ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 2, pp. 173-187 © Pleiades Publishing, Ltd., 2025.
Published in Russian in Biokhimiya, 2025, Vol. 90, No. 2, pp. 189-206.
173
REVIEW
Techniques for Selective Labeling
of Molecules and Subcellular Structures
for Cryo-Electron Tomography
Evgeny P. Kazakov
1,2,a
*, Igor I. Kireev
1,2
, and Sergei A. Golyshev
1
1
Belozersky Research Institute of Physico-Chemical Biology,
Lomonosov Moscow State University, 119991 Moscow, Russia
2
Department of Cell Biology and Histology, Faculty of Biology,
Lomonosov Moscow State University, 119991 Moscow, Russia
a
e-mail: kazakov.evgeny.2016@post.bio.msu.ru
Received November 7, 2024
Revised January 9, 2025
Accepted January 20, 2025
Abstract— Electron microscopy (EM) is one of the most efficient methods for studying the fine structure of
cells with a resolution thousands of times higher than that of visible light microscopy. The most advanced
implementation of electron microscopy in biology is EM tomography of samples stabilized by freezing with-
out water crystallization (cryoET). By circumventing the drawbacks of chemical fixation and dehydration,
this technique allows investigating cellular structures in three dimensions at the molecular level, down to
resolving individual proteins and their subdomains. However, the problem of efficient identification and local-
ization of objects of interest has not yet been solved, thus limiting the range of targets to easily recognizable
or abundant subcellular components. Labeling techniques provide the only way for locating the subject of
investigation in microscopic images. CryoET imposes conflicting demands on the labeling system, including
the need to introduce into a living cell the particles composed of substances foreign to the cellular chemis-
try that have to bind to the molecule of interest without disrupting its vital functions and physiology of the
cell. This review examines both established and prospective methods for selective labeling of proteins and
subcellular structures aimed to enable their localization in cryoET images.
DOI: 10.1134/S0006297924604015
Keywords: electron microscopy, cryo-electron tomography, correlation microscopy, genetically encoded tags,
gold nanoparticles, quantum dots, ferritin, metallothionein, encapsulins
Abbreviations: cryoEM, cryo-electron microscopy; cryoET,
cryo-electron tomography; EM, electron microscopy;
FKBP, FK506-binding protein; FRB, FKBP-rapamycin bind-
ing domain; NP, nanoparticle; QD, quantum dot; GFP,
green fluorescent protein.
* To whom correspondence should be addressed.
INTRODUCTION
Electron microscopy (EM) has proven to be a
reliable and efficient tool for studying the structure
of cells with a high resolution unavailable in visible
light microscopy. However, preparation of ultrathin
sections of cells requires chemical fixation that of-
ten greatly changes the fine structure of the sample.
Even fixation with aldehydes and osmium tetroxide,
which is considered as the “gold standard” of sample
preparation, is not completely free of artifacts [1-3].
Cryofixation, i.e., sample stabilization by rap-
id cooling under conditions preventing formation
of ice crystals, has come a long way and become
an efficient alternative to chemical fixation. When
a sample is rapidly frozen by immersion in a liq-
uid coolant with a high heat capacity [4] or frozen
under high pressure, diffusion processes in the cell
stop almost instantly [5, 6] and individual protein
molecules and macromolecular complexes are stabi-
lized in their native state, the main characteristic of
which is preservation of hydrated state of biomole-
cules [7, 8].
KAZAKOV et al.174
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
Similar to visible light microscopy, image res-
olution in EM depends on the sample thickness.
The optimal sample thickness in EM is determined
by the mean free path of electron in the sample ma-
terial [9]. For cryofixed hydrated cells and tissues,
this value is 300-400 nm at the accelerating voltage
of 300 kV. Consequently, cell biology studies can
only be conducted in thin peripheral extensions of
cells cultured on substrates transparent to electrons
or require advanced techniques, such as obtaining
sections of cryostabilized specimens with a cryo-ul-
tramicrotome (CEMOVIS, cryo-electron microscopy
of vitrified sections) [10-12] or by milling cryofixed
samples in focused ion beam/scanning electron mi-
croscope (FIB-SEM) to produce a single lamella plate
approximately 200-300 nm thick [13-15]. The sections
obtained by these methods are further examined by
electron microscopic tomography methods.
Tomography is a reconstruction of a three-di-
mensional image based on a series of two-dimension-
al projections of an object obtained at different an-
gles, known as tilt-series [16]. Tomography not only
improves the axial resolution, but also significantly
increases the contrast of resulting images due to the
multiple averaging of brightness values of each vol-
ume element (voxel) in the three-dimensional image.
Cryo-electron tomography (cryoET) allows to study
the structure of cells, down to the three-dimension-
al structure of individual protein molecules in their
native intracellular environment with a resolution
of ~3-5 Å, which has been already achieved by using
the subtomogram averaging technique [17, 18].
An EM image simultaneously shows lipid, pro-
tein, and nucleoprotein structures, aqueous contents
of the vacuoles, and polysaccharides of the cell walls,
so a researcher often faces the problem of detecting a
molecule-sized object of interest against this complex
and detail-rich background. This problem is solved
relatively easily if this object is abundant (ribosomes),
stands out from the background by its geometry (pro-
teasomes and subcellular formations of viral origin),
or is associated with certain membrane or cytoskel-
etal components. At the time of writing this review,
according to PubMed, the studies on such objects
accounted for 42% (732 out of 1728) of publications
using cryoET). However, when the goal is to localize
less represented proteins or to detect zones where the
processes under study occur, researchers often find
themselves in a difficult situation [18-20].
The problem of the object localization in a micro-
scopic image can be solved by using labeling methods.
Ongoing progress in the methodology of cell ultra-
structure visualization by the cryo-electron microsco-
py (cryoEM) requires the development of compatible
labeling methods [20-22]. Localization of proteins by
visible light microscopy has been achieved by using,
first, fluorescently labeled antibodies in fixed cells
and, then, genetically encoded fluorescent labels in
living cells. The problem of protein localization at the
ultrastructural level lacks such elegant solutions even
for the EM of ultrathin sections. Immunogold tech-
niques, enzyme labels, and selective contrasting by
photooxidation are employed in this field with vary-
ing success. However, all these methods rely on chem-
ical fixation, which is sometimes extremely destruc-
tive for cellular substructures, as it has been shown
in the studies that used cryo-methods to investigate
the three- dimensional structure of chromatin [23, 24].
The development of efficient labeling tools for
EM of cryofixed cells would significantly expand the
range of experimental tasks and allow for a more
complete implementation of such advantages of cryo-
fixation as the absence of non-specific aggregation of
soluble proteins and preservation of the native aque-
ous environment of subcellular structures. Selecting
a proper labeling strategy will also allow to obtain
high-resolution data and to fully realize the potential
of cryoET.
However, since the main physical mechanisms
of contrast generation in the formation of an EM im-
age are elastic electron scattering and phase contrast
[9, 25], the main requirement for a labeling particle
indicating the location of a studied protein is its abil-
ity to efficiently scatter electrons to make it clearly
distinguishable against the background of intracellu-
lar structures despite the small size (2-10 nm). There-
fore, it should be composed of heavy elements, most
of which are normally absent in living cells.
A labeling technique compatible with cryofixation
should solve a multi-faceted problem: introduction of
particles containing heavy elements into a living cell
and their binding to the target molecules should not
disrupt the functioning of these molecules, minimally
affect their structure, and have no negative effects on
the cell physiology, which looks like mutually exclu-
sive requirements [26]. At the same time, it is highly
desirable to have the ability to pre-identify labeled
cells and areas of interest in them by visible light mi-
croscopy to implement various variants of cryo-cor-
related light and electron microscopy (cryoCLEM),
which is especially important in the ultrastructural
analysis of rare cellular events [27] and production of
lamellae using the FIB-SEM method. Obviously, these
requirements lead to compromises in the practical
implementation of the methods for molecule labeling
for cryoET.
This review analyzes implemented and prospec-
tive methods for selective labeling of proteins and
subcellular structures for their further localization
in cell cryosections, as well as the systems that are
potentially applicable for solving this problem but
have not yet been formalized as protocols.
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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
NANOPARTICLES
For many decades, colloidal gold particles (5-
30nm) conjugated with antibodies have been success-
fully used as labels in the EM of ultrathin sections
with chemical fixation (immunogold labeling method)
[28, 29]. Gold particles stand out against the back-
ground of cellular components due to their high elec-
tron density, while the possibility of detecting proteins
on the surface of prepared ultrathin sections elimi-
nates the need for permeabilization of membranes for
the delivery of antibodies into the cell [30].
Can gold-conjugated antibodies be used for label-
ing cellular components in cryoET? Yes, due to the
chemical inertness of gold, its low cytotoxicity [31],
and high electron density that distinguishes it from
the background of vitrified cellular contents. Gold
particles conjugated with antibodies can be used in
cryoEM/ET for detection of objects on the cell surface,
vesicles, and virions [32-36] (Fig. 1a). This approach
can be called “live immunogold labeling”.
The question arises of whether it is possible to
deliver gold-labeled antibodies into a living cell with-
out significantly disturbing the cellular physiology.
Such experiments have been successfully performed,
although their results were assessed by the EM of
chemically fixed ultrathin sections. Loading antibodies
conjugated with an electron-dense label into a living
cell can be done in three ways. The first method is
microinjection [37]. For example, anti-GFP antibodies
conjugated with gold nanoparticles (NPs) were inject-
ed into the cell nucleus to visualize and locate gene
loci containing artificially introduced repeats of the
bacterial lac operator labeled with GFP-LacI and rep-
lication zones labeled with GFP-PCNA (proliferating
cell nuclear antigen).
The second method is loading antibodies con-
jugated with gold NPs into the cell using lipophilic
transfection agents. This approach was used to solve
the problem of mapping transcription sites at the ul-
trastructural level. The authors of [38] used Fab′ frag-
ments of monoclonal antibodies against the C-terminal
domain of RNA polymeraseII conjugated with 0.8-nm
gold NPs [38]. Although only ~10% of available target
molecules detected by other methods were labeled,
the authors believed it to be a satisfactory result for
the experimental task as they were able to demon-
strate the absence of RNA polymerase clustering in the
nucleus, which, albeit indirectly, favors the mobility
of transcription complexes and is against the “tran-
scription factories” concept.
The third method is the delivery of antibodies
into the cytoplasm using streptolysin O, a bacterial
peptide that forms temporary pores in the cell mem-
brane. The possibility of loading small organic mol-
ecules (for example, HaloTag ligands), single-domain
antibodies against PCNA, and whole IgG molecules (up
to 150 kDa) using this method has been demonstrated
in [39, 40]. It should be noted once again that at the
time of writing this review, none of these methods for
loading antibodies into cells had been used in cryoEM
studies; however, there are no fundamental obstacles
to adapting these methods for cryofixation.
All the above-described methods for delivering
labeled antibodies are to some extent harmful to
the cell, but this disadvantage is impossible to avoid.
Aserious limitation in the use of transfection reagents
and streptolysinO is that the label is delivered to the
cytoplasm. If the target is located in the nucleus, the
labeling method should include an additional mecha-
nism for transporting the label through nuclear pore
complexes.
The methods for in vivo labeling of intracellular
structures, in which an electron-dense label (e.g., an
antibody with a bound NP) and a target molecule co-
exist separately for some time inside the cell general-
ly provide no opportunity to distinguish between the
target-bound and unbound labels [41]. This is not a
serious problem when the targets are located in the
membrane and exposed to the external environment,
because the unbound label can be washed off before
cryofixation.
Certain species of gold NPs fluoresce [42, 43],
which adds to the label versatility and allows its use
for the colocalization of labeled cells and structures,
although, perhaps, with less efficiency than using spe-
cialized low-molecular-weight fluorochromes.
Due to the achievements in synthetic biochem-
istry, gold NPs can be used as electron-dense labels
not only in conjunction with antibodies. Thus, the
application of functionalized metal NPs is an inter-
esting approach for labeling proteins in situ [22]. The
authors of [22] developed a method for synthesizing
gold nanoclusters about 2 nm in size, the surface of
which was protected by a polyethylene glycol-based
surfactant. Cell nucleus was used as a model labeling
target, so the particles were additionally conjugated
with the NLS (nuclear localization signal) peptide to
facilitate the nuclear import. After being delivered to
the cell by electroporation, such NPs efficiently relo-
cated to the nucleus, while the control particles with-
out the NLS resided in the cytoplasm.
Another promising approach is functionaliza-
tion of NP surface with the human immunodeficien-
cy virus TAT (transactivator of transcription) peptide
to impart gold NPs with the ability to penetrate cell
membranes. Due to the unique mechanism of mem-
brane penetration, the TAT peptide ensures an effi-
cient delivery of NPs into the cell cytoplasm. Various
methods for synthesizing such particles and verify-
ing their ability to penetrate into the cell have been
recently presented in a number of studies [44-47].
KAZAKOV et al.176
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
Fig. 1. Comparative sizes of different labels, methods of their delivery into cells, and applicability for labeling biomolecules,
subcellular structures, and various cell compartments in cryoET. a)Conjugates of antibodies with gold nanoparticles (AuNPs)
or quantum dots label membrane proteins on the cell surface [32-36] and can be delivered to the cytoplasm (tested in com-
bination with chemical fixation and preparation of ultrathin sections [37, 38]). b)Gold nanoparticles (AuNPs) functionalized
with substrates of self-labeling tags penetrate the membrane by electroporation or due to a modified surface and bind
to chimeric target proteins (partially implemented in [22]). c) Cloneable nanoparticles (NPs) (e.g., bacterial ferritin FtnA).
Protein cage that accumulates metal ions is assembled on the chimeric protein [26, 69, 70]. d)DNA origami-based SPOT tag
binds via the RNA aptamer to the chimeric protein that exposes GFP to the extracellular environment [73]. e)Encapsulins
(e.g., GEM2 system [41]). Cell produces FKBP-conjugated encapsulin subunits which oligomerize into particles, and FRB (FKBP
rapamycin binding domain)-containing chimeric target protein. The binding of the particles with the target is induced by
rapamycin. f)Metabolic label (BSA or another carrier associated with an electron-dense particle and a fluorescent marker)
is absorbed by the cells via endocytosis and accumulated in the endosomes [92].
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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
Another advantage of this method is the ability of
the TAT peptide to translocate the associated parti-
cle from the cytoplasm to the nucleus [48], i.e., such
construct can be used as a basis for developing the
systems for labeling nuclear targets, which is a non-
trivial task.
The cited works have been mostly focused on the
use of produced nanoconstructs as vehicles for the
delivery of therapeutic agents into the cell cytoplasm
and nucleus. A combination of this rather gentle de-
livery method with conjugation of functionalized gold
NPs with low-molecular-weight ligands potentially
allows to create a highly efficient system for label-
ing intracellular proteins and subcellular structures
for cryoET (Fig. 1b). As ligands, this method may use
substrates for genetically encoded “self-labeling” pro-
tein markers, like SNAP-tag and HaloTag. SNAP-tag
is a modified O
6
-alkylguanine alkyltransferase (AGT),
an enzyme involved in DNA repair [49]; HaloTag is a
mutant bacterial haloalkane dehalogenase [50]. Both
markers are introduced into cells using plasmids en-
coding a chimeric protein of interest fused with the
marker. The substrate of SNAP-tag is O
6
-benzylguanine,
while the substrates of HaloTag are various modified
chloroalkanes. Both enzymes are modified in a way
that their interaction with the substrate results in the
formation of a covalent bond that irreversibly binds
the substrate to the target carrying the marker. This
allows to create a bond between the labeled protein
and functionalized particle. However, the development
of such a system poses many potential difficulties,
including the already mentioned problem of distin-
guishing between the target-bound and unbound label
particles.
Another type of particles potentially suitable for
the ultrastructural labeling are quantum dots (QDs).
They are fluorescent semiconductor crystals 5-20 nm
in size [51]. QDs emit bright and stable fluorescence
characterized by a large Stokes shift and narrow emis-
sion spectra, which allows simultaneous imaging of a
large number of targets.
QDs used in bioimaging have a core containing
cadmium and other elements that are significantly
heavier than the elements in biomolecules, which
permits direct observation of QDs by EM. Therefore,
QDs are intrinsically bimodal labels [52]. At the same
time, their chemical composition makes them cytotox-
ic, thus limiting their use in biological research. Var-
ious novel types of QDs have been obtained that do
not contain cadmium or do not have the metal core at
all, but they are not as biologically inert as gold [53].
Even more significant problem is that the deliv-
ery of QDs into a living cell is associated with the
same difficulties as the delivery of gold NPs. In gener-
al, QDs are a potential alternative to gold NPs as labels
for cryoET, since they have the advantage of brighter
fluorescence but the disadvantage of high cytotoxicity
at the same time.
CLONEABLE NANOPARTICLES:
METALLOTHIONEINS, FERRITINS, AND OTHERS
The concept of cloneable NPs (by analogy with
cloneable or, in the terms of this review, genetically
encoded labels) implies the creation of a polypeptide
that under certain conditions, forms an electron-dense
particle around itself. Research is underway in this
field, and significant progress has been made.
Metallothioneins are a family of small (~6 kDa)
proteins containing stretches of 20 cysteine residues,
which allow them to bind ions of heavy metals (gold,
silver, cadmium, etc.) [54, 55]. Metallothioneins have
been found in different organisms [56]; they are in-
volved in protection against the toxic effects of heavy
metal salts [57] and oxidative stress [58].
The ability of metallothioneins to bind and con-
centrate metal ions allows their application as ge-
netically encoded labels for identification of protein
complexes isolated from cells [59, 60], as well as for
labeling proteins inside the cells [61, 62] with subse-
quent visualization by various EM methods. To en-
hance the signal, researchers used the systems with
tandemly arranged metallothioneins [63], but due to
the small size of protein monomers (~6 kDa for metal-
lothionein II), the molecular weight of a tandem of
four metallothioneins is comparable to that of GFP.
The labeling with heavy metal salts in living cells is
usually performed for 30 min to several hours [59],
which minimizes the toxic effect of these metals and
makes metallothioneins applicable in cryoEM [59].
After successfully testing the metallothionein la-
bel with several model bacterial proteins [64], Dies-
tra et al. used this technique to study the location of
the Hqf protein in Escherichia coli cells by combining
metallothionein labelling with cryo-substitution and
acrylic resin embedding, as well as with the Tokuyasu
technique (which allowed the use of immunogold as
a control) [65]. Hqf controls the post-translational
fate of mRNAs by organizing their interaction with
special small non-coding RNAs involved in the regu-
lation of the synthesis of bacterial membrane proteins
[65]. This work demonstrated an unusual near-mem-
brane location of Hqf, which in turn, indicated local-
ized translation of membrane proteins and localized
mechanisms for its control, as well as confirmed the
versatility of the metallothionein labeling system.
Hirabayashi et al. [66] used metallothionein label-
ing in combination with the CEMOVIS method to study
the organization of protein complexes controlled by
PSD95 (postsynaptic density 95) protein on the post-
synaptic membranes of neurons [66]. The authors
KAZAKOV et al.178
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
demonstrated that contrasting the ultrathin sections
with heavy metals after chemical fixation did not allow
to resolve the structures formed by this protein, just
as it was impossible to detect them on the uncontrast-
ed cryosections. Using a chimeric construct consist-
ing of PSD95 and a tandem of three metallothioneins
loaded with cadmium ions, the authors were able to
show that the individual PSD95-containing small struc-
tures, which they termed the “cores”, merged into the
lamellar complexes under the postsynaptic membrane.
Metallothioneins were also tested in a model sys-
tem with KRAS GTPase in the cells after cryofixation.
This protein was previously shown to preferentially
localize to the filopodia by ultrathin sectioning using
a protein label incompatible with cryofixation, and
then KRAS was detected in the same cell areas using
a metallothionein label “developed” by loading with
cadmium ions immediately before the plunge-freezing
cryofixation [67]. The authors found that the location
of KRAS was similar in both cases, noting a signifi-
cantly better preservation of the intracellular envi-
ronment during cryofixation, but also pointing out
that the labeling efficiency was low. These examples
demonstrate a potential versatility of metallothionein
labels and their applicability for cryoET imaging.
Metallothionein-based labels have an advantage
over some other systems (described in the text above
and below) in the fact that the electron-dense particle
is generated directly on the labeled molecule, which
eliminates the problem of unbound particles, although
the background signal from endogenous compounds
that bind metal ions cannot be excluded.
Genetically encoded ferritin is another elec-
tron-dense label used in EM. Bacterial ferritin is a
fairly large (approximately 12 nm in diameter), multi-
meric protein complex assembled from 24 FtnA mono-
mers. It forms a cage capable of accumulating ~2000
iron atoms, which provides its high electron density
and makes this protein detectable in EM images [68]
(Fig. 1c). The possibility of using bacterial ferritin for
in situ labeling of proteins in E. coli cells has been
demonstrated in [26]. The authors expressed the re-
combinant genetic constructs encoding ferritin A in
tandem with a membrane-targeting peptide and pro-
teins with known localization, such as ZapA (marks
the septum in a dividing bacterial cells) and CheY
(component of the chemotactic receptor apparatus
of E. coli, which forms clusters on the plasma mem-
brane inner surface). This work showed that ferritin
as a label for cryoET, is suitable for visualization and
three-dimensional reconstruction of various function-
al compartments of bacterial cell. Interestingly, in its
native environment, the ferritin label binds free en-
dogenous ferritin molecules to form a cage (Fig. 1c).
However, expression of ferritin subunits with the
mitochondrial localization signal in eukaryotic cells
has led to undesirable effects, including mitochondri-
al aggregation and non-specific localization of ferritin
particles, which was probably caused by the large size
of protein complexes formed by ferritin and involve-
ment of several labeled molecules in the formation
of a single ferritin cage. This problem was solved by
combining the ferritin label with a rapamycin-induc-
ible system [69]. Ferritin is synthesized as a chime-
ric protein containing FRB (FKBP-rapamycin binding
domain), while the target protein is fused with FKBP
(FK506-binding protein). The binding of the ferritin
label to the target protein can be induced by incuba-
tion with rapamycin for ~15 min only [69], thus min-
imizing the negative effects of prolonged exposure to
rapamycin on the cells. To saturate the ferritin cage
with iron atoms, the cells should be cultured in the
presence of 1 mM Fe
2+
for 16 h, which, according to
the authors, had no toxic effect on the cells. The sys-
tem was named FerriTag. Its operation in eukaryotic
cells requires additional synthesis of unmodified fer-
ritin molecules as a material for forming the bulk of
the particles. A limitation of this technology is the in-
ability to label proteins located inside membrane or-
ganelles, since the membranes prevent the interaction
of FRB-ferritin with the FKBP-target. Additionally, the
FRB-FKBP linkage increases the distance between the
target and the label by approximately 5nm [69].
This system was further optimized for the use
in cryoET. The authors of [70] adjusted several pa-
rameters of FerriTag, in particular, by constructing a
plasmid encoding all the necessary components (tar-
get-FKBP, ferritin light chain-FRB, and unmodified
ferritin heavy chain). The arrangement of the corre-
sponding genes in the plasmid relative to the IRES
(internal ribosome entry site) ensured the correct
quantitative ratio of the produced proteins. The sys-
tem was tested for labeling and localization of the
mitochondrial outer membrane proteins TOM20 and
Bcl-xL and the membrane protein KRAS located on the
cytoplasmic side of the plasma membrane.
In the study of the mechanisms of resistance of
the Pseudomonas moraviensis stanleyae bacterium to
the high concentrations of selenium compounds lethal
to other organisms, it was discovered that bacterial
cells formed selenium nanoparticles almost uniform
in size and with a characteristic non-spherical, but
symmetrical shape [71]. The authors identified the en-
zymes responsible for selenium reduction and repro-
duced the redox process carried out by the bacterium
using glutathione reductase, selenite ion (SeO
3
2−
), and
NADPH. By controlling the reaction through changes
in the selenite concentration, they obtained the par-
ticles ranging in size from 5 to 50 nm that formed
around glutathione reductase molecules, gradually
“entombing” the enzyme. The authors suggested that
glutathione reductase may be one of the candidates
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for the core in a labeling system based on cloneable
nanoparticles.
Another option for the creation of cloneable
nanoparticles involves the use of proteins labeled
with metallothionein-based peptides modified to in-
crease their stability [72]. This method, however, re-
quires cryofixation followed by postfixation via the
cryosubstitution procedure followed by the devel-
opment of the labels through the reduction of gold
ions and formation of particles. This method does
not solve the problem of obtaining a labeling system
that is fully compatible with cryoET, since the redox
conditions required for this reaction (e.g., the use of
borohydride NaBH
4
) are unlikely to be realized in a
living cell. However, this approach may become an
effective replacement for the immunogold labeling, as
it combines the advantages of cryofixation with cryo-
substitution and ultrastructural localization.
We believe that further attempts in creating
cloneable nanoparticles will lead to the emergence of
“an EM analogue of GFP” in the future.
SPOT – PHOSPHORUS-BASED LABEL
Gold NPs, QDs, and metallothionein- and ferri-
tin-based labels necessarily contain metal atoms or
ions in high concentrations, while the representation
of these elements in biomolecules is very low and
their potential toxicity cannot be ignored. Among
the elements normally present in biomolecules, phos-
phorus is the most efficient generator of elastically
scattered electrons. Phosphorus-based labels have
been created using DNA origami and RNA aptamer
technologies. The method was named SPOT (signpost
origami tag) [73].
DNA origami is a technology for creating nano-
structures from a long single-stranded DNA scaffold
molecule and short staple molecules complementary
to specific segments of the scaffold. Interaction with
the staples leads to the reproducible folding of the
scaffold into a pre-designed conformation [74, 75].
DNA origami is a promising basis for creating nanode-
vices and drug delivery systems, although it is gener-
ally considered more as an object than a tool of micro-
scopic research. In microscopy, DNA origami has been
employed to create support films for EM visualization
[76, 77] and to manufacture resolution test samples
for sub-diffraction visible light microscopy [78].
Aptamers are oligonucleotides that selectively
bind certain chemical groups; they represent nucle-
otide-based analogs of antibodies [79]. Aptamers are
widely used as label carriers and are employed in
visible light microscopy, flow cytometry, and various
types of computer tomography in medicine and re-
search [80, 81].
SPOT is a wedge-shaped DNA construct of about
7MDa assembled from a 7.5-kb DNA scaffold and 238
staples. The narrow end of the wedge extends into a
rigid stem, at the end of which sits an RNA aptamer
that recognizes a fluorescent protein. The density of
DNA packing in the SPOT construct ensures a high
local concentration of phosphorus, and the asymmet-
ric structure of the wedge, unlike the overwhelming
majority of nanoparticle tags that have a spherical or
close to spherical shape, indicates the direction to the
target, while the long rigid “stem” separates the mas-
sive body of the label from the tagged structure, thus
minimizing the effect of the label without reducing
the accuracy of localization [73] (Fig. 1d). The authors
tested the resulting construct using fluorescent con-
structs based on the herpes simplex virus glycopro-
tein B located on vesicles and cell membranes and
membrane glycoprotein of murine leukemia virus lo-
cated on viral particles as targets.
The SPOT construct proved to be non-toxic and
clearly visible in cryoET images due to its size, shape,
and electron density. Since SPOT binds to the target
via a fluorescent protein, it is a bimodal label. The
main and unique feature of SPOT is that due to its
shape, it literally points to the exact location of the
target molecule. As the authors of the method claimed,
they were able to trace the transition of the SPOT stem
into the electron density corresponding to the labeled
protein [73].
Despite the listed advantages, the obvious limita-
tion of the SPOT label is its complexity: it is assembled
ex vivo, undergoes complex purification procedure,
and does not penetrate into a living cell. However,
the combination of the SPOT properties, as the au-
thors pointed out, makes this system a highly efficient
tool for studying proteins on the external surfaces of
natural and artificial vesicles, viruses, and cell mem-
branes. At the time of writing this article, we were
unable to find published reports on the experimental
application of SPOT labeling technique.
ENCAPSULINS – VIRUS-LIKE
PROTEIN NANOCOMPARTMENTS
Encapsulins, which are naturally occurring bac-
terial nanocompartments, offer a yet another basis
for developing techniques for labeling subcellular
structures for cryoET. These protein complexes were
initially identified in the Brevibacterium secretions
during investigation of their bacteriostatic properties
[82]. Encapsulins have a significant biotechnological
potential as the vehicles for the delivery of therapeu-
tics into the cells [83].
Encapsulins self-assemble into virus-like icosahedral
structures measuring 22 nm or more in diameter[84].
KAZAKOV et al.180
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
These particles are defined by their triangulation
number, such as T = 1 for encapsulins isolated from
Mycobacterium tuberculosis [85] or T = 3 for nano-
compartments from Myxococcus xanthus. Currently,
the largest known encapsulins, measuring 42 nm in
diameter, have T = 4 and are produced by Quasibacil-
lus thermotolerans [85-87].
Encapsulins accumulate inside the cells without
any significant toxic effect [88], which makes them
suitable for the use as genetically encoded labels in
EM. While their virus-like shape allows identification
in electronograms, their contrast is insufficient for
labeling intracellular protein complexes, necessitat-
ing signal enhancement. By combining various encap-
sulin types with differing amounts of inward-facing
metallothioneins during nanocompartment assembly,
researchers have created a series of large particles ca-
pable of binding metal ions. This advancement result-
ed in the appearance of a novel category of EM labels
known as EMcapsulins [89], which can be used not
only in invitro cultured cells but also in living model
organisms. For instance, researchers employed EMcap-
sulins to label different cell types in the nervous sys-
tems of Drosophila and mice, facilitating subsequent
connectome reconstruction using FIB-SEM [89].
Finally, it was demonstrated that encapsulin-based
tags can be used as selective markers of various cell
compartments in the correlation fluorescence micros-
copy and cryoET. For this purpose, a system called
GEM2 [41] was developed based on Srp1 encapsulin
from Synechococcus elongatus. To prevent aggrega-
tion of the protein labeled with large (25-nm) parti-
cles formed by encapsulins, the authors designed a
system that ensured particle binding to the target
molecule only in the presence of the added ligand.
To accomplish this, encapsulin and the protein of in-
terest were linked to the above-mentioned FRB and
FKBP, respectively, whose binding was initiated by
rapamycin (Fig. 1e). The designed system was tested
in cryoET with several model targets: mitoGFP located
on the outer mitochondrial membrane, mEGFP-Ki-67
located on the surface of chromosomes in a divid-
ing cell, Nup96-eGFP nucleoporin (which faces the
cytoplasm), and Seipin-sfGFP, an endoplasmic reticu-
lum protein that pinches off lipid droplets from the
membranes. In all these cases, the natural contrast
of such large particles was sufficient for localiza-
tion of labeled proteins and automated analysis of
cryotomograms using convolutional neural networks.
The potential of this technology as a selective la-
beling technique for EM is evident. However, it is cru-
cial to acknowledge that despite advancements in the
application of encapsulins, these techniques involve
complex and laborious procedures, including genet-
ic engineering and expression of large genetic con-
structs in the cell. Therefore, meticulous monitoring
is essential to ensure that the structure and functions
of macromolecular complexes labeled through this ap-
proach remain intact [41]. It is also worth noting that
in this multicomponent system, the linker between the
encapsulin particle and the target is approximately
20 nm long, which undoubtedly affects the accuracy
of target localization. Finally, the authors found that
the kinetics of tag binding to the target depends on
the target concentration [41].
METABOLIC LABELING
Metabolic tags are diverse molecules that are
consumed by cells during various cellular processes.
What distinguishes this approach from the above-de-
scribed methods is its focus on targeting subcellular
compartments rather than specific proteins. The use
of metabolic tags, along with immunoEM techniques,
can be considered as one of the first approaches to
imaging subcellular components using EM. For exam-
ple, the Golgi apparatus was specifically labeled at the
ultrastructural level by incubating cells with ceramide
conjugated to a fluorescent molecule. Subsequent pho-
tooxidation of diaminobenzidine upon the tag irradi-
ation with visible light formed the electron density
in the trans-Golgi zone [90].
The target of this labeling procedure is the endo-
somal-lysosomal compartment, which occupies a sig-
nificant portion of the cell’s volume and interacts with
both endoplasmic reticulum and the Golgi apparatus.
The loading of tags into the endosomal compartment
is performed by the natural cellular mechanism of en-
docytosis, which automatically solves the issue of tag
delivery. Either nanoparticles are absorbed directly
or bovine serum albumin (BSA) is used as the label
carrier, which can be conjugated with both fluores-
cent dye and metal particles. A combination of this
approach with light microscopy has been applied to
study the restructuring the cell vacuolar system in
infections caused by Salmonella enterica and Salmo-
nella typhimurium [91]. Recently, bimodal BSA was
obtained, which carried both a fluorescent group
and a gold particle as an electron-dense. It was test-
ed for applicability in several microscopic modalities,
including fluorescence microscopy, correlation light
fluorescence, EM in ultrathin sections with chemical
fixation, and correlative cryoET, in which such bimod-
al labels are in high demand [92]. In addition to the
main function (marking of the endosomal-lysosomal
compartment lumen), contrasting gold particles con-
jugated with BSA can be used as fiducial markers for
the alignment of a series of angular projections during
tomogram reconstruction [92] (Fig. 1e). Moreover,
the proposed design and the method of its synthesis
protect the fluorophore from quenching by closely
LABELING OF SUBCELLULAR STRUCTURES FOR CRYO-ET 181
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
spaced metal atoms, which, as the authors pointed
out, is a serious problem in the design and synthesis
of bimodal labels [92]. Therefore, metabolic labeling
is an efficient and physiological approach to labeling
subcellular compartments in cryoET, even though it is
applicable to a limited range of tasks.
CONCLUSIONS
Kaufman et al. [20] articulated the motivations
behind the development of labeling techniques com-
patible with cryofixation and cryoEM analysis of cel-
lular and tissue samples, including a need to detect
a cell of interest or a rare event in a heterogeneous
cell population, identify a studied molecular ensem-
ble or a specific molecule in an EM image [25], or
reveal a yet unknown process or structure based on
the involvement of the studied molecule in it. And
while the first two tasks can be solved using genet-
ically encoded fluorescent labels and the correlation
approach (CLEM), the remaining tasks require the use
of labeling technologies at a completely different level.
Two questions arise. How does the preservation
of the native state of biomolecules achieved by cry-
ofixation combined with the use of labels affect the
functioning and behavior of the labeled molecule in a
living cell? What new information can be obtained us-
ing compromise solutions to the first of these issues?
The first question is rather difficult to answer
in general because the methods of ultrastructural la-
beling are not yet widely used in the cryo-electronic
format, and their pitfalls will appear gradually with
a wider introduction of such methods into laboratory
practice. Considering the experience accumulated for
the application of GFP and its analogs, there is hope
that possible solutions to emerging problems will be
found quickly, as the studies on the optimization of
such techniques are already underway [66, 86].
What problems requiring the use of cryoET are
difficult to solve without using the labels? The an-
swer is: (i) localization of specific genomic sequences
in the intact nucleus, (ii) obtaining three-dimensional
structures of globular proteins in their native envi-
ronment, and (iii) determination of the orientation
of protein subunits in native multimeric intracellular
complexes.
While having the ability to use an electron-dense
label to mark proteins like TALEs (transcription acti-
vator-like effectors) [93, 94] and dCas [95], researchers
can identify specific sequences in the genome and, by
attaching labels to the regulatory components of mul-
timeric enzymatic complexes, identify areas in which
the studied process (transcription, replication, DNA re-
pair, processing of certain species of RNA) take place.
Such targets are indistinguishable on the chromatin
background, and the use of labels seems necessary to
solve the problem of their localization. The ability to
implement such labeling in combination with cryo-
fixation will significantly expand our understanding
of the molecular mechanisms of these processes be-
cause of the preservation of the native or near-native
structures in which the process occurs and involved
proteins.
Finally, the ability to label a protein in such a
way that its orientation in space can be determined
using the label makes it possible to solve the problem
of determining the position and, possibly, to identi-
fy the role of a given molecule in the functioning of
the entire structure. Such attempts have been made,
although not in intact cells, but in the isolated axon-
eme [96]. Experiments were conducted to establish the
relative position and orientation of protein molecules
using labeling with gold NPs.
Similar attempts have been made with the prepa-
rations of the basal and apical plasma membranes, in
which the N-terminal FerriTag label was used to lo-
calize the Hip1r protein that links the clathrin coating
of endocytic vesicles with actin filaments [97]. Thepo-
sition of the label indicated the parallel orientation
of Hip1r dimers with the C-terminal parts directed
toward the membrane of the forming vesicle and the
labeled N-terminus directed into the cytoplasm, either
perpendicular or at an angle to the clathrin mesh.
Notably, in this study, FerriTag was detected without
loading it with iron ions, but based only on the shape
of the ferritin cage.
None of the described technologies is universal
(Fig. 1, Table 1), especially when compared with flu-
orescent immunocytochemistry and chimeric fluores-
cent protein technology in visible light microscopy.
Each method has a number of features that may seem
as disadvantages, but the variety of labels and meth-
ods of their delivery allows researchers to choose the
most suitable system for solving a specific research
problem, taking into account such factors as known
or supposed location of biological object, its tolerance
to labeling conditions, duration of the experiment, etc.
Many works cited in the review are proof-of-con-
cept studies. Successful application of the described
methods for visualizing a specific target in a specific
cell type does not guarantee the detection of the same
or another target in another type of cells. To transfer
the protocols to other objects, physiological controls
are necessary to evaluate the effect of an excessively
massive or potentially toxic label on cell physiology.
All described methods will undoubtedly benefit
from the integration of a fluorescent component, al-
lowing for the monitoring of the labeling efficiency
and physiological state of cells during the experiment,
selection of cells for the study, and identification ofar-
eas for subsequent lamella fabrication [20].
KAZAKOV et al.182
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
Table 1. Tested labeling methods compatible with cryofixation
Method Label
Label carrier/
label-target linkage
Cell
permeable?
Advantages Disadvantages
Live immunogold
labeling
gold NPs
antibodies/
covalent, ex vivo
no
established
methodology
only for labeling
extracellular
membrane targets
Metallothioneins metal NPs
chimeric protein/
covalent during
synthesis
in the cell
synthesized
in the cell
established
methodology,
small label size,
no unbound label
requires
incubation of cells
with heavy metal
salts
FerriTag
proteinaceous
nanocage +
iron ions
chimeric protein/
rapamycin-induced
binding in the cell
synthesized
in the cell
well-developed
methodology
requires
incubation of cells
with heavy metal
salts
SPOT
wedge-shape
DNA origami
chimeric protein
with GFP/via RNA
aptamer
no
target localization
accuracy, bimodal
system
complex design,
only for labeling
extracellular
membrane targets
Encapsulins
GEM2
protein
nanocage
chimeric protein/
rapamycin-induced
binding in the cell
synthesized
in the cell
do not contain
metals, no effect
on the target
molecule prior to
the rapamycin signal
large size, long
linker, complex
stoichiometry
Metabolic
labeling
gold NPs +
fluorophore
BSA/adsorption+
covalent binding
ex vivo
endocytosis
bimodal, biologically
inert, works
as a fiducial marker
for aligning a series
of sections
only for labeling
the endosome-
lysosomal
compartment
CryoET, as perhaps the most promising and pow-
erful implementation of cryoEM used for solving the
problems of cell biology, has already taken its place
in the arsenal of researchers, but the development of
labeling systems for the cryo-format is slow. This is
largely due to the objective complexity of the problem
itself. The second reason for the slow development
of selective labeling tools may be that the current
demand for transmission cryoelectron microscopes
significantly exceeds the availability of high-tech
equipment for these studies. This puts researchers de-
veloping such labeling systems in a difficult situation
when they have to risk expensive instrument time.
However, the appearance of works, such as recent
preprints by Wang et al. [70] and Sun etal. [97], who
used FerriTag to localize protein molecules in macro-
molecular complexes to establish their structure and
orientation, perfectly illustrates both this thesis and
the general demand for the development in this study
field.
At present, it is difficult to predict the direction
in which the next breakthrough will occur, and wheth-
er it will occur at all. Perhaps, a universal labeling
system for cryoET will never be invented, and re-
searchers will need to choose a method from a set
of specialized techniques. It is also possible that a
completely different solution to the problem of the
object localization will be found, for example, the ap-
plication of the elemental composition analysis for lo-
calization of “elemental labels” [98, 99] (although such
method requires even more specialized equipment) or
by implementing effective algorithms for solving the
inverse problem, namely, identification of a protein
in a cryo-tomogram based on its known or predicted
structure (which no longer can be considered label-
ing). These methods are already entering research
practice, and a still small number of examples of their
application is gradually increasing [100, 101]. Or, per-
haps, the progress will follow the path of improving
the correlation microscopy and will result in the cre-
ation of a visible light fluorescence microscope with
a subdiffraction resolution of about 30 nm or even
less that would operate under cryogenic conditions
[19] and will be supported by the introduction of a
unified format for the data transfer between different
instruments.
LABELING OF SUBCELLULAR STRUCTURES FOR CRYO-ET 183
BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025
Contributions. E.P.K. and S.A.G. developed the
concept and wrote the text of the article; I.I.K. edited
the manuscript.
Funding. The work was supported by the Russian
Science Foundation (project no.23-74-00021).
Ethics approval and consent to participate.
This work does not contain studies involving human
or animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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