two neuronal processes, known as the synaptic cleft
(approximately 20 nm),3,4the endoplasmic reticular lamellae (range: 20 –
60 nm)5and the width of the renal slit diaphragm, the kidney filtering
unit (approximately 40 nm).6The breakthrough of SRM in surpassing
these limitations is therefore of immense importance to achieve a deep
understanding of cellular and tissue-based processes in health and dis-
ease.2,7–11Increasingly, the analysis of cellular ultrastructure also plays
an important role in clinical diagnostics.12,13Therefore, SRM techniques
have become indispensable tools in life sciences, providing key insights
into the molecular architecture and dynamics of cells and tissues, as
many crucial biological structures and interactions occur at these length
scales.14Since the introduction of the first SRM methods,15–21multiple
optical approaches have been developed, including both commercially
available solutions and open-source image enhancement algorithms.22–
29These optical and computational SRM technologies leverage various
principles to achieve resolution beyond the diffraction limit, with resolu-
tions reaching as low as 20 nm26depending on the method and setup.
Furthermore, recent approaches, including Minflux,30–32Minsted,33,34
and RESI35have even reported sub-nanometer resolution.
Less than a decade ago, a radically different approach to achieving
resolutions beyond the diffraction limit called “Expansion Microscopy ”
(ExM)36was developed. Unlike many other methods to break the dif-
fraction limit, ExM is a tissue processing technique that enhances the
resolution of microscopes by physically enlarging biological samples
before imaging.36–40This eliminates the need for specialized SRM equip-
ment, making ExM relatively simple to implement. One of the key
advantages of ExM is its ability to enhance resolution in all three spatial
dimensions (3D) and enable thick sample imaging. However, due to thenecessity of fixation and separation of biomolecules during the process,
ExM is not compatible with live-cell imaging. Since its introduction,
ExM has rapidly gained widespread acceptance within the scientific
community with an increasing availability of specialized protocols,
highlighting its broad applicability as a versatile and valuable tool across
various scientific disciplines. Depending on the ExM protocol and the
achieved degree of tissue expansion, ExM has been shown to enable res-
olutions under 70 nm
36,41using diffraction-limited microscopy systems.
To extend the range of applications, ExM protocols were initially
combined with light-sheet42–45and conventional confocal micro-
scopes.36,46However, the modular nature of ExM as a tissue processing
technique makes it compatible with a wide range of microscopy tech-
nologies, including SRM [ Fig. 1(a) ]. In particular, ExM has been suc-
cessfully integrated with methods such as stimulated emission
depletion (STED),47single-molecule localization microscopy
(SMLM),48structured illumination microscopy (SIM),49and fluores-
cence fluctuation (FF)-based SRM algorithms (FF-SRM),50demon-
strating enhanced resolution and enabling researchers to create novel,
modular, and versatile combined SRM pipelines tailored to their spe-
cific scientific requirements [ Fig. 1(b) ]. Other intriguing approaches
that leverage combinations of methodologies across disciplines further
underscore the potential of integrated techniques, as highlighted in
recent studies on advanced imaging and analysis methods.51–53
However, given the different principles of action and applicability of
these SRM methods, individual considerations, including specificstrategies for troubleshooting and limitations, must be addressed to
achieve optimal results. In this review, we discuss the basic principles
of ExM and SRM methods that have been successfully combined with
ExM, along with their applications, strengths, limitations, and resolu-tion range, thereby guiding researchers in selecting an appropriatecombined method that best suits their scientific needs.
II. EXPANSION MICROSCOPY
ExM is a highly effective specimen processing technique that
physically expands samples, enabling resolutions that surpass the dif-
fraction limit of conventional light microscopes.
36Instead of relying
on advanced optics or computational approaches, ExM achievessuper-resolution by embedding biological samples in swellable hydro-gel polymers. As the hydrogel expands, the biomolecules and theirassociated fluorescent labels are separated, enabling the visualization of
structures below the diffraction limit. This section covers different var-
iations of ExM, their underlying chemistry, and combinations of ExMwith SRM techniques. A summary of available ExM protocols is pro-vided in supplementary material Table 1.
A. Terminology
Early studies on tissue clearing, particularly hydrophilic clearing
protocols, occasionally observed increases in tissue volume during theclearing process.
54For example, in 2011, the Scale brain-clearing pro-
tocol, which uses substances such as urea and Triton X-100, was
observed to cause a 1.25-fold increase in tissue size.55Subsequent clear-
ing protocols, including CLARITY56and CUBIC,57also acknowledged
tissue swelling as a treatment by-product. In these investigations,expansion was often seen as a side effect of the clearing process, some-times discussed as uncontrolled or undesired. Of note, CUBIC-X,
58an
extension of CUBIC, purposefully achieves an approximately 2 /C2linear
expansion by using small molecules such as imidazole and antipyrineto induce tissue swelling. Given these findings in the context of therapidly growing field of ExM, it is important to distinguish betweenclearing-focused protocols that result in smaller degrees of specimenswelling as a side effect and “true”ExM protocols designed to enable
super-resolution through specimen expansion. Thus, “ExM ”generally
refers to methods that involve embedding a biological specimen intopolyelectrolyte hydrogels that are specifically designed and validated tohave high degrees of isotropic swelling capabilities while preserving thenanostructure of the biological specimen.
B. Principles
While many protocol variations exist today, the most widely
adopted ExM protocols
37follow a somewhat similar workflow to
achieve isotropic specimen expansion while preserving structural
information at nanoscopic scales.38Notably, in this rapidly evolving
field with an increasing number of protocol modifications and varia-tions, the general workflow described here might not fully translate toall protocols available to this date.
59–61Classically, molecular anchors
are covalently attached to biomolecules, facilitating their binding to a
swellable hydrogel synthesized across the specimen.37Subsequently,
the specimen is incubated in a monomer solution [containing sodiumacrylate (SA)], allowing the monomers to distribute homogeneously.Next, via free-radical polymerization, a densely cross-linked (via thecross-linker N,N
0-methylenebisacrylamide) polyelectrolyte hydrogel
(sodium polyacrylate) is formed, resulting in a robust mechanicalApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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coupling with the attached biomolecules and fluorescent labels.36,37
The specimen ’s mechanical properties are then homogenized through
either chemical denaturation via heat and detergent treatment, orenzymatic digestion, depending on the specimen texture and experi-
mental requirements. Finally, immersion in de-ionized water induces
isotropic swelling through osmotic force, facilitated by the highlycharged nature of the polyelectrolyte mesh.
36
C./C244-Fold expansion
The original ExM protocol achieved a 4.5-fold linear expansion
with approximately 70 nm lateral resolution.36This foundational pro-
tocol demonstrated the potential of physically expanding specimens toovercome the diffraction limit of conventional light microscopy. Byswelling the hydrogel-embedded sample isotropically, the fluorophorestagging biomolecules are separated, enabling the observation of biolog-
ical structures below the diffraction limit. In the original fourfold
expansion protocol, Chen et al. used a polyacrylamide-based hydrogel
where acrylamide (AAm) serves as the monomer backbone while N,N
0-methylenebisacrylamide crosslinks polymer chains.36Biomoleculesof interest had to be tagged with a gel-anchorable fluorescent label,
which required custom synthesis, posing a hurdle for researchers look-ing to adopt the method. Additionally, another limitation was its
inability to image genetically encoded fluorescent proteins without
antibody labeling. However, since its first report, this protocol hasundergone several advancements and variations to improve flexibility,
applicability, and overall ease of use, aiming to increase accessibility
and enhance its performance in specific settings, which are discussedfurther.
Protein retention ExM (proExM)
37made ExM more available to
the general scientific community by introducing a commercially avail-
able cross-linking molecule (Acryloyl-X, AcX) as a novel strategy to
link specimen proteins to the hydrogel. Additionally, methacrylic acidN-hydroxy succinimidyl ester (MA-NHS) and glutaraldehyde have
also been successfully utilized to bind proteins within the hydrogel.
62
This addressed two of the main drawbacks of the original ExM proto-
col, namely, the requirement for custom-built gel-anchorable fluores-
cent labels and the inability to expand and visualize endogenous
fluorescent proteins such as green fluorescent protein (GFP), while stillachieving a fourfold linear expansion factor. Next, ExPath shares
FIG. 1. Overview of combined methods. (a) Graphical overview of the general workflow of combined methods. Biological samples are fluorescently labeled (1) , followed by
hydrogel embedding (2), and sample expansion (3). The samples are then imaged using different SRM technologies (4), often involving computational p rocessing (5).
(b) Resolution range of SRM modalities alone and when combined with ExM protocols. Generally, FF-SRM and SMLM focus on photo-switchability/photo-c onvertibility while
SIM and STED focus on optical systems optimization strategies. While blue text reflects SRM methods without additional expansion. Magenta text refl ects SRM modalities com-
bined with a fourfold ExM protocol. The cyan text reflects the combination with a higher-fold ExM protocol.Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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many similarities to proExM but is optimized for expanding pathology
tissue specimens,63facilitating the analysis of archival pathology sam-
ples (i.e., tumor biopsies) to understand disease-related changes in sub-cellular tissue-based structures and processes.
In addition to proExM, a new strategy for RNA anchoring to
hydrogels termed ExFISH
64not only provided a new way to anchor
RNA molecules but also allowed high-resolution imaging of specificnucleic acid sequences using fluorescence in situ hybridization (FISH)
in cells and tissues. Importantly, ExFISH also pioneered the strategy ofstabilizing expanded specimens by re-embedding them in unchargedgels, preventing shrinkage in environments with higher osmolarity,such as specialized buffers required for FISH
64and fluorescence in situ
sequencing65in expanded specimens.
D. Beyond 4-fold expansion
For a better resolution using ExM with standard microscopes,
advanced ExM protocols have been developed by increasing theexpansion factor. These protocols include multiple rounds of hydrogelre-embedding or those achieving higher expansion in a single round.In general, these higher-fold ExM protocols have been reported toattain resolutions in the range of /C2425 nm in combination with conven-
tional diffraction-limited microscopes.
Protocols such as Iterative ExM (iExM),
66Pan-ExM,67and
iU-ExM68build upon this concept by involving multiple rounds of
hydrogel embedding and expansion, achieving up to 20 /C2isotropic
expansion. The iExM protocol builds upon the original ExM protocolby introducing a two-step expansion process. After an initial 4.5 /C2
expansion, a second swellable polymer mesh is formed within theexpanded space, allowing for an additional expansion, leading to atotal/C2420/C2expansion. iExM has been successfully applied to experi-
mental tissue specimens, allowing the visualization of synaptic proteinsand dendritic spine architecture in mouse brain circuitry.
66iU-ExM is
built upon the U-ExM protocol69to preserve nanoscale ultrastructure
in cellular organelles. Inspired by the MAP39protocol, iU-ExM opti-
mizes the fixation step using a combination of low concentrations offormaldehyde (FA) and acrylamide and optimizes the homogenizationprocess. Similarly to iExM, iU-ExM involves multiple rounds of hydro-gel embedding and expansion, demonstrating its successful applicationin cellular and tissue contexts.
68Pan-ExM is a protocol variant opti-
mized for the spatial molecular analysis of cultured cells in theirwhole-cell ultrastructural context.
67Pan-ExM involves the incubation
of cells in acrylamide (AAm) and formaldehyde (FA) to prevent inter-protein crosslinks and a homogenization step using sodium dodecylsulfate (SDS) and heat to separate neighboring proteins followed byiterative rounds of hydrogel re-embedding to unmask cellular epitopesfor subsequent staining. One distinct feature of the Pan-ExM workflowis the application of an NHS ester staining to achieve a pan-proteincontrast and provide a general spatial context to specific molecular
markers. Another distinctive aspect of pan-ExM is the use of a cleav-
able amidomethylol bond within the hydrogel ’s backbone, facilitating
subsequent expansion. Delipidation and denaturation, achievedthrough sodium dodecyl sulfate (SDS) and heat treatment, contributeto the preservation of protein content.
In contrast to protocols involving iterative rounds of expansion,
other ExM protocols such as X10-ExM,
70Tenfold Robust Expansion
Microscopy (TREx),71and Magnify72achieve an approximately ten-
fold linear expansion in a single round of gel embedding andexpansion. X10-ExM achieves high degrees of expansion by introduc-
ing an alternative gel chemistry to that of classic ExM protocols likeproExM. While proExM relies on a gel composition consisting of
sodium acrylate (SA), acrylamide, and N,N
0-methylenebisacrylamide,
X10 employs a different formulation based on the introduction of N,N-dimethylacrylamide acid (DMAA) to form polymer chains, com-bined with SA for cross-linking these chains. When exposed to de-ionized water, this distinctive composition enables the resulting gel toexpand over 10 /C2in each dimension. Unlike classical ExM protocols,
X10 requires nitrogen degassing during its gel preparation process toeliminate molecular oxygen, which would otherwise inhibit the poly-merization reaction. X10 and classical ExM protocols share a common
approach for protein retention within the gel, using Acryloyl-X. In the
development of TREx, a systematic investigation compared variouspublished gel protocols, pinpointing the optimal balance betweenexpansion factor and gel stability. By introducing a high monomercontent alongside a reduced initiator concentration, TREx demon-strated an approximate 10 /C2expansion factor in a single round of
expansion while maintaining enhanced mechanical hydrogel stabilityand achieving a slower and more controlled polymerization rate,
which facilitates a homogenous polymerization across the tissue speci-
men. The systematic analysis also addressed variations in expansioncapabilities among different tissue types, proposing a framework forfine-tuning the crosslinker concentration. TREx is compatible withboth the expansion of cells and tissues. Magnify
72is another technol-
ogy that expands the anchoring capabilities of ExM by incorporating anovel hydrogel formula and anchoring strategy capable of preserving adiverse range of biomolecules. In contrast to classical ExM protocols,
which have relied on anchoring molecules specific to particular bio-
molecule classes, such as proteins or RNA, the introduction of metha-crolein represents a major difference. Methacrolein is a small moleculecapable of integrating a diverse range of biomolecules, including pro-teins, nucleic acids, and lipids, all at once into the gel. Methacroleinhas been used in fixation protocols and chemically modifies biomole-cules similarly to formaldehyde, introducing an isopropenyl functionalgroup that actively participates in the in situ polymerization step.
Magnify has been applied to cells and tissues.
E. Potential and challenges
ExM has emerged as a widely adopted and established technol-
ogy, offering extensive validation and boasting a large community ofusers. Its ease of implementation and robustness make it highly attrac-tive, with applications spanning various fields such as microbiology,
clinical pathology, autoimmune disease, cancer biology, neurobiology,
degenerative disease, inflammatory disease, cell biology, and manyothers. As a specimen processing technique, one of the key strengthsof ExM lies in its compatibility with a broad array of additional SRMtechnologies. This versatility allows ExM to be seamlessly integratedinto existing microscopy setups, offering the potential to enhance theresolution of both standard microscopes and super-resolution meth-ods. Despite these advantages, ExM presents several challenges and
potential limitations that may require careful consideration to be suc-
cessfully combined with other SRM methods [ Fig. 2(a) ].
One important consideration in ExM, especially when combined
with additional SRM, is the impact on fluorophores and endogenousfluorescent proteins. It has been observed that signal intensity maydecrease after ExM, primarily due to fluorophore degradation duringApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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free-radical polymerization and dilution of fluorescent dyes, particu-
larly in pre-expansion labeling.37,73,74For instance, a 4 /C2linear expan-
sion results in a 64 /C2dilution of fluorescent dyes. This dilution effect is
more pronounced in ExM protocols that introduce higher degrees ofexpansion. Additionally, not all dyes are compatible with ExM. Forexample, cyanine dyes like Alexa Fluor 647, commonly used in SRMmethods for their blinking capabilities, often fully degrade during thegelation process.
37,75
Another factor to consider, especially for endogenous proteins, is
degradation due to the digestion process. For example, some fluores-
cent proteins are prone to degradation when using Proteinase-based
digestion. While bbarrel-shaped endogenous proteins such as GFP are
generally more protease-resistant, other endogenous fluorescent pro-teins may be more prone to degradation.
37,38,41Photobleaching is
another significant factor in ExM, influenced by degradation, dilution,and the incompatibility of many fluoroprotective mounting mediawith ExM.
76
The classic polyacrylamide-based hydrogels, the most widely
adopted method for ExM, achieve their maximal expansion when
incubated in ddH 2O.36The expansion is reversible and can be adjusted
by adding more osmolytes to the expansion solution. For example,while incubation in ddH
2O can achieve an approximately4/C2expansion factor, incubation in phosphate-buffered saline causes
the expanded gel to shrink to a final expansion factor of around
2–3/C2.41The dependence of some SRM methods on specific buffers
that modify fluorophore states may, therefore, pose a challenge whencombined with ExM.
ExM presents challenges related to the sensitivity of expanded
hydrogels to temperature and hydration. Prolonged imaging sessionscan lead to dynamic distortions, shrinkage, or micromovements in theexpanded samples. Proper mounting of specimens can be challenging,increasing the likelihood of lateral movements during imaging.
77
Linkage error is an important consideration in ExM, referring to
the systematic offset between the fluorescent probe ’s observed location
and the target protein ’s actual position introduced by the labeling strat-
egy [ Fig. 2(b) ]. For indirect immunolabeling using primary/secondary
antibody complexes, this offset can be as much as 15 –20 nm spa-
tially.78–81Strategies to reduce linkage error in SRM and ExM include
nanobody-based labeling approaches, genetic modifications of thespecimen to express fluorescent proteins, self-labeling protein tags,grafting of peptide ligands, or post-expansion labeling in expansion-
based methods.
22,48,82 –85Additionally, the degree of fluorescent label-
ing (DOL) of each tag is crucial to consider. Indirect immunolabelingwith secondary antibodies carrying two to five fluorescent markers can
FIG. 2. Expansion workflow and challenges when combining ExM with additional SRM. (a) Graphical overview of the ExM workflow, including critical steps and p otential limita-
tions when combining ExM with SRM. After fluorescent labeling, anchoring treatment is performed followed by monomer infusion and polymerization re sulting in a sample-
hydrogel hybrid. The sample is next homogenized and expanded by incubating the specimen in de-ionized water. The expanded specimens are then mounted and imaged.
Potential challenges and limitations in the ExM workflow when combined with other SRMs are highlighted stepwise through numbers (1) –(7). (b) Illustration of linkage error with
pre-ExM labeling and fluorophore dilution in non-expanded (pre-ExM) and expanded (post-ExM) samples. Expansion results in increased linkage erro r as well as a lower num-
ber of fluorophores per field of view. (c) Lateral drifts and hydrogel distortions during image acquisition result in diminished image quality and ar tifacts particularly when com-
bined with methods requiring time-stacked image acquisition. Reproduced with permission from Kylies et al. , Nat. Nanotechnol. 18, 336 –342 (2023). Copyright 2023 Authors,
licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ .Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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result in a higher fluorescence signal, while smaller tags with DOL
close to one yield lower signal levels.
Another factor to consider when combining ExM with other
SRM methods is the spatial heterogeneity of expanded gels present at
the nanoscale. Conventional polyacrylamide-based hydrogels may
exhibit reduced fidelity at scales below 20 nm, which could be a signifi-
cant factor in their combination with SRM techniques.86,87
One more challenge arises when combining Expansion
Microscopy (ExM) with super-resolution microscopy (SRM) for 3D
imaging. While the transparency and refractive index homogeneity of
ExM hydrogels mitigate issues like light scattering, careful consider-
ation must still be given to sample thickness, lens compatibility, and
chromatic aberrations, especially at greater imaging depths.Techniques such as STED are well-suited for imaging expanded sam-
ples due to their longer working distances, particularly when paired
with water immersion objectives.
88,89These objectives enhance imag-
ing depth and minimize refractive index mismatches with the hydro-
gel. While effective for thinner samples, SIM may suffer fromchromatic aberrations as imaging depth increases.
90Methods like pho-
toactivated localization microscopy (PALM) and stochastic optical
reconstruction microscopy (STORM) are most effective for thin sam-ples (under 10 lm) within a single imaging plane, as they are highly
sensitive to optical distortions.
91To address the challenges posed by
thicker expanded samples, lower numerical aperture (NA) water
immersion lenses were suggested to improve light penetration and
reduce aberrations, though with some loss in resolution.47,49
Alternatively, cryosectioning expanded tissues can produce thinner
sections that are compatible with high-resolution imaging techniques
while preserving structural integrity,92albeit at the cost of increased
sample preparation complexity.
III. COMBINING ExM WITH SRM
Integrating ExM with SRM techniques offers the potential for sig-
nificant enhancements in resolution. For instance, combining the
widely adopted fourfold ExM protocols with open-source super-reso-
lution technologies could yield resolutions comparable to expensivecommercial SRM systems, all while retaining the flexibility and effi-
ciency of conventional setups. Moreover, pairing advanced expansion
protocols, which achieve higher degrees of expansion, with SRM meth-ods or employing sophisticated optical SRM techniques alongside
ExM can push resolution boundaries into the single-digit nanometer
range.
The modular nature of these combined methods allows research-
ers to tailor their SRM pipeline to meet their specific experimentalneeds, while at the same time using familiar equipment. This facilitates
data interpretation and troubleshooting and enables the creation of
custom SRM workflows. Furthermore, the addition of ExM can
enhance resolution both, laterally and in the z axis, significantly
benefiting 3D SRM imaging, regardless of the chosen secondary SRMmethod.
However, SIM and STED, relying on optical system optimization,
and SMLM and FF-SRM, which depend on fluorophore properties
like photo-switchability and photo-convertibility, present different
challenges when combined with ExM. This section explores the princi-ples and applications of combined ExM and SRM approaches, empha-
sizing their strengths, challenges, limitations, and potential
troubleshooting strategies. Its goal is to offer a comprehensive under-
standing of these methods, assisting readers in choosing a combinedpipeline for their research requirements. The methods are ordered by
the level of achieved resolution, and Table I offers an overview of com-
bined ExM and SRM techniques. Supplementary material Fig. 1 fur-
ther illustrates the challenges and pitfalls of the various combined
methods.
A. Fluorescence fluctuation-based super-resolution
microscopy
Fluorescence fluctuation-based super-resolution microscopy
(FF-SRM) relies on analyzing fluorescence fluctuations over time in a
continuous high-speed time-lapse image series, in some aspects similarto the workflow of SMLM. However, FF-SRM generally requires aconsiderably lower number of frames (typically in the orders of10–100/C2less) to render a super-resolution image compared to SMLM,
which typically requires the acquisition of several thousands of frames.Additionally, unlike SMLM, which generally requires discrete photo-switching of fluorophores, FF-SRM methods are compatible with bothefficient and non-efficient photoswitching fluorophores. This makes
the method compatible with a wider range of fluorescent dyes and
requires less complex sample preparation.
27,29In most applications, on
its own, FF-SRM achieves a resolution in the range of 50 –150 nm
depending on the algorithm and microscope used as well as on the par-ticular experimental setup.
26,27,103FF-SRM methods are compatible
with a broad spectrum of microscopes, including epifluorescence, con-focal, and total internal reflection fluorescence (TIRF) microscopes andwith both fixed and live samples in a multicolor setting.
27,103
One of the first FF-SRM methods, Super-Resolution Optical
Fluctuation imaging (SOFI), was introduced in 2009 and has sincebeen widely adopted by the community.
27,28,104Among other FF-SRM
methods developed,27,105 –111another widely adopted FF-SRM algo-
rithm, Super-Resolution Radial Fluctuations (SRRF), was introducedin 2016.
29The SRRF framework stands out for its user-friendliness,
resolution, fidelity and modular implementation of computation qual-ity control algorithms.
112Of note, an updated version called enhanced
SRRF (eSRRF) with increased resolution and fidelity, as well as with an
added 3D image data analysis feature, was recently introduced.113
B. Combining FF-SRM with ExM
FF-SRM algorithms are promising SRM tools for the combina-
tion with ExM achieving resolution ranges of 25 nm when combinedwith a fourfold ExM protocol
50,93a n dh i g h e rw h e nc o m b i n e dw i t ha
higher-fold ExM protocol.72,94Both FF-SRM algorithms and ExM pro-
tocols are open-access and can be easily implemented across variousmicroscopy setups. They offer flexibility in both sample preparationand data analysis, thus supporting the wider accessibility of higher res-olutions in SRM. Unlike some techniques, FF-SRM algorithms do notrequire specialized fluorophores or buffer systems, allowing forstraightforward application with standard immunofluorescence proto-cols.
27Many FF-SRM algorithms were originally designed to be com-
patible with live-cell imaging103and therefore do not require high
illumination. This makes them well-suited for use with ExM as it
reduces photobleaching and laser-induced gel distortions in expandedhydrogels. In addition, their data analysis workflows are often inte-grated into user-friendly platforms such as FIJI/ImageJ plugins.
29,113
Despite these advantages, combining FF-SRM algorithms with
ExM can still present several challenges that need to be addressed.Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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TABLE I. Overview of the combined methods.
SRM
categoryProtocols,
references ExM and SRM methodsLateral resolution and applied
sample type Advantages and challenges
FF-SRM ExSRRF50/C244-Fold ExM þSRRF /C2425 nm resolution
Clinical and experimental
tissuesAdvantages :
1. No special buffers and fluorophores
are required
2. No specialized microscope
is required
Challenges and limitations :
1. Motion artifacts during time-stack
image acquisition
2. In general, lower lateral resolution
compared to ExM þSTED and SMLMExFEAST93/C244-Fold
ExMþAiryscan þSRRF/C2425 nm resolution
Cells and clinical tissues
Magnify72/C2410-Fold ExM þSOFI /C2415 nm resolution
Human lung organoids
ONE94/C2410-Fold ExM þSRRF Resolution not explicitly men-
tioned but single-digit nanome-
ter resolution implied.
Isolated proteins, cells, and
experimental tissues
SIM ExSIM49,92,95/C244-Fold ExM þSIM /C2425–30 nm resolution
Microorganisms,49
Drosophila ,92and cells95Advantages :
1. No special buffers and fluorophores
are required
2. High image acquisition speed
Challenges and limitations :
3. Specialized microscope required
1. Low signal to noise ratio
2. Limited to thin samples, serial cryosec-
tioning for thicker samples required
3. Spherical aberrations
4. Adjustments of the refractive index of
oil and water necessary
STED ExM þSTED96–98
U-ExM þSTED69
ExSTED47/C244-Fold ExM þSTED /C2410–20 nm resolution
Experimental tissues,96,98
cells,47,98isolated organelles,69
and microtubules97Advantages :
1. High lateral resolution (below 10 nm)
2. No special buffer is required
Challenges and limitations :
3. STED-compatible fluorophores
required
4. Increased photobleaching
5. Low signal-to-noise ratio
6. Signal amplification strategies may
increase linkage errorX10ht-STED99/C2410-Fold ExM þSTED
(cells and vesicles)
/C246-Fold ExM þSTED
(tissues)/C246–8 nm resolution
Cells, vesicles, and experimen-
tal tissues
SMLM Ex-SMLM48
ExSTORM100
LR-ExSTORM101/C242–3.4-Fold
ExMþSTORM/C244–10 nm resolution
Microtubules and centrioles,48
meiotic chromosome100
Clathrin-coated pits101
Technical validation of hydro-
gel expansion fidelity87Advantages :
1. High lateral resolution (below 10 nm)
Challenges and limitations :
1. SMLM-compatible fluorophores
required
2. Specific buffers required that
may limit the expansion factor
3. Preserving epitopes for post-expansion
labeling, which is required to avoid loss
of fluorescence
4. Protein digestion step should be
adjusted to preserve fluorescent proteinsEx-PALM
(SExY)102/C245-Fold ExM þPALM YeastApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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Specifically, issues may arise from artifacts generated by lateral drifts
and hydrogel distortions during the time-lapse image acquisition
required for FF-SRM algorithms [ Fig. 2(c) ], or from analytical defects
introduced by the algorithms themselves.112Furthermore, the addi-
tional resolution gained when combining ExM with FF-SRM is gener-
ally lower than when combined with other SRM methods such as
SMLM and STED. Despite these challenges, the potential of combining
FF-SRM with ExM has recently increasingly been recognized and
implemented in different scenarios.50,72,93,94
Kylies et al. developed a novel, modular, and open-source super-
resolution pipeline termed expansion-enhanced super-resolution
radial fluctuations (ExSRRF)50that is optimized for the analysis of
clinical and experimental pathology tissue specimens, leveraging the
most widely adopted fourfold ExM protocol for pathology speci-
mens37,63in combination with the FF-detection algorithm SRRF.29
ExSRRF achieves a lateral resolution of 25 nm and, when used withconventional widefield microscopes, allows for imaging at various
scales, from entire tissue overviews to nanoscale compartments [see
Fig. 3(a) ]. This facilitates molecular profiling of subcellular structures
in archival formalin-fixed paraffin-embedded tissues from complex
clinical and experimental specimens, including those from ischemic,
degenerative, neoplastic, genetic, and immune-mediated disorders.
Furthermore, the authors have shown the potential to combine
ExSRRF with customized and open-source image segmentation and
analysis algorithms, making it a flexible, robust, scalable, accessible,
and adoptable open-source SRM platform.
Another example of combining the FF-based SRM algorithm
SRRF with a fourfold ExM protocol is ExFEAST [Expansion
fluctuation-enhanced Airyscan technology (FEAST)] developed by
Wang et al.
93In contrast to ExSRRF, which focused on the applicabil-
ity of tissue analysis in a clinical and experimental pathology context,
therefore combining ExM and SRRF with LED-based WF microscopes
as they represent the most commonly used microscopes in clinical andexperimental pathology, ExFEAST applied a similar principle to laser-
based confocal microscopes using the Airyscan technology,
25achieving
a lateral resolution of 26 nm. This modular technology was applied to
resolve subcellular architecture, such as the cytoskeleton of cells and in
human breast cancer samples, where it aided in enhancing the accu-
racy of counting the average human epidermal growth factor receptor
2 (HER2) copy number for diagnostic purposes.
Klimas et al. demonstrated that the combination of SOFI and the
novel ExM protocol Magnify achieves an 11-fold expansion factorwhile retaining nucleic acids, proteins, and lipids without requiring a
separate anchoring step.
72While using only diffraction-limited micro-
scopes, Magnify achieves a lateral resolution of 25 nm. However, when
combined with SOFI (Magnify-SOFI) the authors reported a theoreti-
cal lateral resolution of around 13 nm under optimal conditions.
Among other applications, one example of the improved effective reso-
lution was demonstrated by the visualization of basal bodies in humanbronchial basal stem-cell-derived lung organoids that were resolved
with Magnify-SOFI but challenging to resolve with Magnify alone
[Fig. 3(b) ].
The one-step nanoscale expansion (ONE) microscopy method by
Shaib et al. combines tenfold expansion of the specimen with the fluo-
rescence fluctuation detection algorithm SRRF, enabling the detailed
description of cultured cells, viral particles, molecular complexes, andeven the structure of single proteins such as antibodies [ Fig. 3(c) ].
94Furthermore, while not the main focus of the ONE microscopy manu-
script, its applicability was also demonstrated in tissues. In comparison
to other methods that combined FF-detection with ExM, ONE micros-
copy requires a larger number of frames (1000 –2000) to generate a
reconstruction, needed for the high-order correlations used. In con-trast, ExSRRF was shown to use as low as 100 frames for a reconstruc-tion. A lateral resolution of around one nanometer was reported.
94
Taken together, these studies highlight the potential of combining
ExM with FF-SRM to achieve nanoscale resolution across a wide vari-ety of sample types and research questions, ranging from pathology
specimens and cells to the structural analysis of proteins at a
nanoscale.
C. Structured illumination microscopy
Structured illumination microscopy (SIM) is a commonly used
technique for enhancing resolution. For simplicity, we here refer to
SIM as an SRM (super-resolution microscopy) technology. While clas-sical SIM is still fundamentally bound by diffraction principles it canstill double the resolution of conventional microscopy.
114
In life sciences, SIM has been applied for live cell and tissue imag-
ing, achieving a resolution in the range of 100 –120 nm.16,17,115 –119The
key principle behind SIM is the excitation of the sample with patternedillumination. These patterns interact with the fluorescently labeled
sample, creating a moir /C19e effect that encodes high-frequency informa-
tion into the resulting image.
16By capturing multiple images with the
illumination pattern shifted or rotated and then computationally proc-essing these raw images, it is possible to reconstruct a super-resolved
image that effectively doubles the lateral resolution compared to a con-
ventional widefield. There are two main variants of SIM: optical sec-tioning SIM (here termed OS-SIM) and enhanced-resolution SIM(here termed SR-SIM).
90OS-SIM uses structured illumination to sec-
tion the sample optically, removing out-of-focus light and improving
contrast,120,121while SR-SIM specifically aims to enhance resolution
beyond the classical diffraction limit.122SR-SIM requires that the emit-
ted light is incoherent from the excitation, which is most commonlyused in fluorescence microscopy. SR-SIM works by controlling theexcitation in the sample plane, often by generating interference pat-
terns with a periodicity near the diffraction limit that can be readily
combined as a postprocessing step.
122Therefore, image postprocessing
is a critical step for SR-SIM.90,122SIM is a relatively accessible SRM
approach that can be implemented as an upgrade to existing widefield
microscope platforms.123Advancements in SIM hardware and recon-
struction algorithms continue to expand its capabilities, making it anindispensable tool for modern microscopy.
90,123 –126However, SIM is
sensitive to sample movement and requires careful alignment of theillumination patterns.
90,127The computational processing required to
reconstruct the final enhanced-resolution image can be time-
consuming and will introduce artifacts if not performed correctly.128
D. Combining SIM with ExM
SIM technology is well-suited for combination with ExM due to
its ease of use and compatibility with multicolor imaging. SIM does
not require specialized fluorophores or buffers, making it highly versa-tile. It offers fast image acquisition compared to other SRM methods,and its data analysis is relatively straightforward.Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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FIG. 3. ExM combined with FF-SRM and SIM. (a) ExSRRF allows nanoscale analysis in clinical and experimental tissues using conventional widefield microscop es by combin-
ing a classical fourfold ExM protocol with SRRF, crossing scales from tissue overviews to nanoscale compartments, allowing the visualization of mit ochondria and their cristae.
(b) The addition of SOFI to the tenfold ExM protocol Magnify further enhances the resolution allowing a clear visualization of basal bodies in human br onchial basal stem-cell-
derived lung organoids. (c) The ONE microscopy technology combines a tenfold ExM protocol with SRRF allowing the visualization of nanoscale structu res such as antibodies
labeled with fluorescent NHS-ester. (d) Combining ExM with SIM enabled the visualization of the adhesive disk and flagellar axonemes in Giardia lamb lia. (e) ExM and SIM
resolved mitochondria at visually higher resolutions than confocal microscopy, SIM and ExM alone. Reproduced with permission from (a) Kylies et al. , Nat. Nanotechnol. 18,
336–342 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ ; (b) Klimas
et al. , Nat. Biotechnol. 41, 858 –869 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/
licenses/by/4.0/ ; (c) Shaib et al. , Nat. Biotechnol. (2024). Copyright 2024 Authors, licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License http://creativecommons.org/licenses/by-nc-nd/4.0/ ; (d) Halpern et al. , ACS Nano 11(12), 12677 –12686 (2017). Copyright 2017 American Chemical
Society; (e) Kunz et al. , Front. Cell Dev. Biol. 8, 617 (2020). Copyright 2020 Authors, licensed under a Creative Commons Attribution License (CC BY) https://creativecom-
mons.org/licenses/by/4.0/ .Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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In general, the resolution range achieved when combining SIM
with ExM has been reported to be 25 –30 nm.49,92Some authors have
discussed the potential advantages of combining ExM and SIM
(ExM-SIM) over other established SRM techniques, particularly
SMLM without ExM, as their resolution ranges are similar.49One
potential advantage of combining ExM with SIM is the easier imple-
mentation of 3D imaging. An advantage is the potentially better reso-
lution and fidelity of ExM-SIM in situations with high labeling densitycompared to SMLM or FF-SRM methods. While SMLM and FF-SRM
perform optimally when imaging sparsely labeled specimens such as
thin layers of microtubules or nuclear pores,
129,130they face challenges
with dense samples, leading to artifacts and worse spatial resolution in
dense objects.49,131,132Additionally, ExM-SIM has a considerably
shorter data acquisition time ( <1 min/channel) compared to SMLM
(>20 min/channel) due to the lower number of images required per
image stack. It has also been discussed that ExM-SIM is compatible
with a wider range of fluorophores than SMLM, which often requires
fluorophores with photoswitching abilities.26,49However, challenges
remain. Sample preparation and data acquisition parameters should beadjusted to keep photobleaching artifacts to a minimum since
expanded specimens are substantially dimmer than unexpanded ones
and since 3D SIM requires 15 exposures of the sample per focal
plane.
49Most commercial SIM systems use oil immersion objectives,
which can introduce artifacts when imaging deeper into hydrogels due
to refractive index mismatches.49,90
Halpern et al. employed a hybrid imaging approach, termed
ExSIM, combining expansion with SIM, achieving a lateral and axial
resolution of approximately 30 and 75 nm, respectively.49This method
was applied to investigate the cytoskeleton of Giardia lamblia, a human
pathogen, focusing on the adhesive disk and flagellar axonemes,
highlighting ExSIM as a simple and robust modular method for study-
ing biological specimens [ Fig. 3(d) ]. Due to the limitations mentioned
above, the authors applied this method to modestly thick specimens,
approximately 8 lm in post-expansion dimensions. While similar to
many classical ExM protocols,37t h eE x Mp r o t o c o la p p l i e di nt h i ss t u d y
was modified to contain an increased amount of acrylamide to achieve
enhanced mechanical stability while still expanding isotropically,
resulting in a final expansion factor of approximately 3.5-fold. This
enhanced mechanical stability facilitated the reliable detaching of
adherent gel-embedded specimens from their original substrates and
overall handling without introducing damage. The authors also intro-
duce a poly- L-lysine strategy for enhanced attachment of hydrogels to
cover glass substrates, applied in other workflows.50
Cahoon et al. combined a fourfold ExM protocol with SIM to
enable 3D analysis of the drosophila synaptonemal complex (SC),which has previously proposed a challenge even for advanced SRM
m e t h o d ss u c ha sS T E D .
92By combining ExM with SIM, the authors
took advantage of the resolution increase in all three spatial dimen-
sions that ExM provides, thereby enabling sufficient Z-resolution for
3D analysis of the SC. To overcome the limitations in the working dis-
tance of the SIM microscope in large and expanded specimens,
Cahoon et al. proposed a method of cryosectioning. This involved
dehydrating the ExM hydrogels to induce shrinkage, embedding them
in tissue freezing media, cryosectioning into 10- lm-thick slices, and
re-expanding the sectioned hydrogels. This approach produced sam-
ples with a post-expansion thickness of approximately 40 lm, compat-
ible with the SIM microscope ’s working distance, thereby facilitating3D analysis using ExM-SIM. The authors reported a lateral resolution
of/C2425 nm and an axial resolution of /C2450–60 nm with the ExM-SIM
approach.
Kunz et al. combined ExM with SIM to study morphological
changes of the mitochondrial cristae as well as the localization of mito-chondrial proteins relative to the mitochondrial cristae [ Fig. 3(e) ].
95In
this study, the authors showcase the utility of a mitochondrial creatinekinase (MtCK) construct linked to a green fluorescent protein (GFP)as a marker for mitochondrial cristae. This MtCK –GFP fusion protein
localizes to the space between the outer and inner mitochondrial mem-
branes, making it a reliable marker for cristae. By applying ExM incombination with SIM to mitochondria labeled with this construct, theresearchers achieved around 30 nm resolution and were able to visual-ize morphological changes in cristae.
All these studies demonstrate the potential of combining ExM
with structured illumination microscopy (SIM) for addressing complexbiological questions. This approach offers significant advantages, suchas enhanced resolution and 3D imaging capabilities.
E. Stimulated emission depletion
Stimulated emission depletion (STED) microscopy is a powerful
super-resolution imaging technique. Introduced in the 1990s by Hellet al., it became one of the earliest approaches to overcome the diffrac-
tion limit of conventional optical microscopes.
15,133STED microscopy
relies on two lasers that form a donut shape. The first laser, known asthe depletion laser, switches off the fluorescence of dye molecules at
the edges of the excitation laser ’s focal point. Consequently, only the
fluorophores near the center of the depletion beam are allowed to emitfluorescence in their original spectral emission range, reducing theeffective size of the detected point spread function (PSF). This processeffectively enhances the resolution of the microscope. By carefully con-trolling the intensity and shape of the depletion laser, state-of-the-art
STED microscopes have been shown to achieve lateral resolutions in
the range of 20 –30 nm.
15,133 –135
STED microscopy has been widely adopted in various fields of
biology, from cell biology to neuroscience, enabling researchers to
visualize intricate cellular structures, track the dynamics of proteins
and organelles, and study the organization and function of biologicalsystems with unprecedented detail.
136,137Examples of the successful
application of STED include the study of synaptic structure and func-tion, as well as the investigation of membrane-associated proteins andtheir interactions. Additionally, STED has also been applied for in vivo
imaging.
138–141Key advantages of STED microscopy include its high
spatial resolution and compatibility with 3D image acquisition.Limitations and challenges of STED microscopy include the require-ment of specialized and expensive equipment, including a high-powerpulsed laser for the depletion beam and complex optical setups.
142
Additionally, the high-intensity depletion laser can photodamage the
sample.143Despite these challenges, STED microscopy continues to be
a valuable tool in modern biological imaging techniques ’arsenal, help-
ing researchers achieve a deeper understanding of the complex pro-cesses in cells and tissues.
F. Combining STED with ExM
STED microscopy generally provides higher spatial resolution
than FF-SRM and SIM, unlike SMLM, does not require special buffersApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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and eliminates out-of-focus light with its confocal setup.
Combinations of ExM and STED have been reported to achieve lateral
resolutions below 10 nm.47,99However, fluorophore degradation, dilu-
tion, and the lack of photoprotective mounting media can interfere
with STED imaging when combined with ExM. Therefore, the key
challenges are increasing and retaining fluorescent signals in expanded
samples.
Unnersj €o-Jess et al. demonstrated the applicability of a combina-
tion of the ca. Fivefold expansion protocol MAP and STED in intact
tissue specimens.96Through a combination of ExM and STED, the
authors were successfully able to resolve two proteins of the renal slit
diaphragm simultaneously at resolutions of below 20 nm, highlighting
the robustness and versatility of this combined approach in tissues
[Fig. 4(a) ].
Gao et al. applied a combination of a fourfold ExM with STED to
resolve the cytoskeleton of cells at high spatial resolution47[Fig. 4(b) ].
To overcome the limitations of low signal intensity after specimen
expansion, a combined and optimized labeling method to maximize
epitope coverage was introduced. This involved the expression of
a-tubulin –GFP in combination with the use of antibodies against GFP,
a-tubulin, and b-tubulin. This approach resulted in approximately a
fourfold increase in fluorescence signal for microtubule labeling after
gel expansion. In order to achieve better axial resolution, the authorsfirst, used the oil immersion lens close to the surface ( <5lm). In this
configuration, they achieved an isotropic resolution of 45 64n m .
After overcoming the limitations of mismatched refractive indices
between the hydrogel and objective immersion oil, the authors
employed two strategies: the first strategy involved the use of a 1.2 NA
60/C2water objective, which slightly reduced the isotropic resolution to
70 nm but allowed for the complete resolution of the microtubule net-
work. The second strategy involved immersing the sample in a sucrose
solution to match the refractive indices of the hydrogel and oil, though
this caused the shrinking of the gel reducing the expansion factor by
approximately 10%. Ultimately, the resolution achieved was similar for
both approaches. Interestingly, it was reported that the use of sucrose
was associated with a lower rate of photobleaching. The lateral resolu-tion in this protocol was reported to be below 10 nm and 50 –70 nm in
the z axis. Limitations to this approach discussed by the authors
include photobleaching, and long image acquisition times, especially
for the 3D image acquisition of large expanded volumes.
47
Another attempt to combine ExM with STED was performed to
reveal the molecular architecture of centrioles, by using the protocol
for preserving ultracellular structure (U-ExM).69The authors used the
adaptive-illumination scan technique DyMIN (Dynamic Intensity
Minimum), which uses reduced light dose, decreasing photobleaching.For labeling, immunostaining with antibodies conjugated to STAR
FIG. 4. ExM combined with STED and SMLM. (a) Application of combined ExM and STED in kidney tissues. The combination of ExM and STED visually achieved higher re so-
lutions than the combination of ExM and confocal and enabled the localization of single podocin and nephrin molecules in the filtration slit. (b) ExST ED visualizes cilia in mam-
malian epithelial MDCK cells. Schematic of motile cilia and cross-sectional and longitudinal views of motile cilia resolved with ExSTED. (c) Compar ative analysis of the meiotic
chromosome resolved with STORM and ExSTORM revealed significantly higher resolutions for ExSTORM images as shown by sharper peak distributions in t he intensity line
profiles. (d) Centrioles resolved by Ex-SMLM. 3D dSTORM of expanded Chlamydomonas centrioles reveals the ninefold symmetry at higher resolution an d fidelity compared to
conventional dSTORM without expansion. Reproduced from (a) Unnersj €o-Jess et al. , Kidney Int. 93(4), 1008 –1013 (2018). Copyright 2018 Authors, licensed under a Creative
Commons CC-BY-NC-ND license https://creativecommons.org/licenses/by-nc-nd/4.0/ ; (b) Gao et al. , ACS Nano 12(5), 4178 –4185 (2018). Copyright 2018 Authors, licensed
under a Creative Commons CC-BY license https://creativecommons.org/licenses/by/4.0/ ; (c) Xu et al. , Proc. Natl. Acad. Sci. U. S. A. 116(37), 18423 –18428 (2019). Copyright
(2019) Authors, licensed under National Academy of Sciences https://www.pnas.org/pb-assets/authors/authorlicense-1633461587717.pdf ; (d) Zwettler et al. , Nat. Commun. 11,
3388 (2020). Copyright 2020 Authors, licensed under a Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ .Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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Red/STAR 580 was used. These dyes are known for their brightness
and photostability. Combining these properties, U-ExM coupled with
STED imaging demonstrated the chirality of the centriole.
Liet al. developed an enhanced labeling strategy to facilitate the
combination of ExM and STED.98Here, the authors re-label the sam-
ple after expansion using biotin –streptavidin interaction. Initially, the
protein of interest was labeled with a biotinylated primary antibody
and a fluorescently labeled secondary antibody. After expansion, fluo-
rescently labeled streptavidin was added to the sample, increasing the
signal brightness, and thereby facilitating the combination of ExM and
STED.
The necessity to implement enhanced labeling strategies is a
known requirement. For example, a labeling strategy uses biotin –
avidin signal amplification,97which exploits the tetrameric structure of
avidin to boost signal strength. Standard immunostaining was per-
formed using biotinylated secondary antibodies and streptavidin con-
jugated with AF488. After expansion, the sample was labeled again
with secondary antibodies conjugated with both biotin and AF488.
Using biotin as a linker for dye-conjugated streptavidin, this strategyboosted the number of fluorophores attached to each antibody target-
ing tubulin, thereby increasing the fluorescent signal sufficiently to use
the highest depletion intensity in STED. Two signal amplification
cycles were needed to achieve sufficient signal for 100% depletion
intensity, resulting in a resolution better than 9 nm. However, the link-
age error increased with each signal amplification cycle, reaching
12 nm. For this reason, AviTag was used to directly biotinylate the tar-get protein. Despite this, depletion intensity greater than 50% could
not be achieved even after two signal amplification cycles.
Saal et al. combined STED with a 10 /C2ExM protocol.
99In order
to overcome limitations of reduced fluorescence intensity in expanded
specimens, strategies to achieve optimized retention of fluorescence
intensity, epitope preservation and label retention were implemented.
A modified digestion step used heat homogenization at 135/C14Cu n d e r
alkaline conditions as opposed to a commonly used proteinase-based
digestion, as well as an optimized anchoring through applying anincreased concentration of the anchoring agent Acryloyl-X. This strat-
egy allowed an up to 11-fold unidimensional expansion under optimal
conditions, and the use of nanobodies as labeling agents by preventing
their washout, enabling multicolor STED imaging at lateral resolutions
below 10 nm. Despite these optimizations, however, the expanded
samples were still significantly dimmer, which can partially be attrib-
uted to the 1000-fold dilution of fluorophores in the expanded volume.Saalet al. devised several methods for post-expansion signal amplifica-
tion to amplify signal intensities. The most effective signal amplifica-
tion, up to sevenfold, was achieved using primary nanobodies carrying
the ALFA-tag, detected by an anti-ALFA nanobody (NbALFA) fused
to a FLAG-tag spaghetti monster (SpaMo36), an engineered GFP with
seven FLAG-tags. These tags were further detected by anti-FLAG anti-
bodies followed by secondary antibodies. The resolution achieved byX10ht-STED is suitable for investigating small structures such as pro-
tein complexes. However, amplification systems can introduce locali-
zation errors, which depend on the type, number, and timing (pre/
post-expansion) of labeling tools (ie. nanobodies and antibodies).
Consequently, the effective resolution varies based on the specific
labeling method.
STED has also successfully been applied as a technical method to
determine the homogeneity of sample expansion.
43,69,74G. Single-molecule localization microscopy
Single-molecule localization microscopy (SMLM) refers to SRM
methodologies that achieve nanoscale resolution by pinpointing the
center of emission of a single fluorescent molecule. This is achieved byinducing fluorophores to switch between transient “on”and “off”
states, a process often termed “blinking. ”These blinking events are
captured in continuous time-lapse image acquisition of typically sev-eral thousand individual frames. The subsequent computational detec-tion of the positions of blinking molecules leads to a high-resolution
image reconstruction of their position in space. Because within each
frame, only a small fraction of the fluorescent dyes is in the “on”state,
the likelihood of two fluorophores spatio-temporally overlapping their
transient emissions is very low. Therefore, each signal detected can be
interpreted as belonging to an individual molecule even when imagedusing a diffraction-limited microscope. Under optimal conditions,SMLM can achieve very high lateral resolutions in the range of as low
as 10 –30 nm. However, SMLM often requires specialized fluorophores
that can be converted into a blinking state through the implementationof tailored imaging buffers, and an optimized labeling density to mini-
mize the spatial overlap of blinking events, which hinders the detection
of individual molecules.
26
Some of the earliest implementations of single-molecule localiza-
tion techniques include the introduction of photoactivated localization
microscopy (PALM) in 2006 by Betzig et al.18Generally, PALM relies
on using photoactivatable or photo-convertible fluorescent proteins
that can be induced to switch into a spectral channel, imaged one at a
time, localized, and then bleached.144–147Stochastic optical reconstruc-
tion microscopy (STORM) leverages similar principles as PALM.However, instead of using fluorescent proteins, in its initial implemen-
tation, STORM used pairs of Cy3 –Cy5 dyes to achieve switchable
behavior.
19A STORM variant dubbed direct stochastic optical recon-
struction microscopy (dSTORM) furthered the concept that conven-
tional photoswitchable fluorescent dyes such as Cy5 and Alexa Fluor
647 can also be reversibly cycled between fluorescent states withoutneeding a paired dye such as Cy3.
21Despite their differences, STORM,
dSTORM, and PALM share the fundamental principle of manipulat-
ing the fluorescence of individual molecules to bypass the diffraction
limit, enabling researchers to visualize cellular structures and processeswith unprecedented detail. The choice of technique often depends on
the specific requirements of the research question and the available
instrumentation and expertise.
H. Combining SMLM with ExM
Combining SMLM with ExM introduces several relevant chal-
lenges that need to be addressed to enable optimal functionality. For
example, the application of blinking buffers that are required in manySMLM methods can interfere with the expansion factor of the hydro-
gels that achieve maximal expansion factors through incubation in de-
ionized water and can shrink when in contact with solutions withhigher osmolarity. Another challenge is the need for specialized fluoro-
phores that can be converted into a blinking state in a hydrogel envi-
ronment and survive the expansion process. Despite these challenges,efforts have been made to combine ExM with STORM and PALM.
Xuet al. combined ExM with STORM to study the molecular
organization of the mammalian meiotic chromosome axis,
100achiev-
ing resolutions in the range of 10 –20 nm and thereby a threefold
improvement in higher resolutions in their combined approachApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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compared to STORM alone [ Fig. 4(c) ]. Additionally, the authors con-
firmed the fidelity of this combined approach using the vector field
method to measure distortion introduced by hydrogel expansion,
revealing only minor spatial distortions in the range of 1% –2% at dif-
ferent scales. To prevent gel shrinkage caused by ionic switching buf-
fers, the authors expanded the gel into a low ionic strength buffer,
achieving a reduced but effective expansion of approximately threefold.
Therefore, the authors demonstrate the possibility of combining ExM
with STORM to increase the resolution.100
Zwettler et al. successfully combined ExM with STORM by
employing a different approach to use blinking buffers in conjunctionwith expanded hydrogels.
48To prevent interactions between ions in
the photoswitching buffer and ionic side groups of the gel that cause
gel shrinkage, the authors re-embed the expanded charged hydrogel
into an uncharged polyacrylamide gel. This stabilized the expanded
hydrogel, albeit at the cost of a slight reduction in the gel expansion
factor of approximately 20%.48,64Overall, this strategy resulted in an
effective increase in spatial resolution as compared to STORM alone
[Fig. 4(d) ]. In addition, Zwettler et al. also tested an alternative strategy
to enable the combination of ExM with STORM by using a spontane-
ously blinking Si-rhodamine dye (HMSiR) to avoid the need for a pho-
toswitching buffer and subsequent re-embedding of the gel, thus in
theory circumventing hydrogel shrinkage. However, the pH of the
double-de-ionized water of /C207.0 adversely affected the blinking char-
acteristics of HMSiR, rendering it insufficient for SMLM. While the
addition of PBS to the expanded hydrogel mitigated this limitation, it
resulted in a considerably lower expansion factor of only around two-
fold, again limiting the spatial resolution of the hydrogel. It should be
mentioned that the authors used post-labeling ExM to reduce linkage
error and increase resolution. In this article, the authors hypothesized
that after 4 /C2expansion, the immunolabeling linkage error of 17.5 nm
(due to primary and secondary antibodies) would reduce to 4.4 nm,
the size of a tubulin monomer. Thus, combining single-molecule local-
ization microscopy (SMLM) with post-expansion labeling could
reduce the linkage error by the expansion factor, enabling fluorescence
imaging with molecular resolution and re-embedding the gel allows
for performing STORM on the expanded sample. The authors success-
fully applied this approach to visualize microtubules and centrioles
using organic fluorophores, achieving minimal linkage error.48
To overcome the problem of loss of fluorescence during expan-
sion, Shi et al. created a trifunctional anchor, a molecule with one arm
for binding to antibodies or SNAP and CLIP tags, a second arm with
methacrylamide for anchoring into the gel, and a third arm with biotin
or digoxigenin for conjugation to an organic dye after expansion.101
This method preserved up to six times higher fluorescence signal com-pared to proExM. The high level of label retention with trifunctionalanchors, along with high labeling efficiency, allowed the authors to
achieve 34 nm resolution with SIM and up to 4 nm effective localiza-
tion precision with STORM and visualize in high detail clathrin-
coated pits. The authors examined commonly used photoswitchable
dyes, such as Cy5, Cy5.5, and AF647, and no loss of brightness or pho-
toswitching kinetics was observed. However, the necessity of using a
photoswitching buffer caused a lower expansion factor of 3 –3.3.
Nevertheless, the authors demonstrated that LR-ExSTORM achieved
higher resolution and revealed far more details than STORM alone.
Vojnovic et al. recently established a specialized method for the
super-resolved analysis of yeast termed “Single-molecule andExpansion microscopy in fission Yeast ”(SExY), combining ExM with
the SMLM technology Photoactivated Localization Microscopy
(PALM).
102The combination of ExM and PALM was chosen for its
use of fluorescent proteins as labeling agents, allowing for high labeling
efficiency and specificity while eliminating the need for a photoswitch-
ing buffer. However, one challenge of this approach of combiningPALM with ExM was the degradation of fluorescent proteins during
the overall expansion process. To overcome these challenges, the
authors implemented an optimized version of the proExM protocol,
significantly enhancing the retention of fluorescent signal from 22% to
around 50% while enabling a fivefold unidimensional expansion. Thiswas achieved by sequential incubation in monomer solutions: starting
with incubating the specimen in a monomer solution that did not con-
tain the initiator Ammonium Persulfate (APS), followed by adding an
APS-activated monomer solution. The sample was imaged at 10 –
80lm depth.
In addition to biological applications, a combination of ExM with
STORM was also used as a validation tool to measure the single-
molecule distortions resulting from hydrogel embedding itself. The
authors used DNA origami technology and STORM to evaluate struc-
tural preservation in hydrogels with different compositions: polyacryl-amide, the most commonly used material in ExM, and tetra-gel, a
hydrogel that does not rely on free-radical chain-growth polymeriza-
tion. The results showed that tetra-gel is more accurate and effective at
preserving molecular structures during the expansion process.
87
IV. DISCUSSION AND OUTLOOK
ExM is an easy and widely used method for achieving nanoscale
imaging using conventional microscopes. To further enhance achiev-able resolution, researchers worldwide are exploring the potential of
combining ExM with additional SRM techniques. Numerous papers
have been published on attempts to combine ExM with SIM, STED,
SMLM, and FF-based SR techniques so far. As it was seen, the success-
ful integration of ExM with SRM is not without challenges. One majorconsideration is the impact on fluorophores and endogenous fluores-
cent proteins, which can suffer from degradation during the gelation
process, partial loss of the protein of interest and fluorophores due to
insufficient anchoring to the gel, and dilution of fluorescent signal due
to the physical expansion of the specimen. This issue is particularlypronounced in higher-fold expansion protocols and can limit the
achievable resolution. Rapid photobleaching of fluorophores and
poorer photophysics of organic dyes in the hydrogel ’s aqueous envi-
ronment also occur, while many SRM techniques rely on bright and
photostable fluorophores. Moreover, linkage errors become a signifi-
cant challenge as resolution increases. Consequently, improving label-
ing strategies is crucial. Another challenge arises from the sensitivity of
expanded hydrogels to specific buffer conditions required by SRMmethods, including SMLM, which can cause the gels to shrink and
compromise the expansion factor.
To address these limitations, various strategies have been devel-
oped. For example, optimized labeling techniques such as post-
expansion labeling and using smaller probes like nanobodies can helpminimize linkage errors and improve signal retention. Moreover, dif-
ferent signal amplification approaches were explored to enhance the
fluorescence signal by maximizing the fluorophores associated with
the molecule of interest.
97,99Finally, to decrease linkage error and pre-
serve fluorescence different tri- and tetrafunctional anchor probeswere developed.
75,101,148 –151Re-embedding expanded gels inApplied Physics Reviews REVIEW pubs.aip.org/aip/are
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uncharged hydrogels has also been employed to stabilize the samples
in the presence of SRM buffers. Additionally, novel gel chemistries and
digestion methods have been explored to enhance the mechanical sta-
bility and fluorophore compatibility of expanded specimens.
Despite these challenges, the combination of ExM with SRM has
already demonstrated its potential to revolutionize our understanding
of biological systems at the nanoscale. From resolving the molecular
organization of the mammalian meiotic chromosome axis100to visual-
izing the cytoskeletal ultrastructure of cells48,97and tissues,50,72,93,99
these hybrid imaging approaches provide unprecedented access to theintricate details of life. As the field continues to evolve, furtheradvancements in labeling strategies, gel compositions, and imaging
technologies are expected to enhance the performance and applicabil-
ity of ExM-SRM pipelines. Developing novel probes, such as trifunc-
tional anchors and self-labeling protein tags, holds promise for
improving signal retention and reducing linkage errors.
Moreover, exploring alternative SRM techniques, such as DNA-
PAINT
152,153and MINFLUX154in conjunction with ExM may open up
new avenues for achieving even higher resolutions and multiplexing
capabilities. Combining DNA-PAINT with ExM presents specific chal-
lenges due to the physical properties of DNA and the requirements of
each technique. DNA-PAINT relies on DNA hybridization, which
requires the presence of metal ions in the solution to stabilize the DNA
duplex. In contrast, the standard protocols for ExM generally involve
removing ions to facilitate the swelling and uniform expansion of the
gel. Therefore, the challenge in combining DNA-PAINT with ExM
arises from the need to balance these requirements —metal ions for
DNA-PAINT and ion-free conditions for optimal gel expansion. The
absence of metal ions during the expansion process could significantly
hinder DNA hybridization, leading to reduced binding speed and poten-
tial misfolding of DNA strands, while using saline buffers necessary for
DNA hybridization could affect the expansion factor, potentially result-
ing in suboptimal sample expansion. Research is needed to identify the
minimum concentration of metal ions that allows DNA-PAINT to
function effectively without causing significant gel shrinkage. This opti-
mization would be crucial for maintaining both the integrity of the
expanded hydrogel and the efficiency of DNA hybridization.
The super-resolution MINFLUX approach has been recently
developed to achieve high-resolution imaging with minimal phototox-
icity and photobleaching. It is based on minimizing the fluorescence
signal from a single molecule by precisely controlling the position of a
donut-shaped excitation laser. This approach allows for the localiza-
tion of individual molecules with high accuracy, enabling the recon-
struction of high-resolution images. MINFLUX has been shown to
achieve resolutions down to 1 –2 nm, making it one of the highest reso-
lution super-resolution techniques currently available.154Therefore, in
theory, combining ExM with MINFLUX has the potential to achieve
Angstrom-level resolution. However, despite this promising prospect,
no work has been published to date on this combination. This lack of
research may be attributed to the high cost and limited accessibility of
MINFLUX technology. Additionally, at such high resolution, sample
heterogeneity and distortion within the hydrogel might become more
apparent, potentially leading to inaccurate data. In this context, tetra-
gels might be more suitable for combining ExM with MINFLUX.
However, the use of these novel gel chemistries is not yet widespread,
due to limitations, such as the need for customized synthesis of the gel
monomers. Another limitation again arises from the labeling strategy,as linkage errors become of increasing concern with higher resolutions
and therefore a significant challenge at Angstrom-level resolution. A
rapidly developing approach involving direct protein labeling with
unnatural amino acids, which can be tagged with any fluorophore
through click reaction may offer promising potential for combiningExM with MINFLUX.
155
Thereby, further improvements in combining ExM with addi-
tional SRM techniques still require the development of new gel chem-
istries for improved structure preservation, enhanced labeling
strategies to minimize or eliminate linkage errors, and fluorophores
capable of withstanding expansion while maintaining their photophys-
ical properties within the hydrogel environment.
Even at its current stage, the synergistic integration of ExM with
SRM offers a powerful and versatile toolbox for nanoscale biological
imaging. By leveraging the strengths of both methods, researchers can
visualize cellular and tissue structures with unprecedented detail, open-
ing the door to groundbreaking discoveries across fields ranging from
neuroscience to pathology. As these techniques continue to mature
and evolve, their potential applications in basic and translational
research are set to expand, transforming our understanding of the
complex processes that govern life at the nanoscale.
SUPPLEMENTARY MATERIAL
See the supplementary material for details as follows: Fig. 1
(workflow and pitfalls of combined methods) further illustrates various
combined workflows and their challenges. A figure legend is embedded
within the supplementary material .T a b l e1( s u m m a r yo fe x p a n s i o n
microscopy protocols) provides further information on different ExM
protocols, including their maximal expansion factor and achieved lat-eral resolution.
ACKNOWLEDGMENTS
This work was supported by the following grants. (1) The Novo
Nordisk Foundation (Young Investigator Award; NNF21OC0066381)
to V.G.P.; (2) The German Research Foundation: CRC/1192 to V.G.P.;
(3) German Federal Ministry of Education and Research: eMed
Consortia Fibromap to V.G.P.; STOP-FSGS-01GM2202A to V.G.P.;
(4) The Else-Kr €oner-Fresenius-Stiftung and the Eva Luise und Horst
K€ohler Stiftung (2019_KollegSE.04 RECORD) to D.K.; (5) The
doctoral college “innovative Promotionsf €orderung im Bereich
translationale Entz €undungsforschung ”(iPRIME) of the Else-Kr €oner-
Fresenius Foundation to M.K.; (6) Intramural funding Clinician
Scientist program to D.K.; (7) the Gulbenkian Foundation (Fundac ¸~ao
Calouste Gulbenkian, H.S.H., D.A., M.D.R., and R.H.); (8) the
European Research Council under the European Union ’sH o r i z o n
2020 research and innovation program (Grant Agreement No.
101001332 to R.H.) and the European Commission through the
Horizon Europe program (AI4LIFE project with Grant Agreement No.
101057970-AI4LIFE and RT-Super-ES project with Grant Agreement
No. 101099654-RT-Super-ES to R.H.); funded by the European Union.
Views and opinions expressed are, however, those of the authors only
and do not necessarily reflect those of the European Union. Neither the
European Union nor the granting authority can be held responsible
for them. (9) the European Molecular Biology Organization
(EMBO-2020-IG-4734 to R.H.); (10) the Fundac ¸~ao para a Ci ^encia e
Tecnologia, Portugal (FCT fellowship CEECIND/01480/2021 to
H.S.H. and Associate Laboratory LS4FUTURE (LA/P/0087/2020,Applied Physics Reviews REVIEW pubs.aip.org/aip/are
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DOI:10.54499/LA/P/0087/2020) to R.H.); (11) the Chan Zuckerberg
Initiative Visual Proteomics Grant (vpi-0000000044 with https://doi.
org/10.37921/743590vtudfp ); and (12) Aarhus University Research
Foundation, AUFF recruiting Grant No. AUFF-E-2022-7-13 to V.G.P
and the Chan Zuckerberg Initiative Essential Open Source Software for
Science (EOSS6-0000000260) to R.H. Schematics were created with
BioRender.com.
AUTHOR DECLARATIONS
Conflict of Interest
The authors have no conflicts to disclose.
Author Contributions
Daria Aristova and Dominik Kylies contributed equally to this
work as co-first authors, and Ricardo Henriques and Victor G. Puelles
contributed equally to this work as co-senior and corresponding
authors.
Daria Aristova: Conceptualization (equal); Formal analysis (equal);
Methodology (equal); Visualization (equal); Writing –original draft
(equal); Writing –review & editing (equal). Dominik Kylies:
Conceptualization (equal); Formal analysis (equal); Methodology
(equal); Visualization (equal); Writing –original draft (equal); Writing
–review & editing (equal). Mario Del Rosario: Methodology (equal);
Visualization (equal); Writing –review & editing (equal). Hannah S.
Heil: Methodology (equal); Visualization (equal); Writing –review &
editing (equal). Maria Schwerk: Methodology (equal); Writing –
review & editing (equal). Malte Kuehl: Conceptualization (equal);
Funding acquisition (equal); Methodology (equal); Visualization
(equal); Writing –review & editing (equal). Milagros N. Wong:
Methodology (equal); Supervision (equal); Writing –review & editing
(equal). Ricardo Henriques: Conceptualization (equal); Funding
acquisition (equal); Methodology (equal); Supervision (equal); Writing
–original draft (equal); Writing –review & editing (equal). Victor G.
Puelles: Conceptualization (equal); Funding acquisition (equal);
Supervision (equal); Visualization (equal); Writing –original draft
(equal); Writing –review & editing (equal).
DATA AVAILABILITY
Data sharing is not applicable to this article as no new data were
created or analyzed in this study.
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