Chemical Physics Impact 2 (2021) 100013

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Chemical Physics Impact

journal homepage: www.elsevier.com/locate/chphi

Viral particle imaging by super-resolution fluorescence microscopy

Stefania Castelletto a,∗, Alberto Boretti b
a
RMIT University, Melbourne, Victoria, Australia
b
Prince Mohammad Bin Fahad University, Al Khobar, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Keywords: Fluorescence microscopy provides a facile imaging methodology and potentials for imaging viruses in vivo. How-
Fluorescence microscopy ever, it is limited by the diffraction limit of light microscopy which is above the size of the virus particles. Flu-
Virus entry orescence super-resolution (FSR) microscopy circumvents the diffraction limit. These techniques, an extension
Super-resolution imaging
of fluorescence microscopy and alternative to electron microscopy, established over the past twenty years, have
permitted the visualization of some virus components. Here, we summarize the principles of different FSR tech-
niques and single virus tracking, and their current applications to virology studies, focusing on some limitations
impeding their widespread use in virus replication and infection mechanism studies in living cells. In the spe-
cific context of virology studies and live-cell imaging, the advantages and disadvantages of the most known FSR
methods are outlined with a focus on their current limitations such as labeling, imaging speed, photo-bleaching,
depth of imaging, and the current attempts to overcome them. Some of the recent demonstrations encompassing
the combination of different super-resolution (SR) approaches have provided answers to open questions as an
example in the human immunodeficiency virus replication and from these experiences, further progress can be
envisaged to reveal other viruses.

Introduction
Viruses also referred to as viral particles or virions are nanoscale
genetic entities that can replicate only within cells of plants, fungi, ani-
mals, or humans. Viruses cores are made of a single nucleic acid strain
(RNA or DNA) also referred to as viral genome within a capsid, enclosed
by a coat covered by proteins, sometimes enzymes, and in case of en-
veloped viruses, by a lipid membrane. The virus enclosure can vary and
undergo assembly to initiate viral infection and replication. Outside a
living cell, viruses cannot reproduce but are however capable of infect-
ing the cells and rely on the host cells for the completion of their life
cycle. Retroviruses like HIV enter in hosts’ cells to reproduce by integrat-
ing their viral genome into the host genome, and their replication occurs
along with the host genome replication during each cell cycle. This is not
the case with other viruses such as Hepatitis, Influenza, or Polio. In gen-
eral, the entry of a virus into a cell starts with attachment to receptors.
This is then followed by conformational changes of viral proteins, and
in case of non-enveloped viruses penetration-through cell membranes,
or cases of enveloped viruses fusion with cellular membranes. Transfer
of viral genomes inside host cells eventually occurs.
Virus components are generally much smaller and their identifica-
tion and visualization are critical in following the virus lifecycle. Virus
lifecycle involves complex interactions between the virus and the host
cell, to achieve the infection. They can be summarized in the following
steps whose details depend on the specific virus [1]:
1. diffusion on the surface of the cell and binding to cells receptors or
host cells membrane;
2. enter in the cell through endocytosis;
3. viral membrane fusion (most enveloped viruses) or cellular mem-
brane fusion (most non-enveloped viruses). Here the viral genome is
released in the cell;
4. replication of the viral genome and translation of additional viral
proteins required for new rounds of replication;
5. assembly and packaging of the new viral genomes into virus parti-
cles;
6. budding where the new virus prepared to be released and ready to
infect new cells;
7. maturation.
The details of the human immunodeficiency virus (HIV) life cycle is
provided in Fig. 1 (a) and also summarized in ref. [2], while Fig. 1 (b)
shows the six steps entry of the Influenza A virus (IVA) as studied in ref.
[3]. Both viruses were further studied using super-resolution methods.
Cell and molecular biologists, virologists, and immunologists need
novel technologies to better understand the above mechanisms at the
single virus level that are specific to each virus. Visualization of the
way the virus enters the cell and transports the viral genome needed

∗
Corresponding author.
E-mail addresses: stefania.castelletto@rmit.edu.au (S. Castelletto), a.a.boretti@gmail.com (A. Boretti).

https://doi.org/10.1016/j.chphi.2021.100013
Received 17 September 2020; Received in revised form 1 February 2021; Accepted 18 February 2021
2667-0224/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
S. Castelletto and A. Boretti
for its reproduction is of paramount importance to viral studies, gener-
ally studied via ensemble biochemistry providing a statistical approach.
However, while a single virus can infect a cell, only a small percentage
of them, for example for retroviruses as HIV that bind to the cell sur-
face, are eventually able to infect the host cell with replication of their
genome [4]. There is therefore significant interest in imaging and track-
ing of viruses at the single virus level to understand their life cycle. Most
current studies on virus imaging are focused on uncovering the mecha-
nism of assembly and cell interaction and infection and the single virus
imaging can provide a novel understanding of these mechanisms. Single-
virus and single-molecule analyses can reveal which different steps and
host factors are required for a successful infection. The recent outbreak
of SARS-CoV-2 has brought even more attention to the need for imag-
ing virus with the desired resolution to uncover the specific mechanisms
associated with the cell infection process [5].
Viruses such as HIV, influenza, and SARS-CoV-2 are approximately
100 nm in diameter, thus considerably smaller than cells even if some
of them can reach the size of bacterial cells [6]. Their constituents
are therefore much smaller than 100 nm. Due to this small size super-
resolution (SR) imaging techniques, as well as high localization for their
tracking, is necessary. A better knowledge of the mechanisms of virus
assembly and infection is needed for prophylaxis and treatment by an-
2
Chemical Physics Impact 2 (2021) 100013
Fig. 1. (a) Illustration shows the HIV replica-
tion cycle, beginning when HIV fuses with the
surface of the host cell, replication, assembly
and packaging, budding and maturation Credit:
NIAID (b) Sequential events during cells entry
for influenza A virus (IAV) obtained by con-
focal fluorescence and machine learning meth-
ods from [3]. Creative Commons Attribution Li-
cense.
tivirals. The ultimate quest is to image a single virus with high resolu-
tion and to track single virus dynamics in living cells. For this purpose,
single-molecule fluorescence microscopy can reveal virus infections and
biology that are inaccessible otherwise. A recent review has focused on
the key mechanism in virus-cell interaction, virus cell entry, and trans-
port observed under various single-molecule fluorescence microscopy
and associated SR techniques [1].
To this end, several methods have been deployed which can address
either resolution or tracking, either in ensemble measurements or at the
single level, mostly in vitro. These methods are based mainly on electron
microscopy which provides enough resolution with however a lack of
dynamic imaging and limited applicability to in vivo imaging due to the
vacuum operation conditions or complex sample preparation. Later fluo-
rescence microscopy, either wide-field or scanning confocal microscopy,
has been applied to the virus, however, these methods lack enough reso-
lution, while having the possibility of single virus imaging and tracking
in principle in vivo. More recently fluorescence super-resolution (FSR)
methods have been applied to virus imaging with the potential of in vivo
applications. At present, none of the imaging and tracking techniques
can accomplish all the above requirements. However, by learning from
the current demonstrations of FSR techniques in specific viruses, further
development can be envisioned to accomplish some of these quests.
S. Castelletto and A. Boretti
The virology world’s major quest of observing in vivo and real-time
the interaction of the nanometric scale virions with the molecular cell
biology is currently barely within reach. This is because of the differ-
ent spatial scales involved, the time scale within which current high-
resolution imaging can be achieved, and the difficulty to image for a
long time due to either methods’ toxicity responsible for cells’ death or
tags photo-bleaching.
Regardless of the possibility of live-cell imaging studies, most to-date
virus studies using FSR microscopy and single virus tracking have been
carried out only on fixed samples. For virology research in vivo, these
technologies necessitate in addition to a higher resolution than current
capabilities for in vivo imaging, also low photo-bleaching and photo-
toxicity, higher imaging speed, and the possibility to achieve imaging
of the sample with depth penetration and specific labeling. In particu-
lar, the acquisition of cells and virus dynamics in the time scale of ms,
requires high imaging speed, while their interaction should allow long
tracking times. This is everything but simple to achieve due to short life
fluorescent labels and photo-toxicity leading to cells death.
This paper focus on the latest developments FSR microscopy has
achieved and may perform in the future regarding viral particle stud-
ies.
Here, we briefly summarize the historical approaches to image
viruses and the latest attempts by using the most popular FSR meth-
ods. We then focus on their current achievements and limitations with
regards to open problems associated with these methods impeding to
reach in vivo single virus imaging [7]. In particular, the development
of FSR methods for virus imaging, while have reached a high level of
resolution ideal for viruses, has been impacted by common open limi-
tations such as accurate labeling, imaging speed, sample depth penetra-
tion, photo-bleaching, and photo-toxicity.
Fluorescence microscopy methods for viral particle imaging
Due to the nanometric virus size, early imaging methods and cur-
rently commonly used methods to achieve static images of viruses, are
based on conventional electron microscopy such as scanning electron
microscopy (SEM) and transmission electron microscopy (TEM) [8],
with viruses, imaged emerging from cultured cells in the laboratory.
However, these methods, while they can provide high resolution (sub
nanometric) in the imaging, are onerous from the sample preparation
point of view, can preclude live imaging, and have limited opportunity
for molecular identification [9]. Other methods such as atomic force mi-
croscopy [10] or optical tweezers [11] can be employed to study prop-
erties of the virus at the nanoscale, however with limited cases applica-
bility and yet far from common.
Fluorescence microscopy [12] is instead more commonly used and
it has permitted virologists to study the virions’ interaction with cell
surface while entering the cell, using wide-field and scanning confocal
fluorescence microscopy due to the relatively fast and easy methodol-
ogy. On the other hand, the resolution of a fluorescence microscope is
𝜆
physically limited by the Abbe diffraction limit [13] given by 2 𝑁𝐴 ~ Total Internal Reflection Fluorescence microscopy (TIRF) [29], con-
190 in the plane of focus (xy) of an oil immersion objective with nu-
merical aperture NA=1.4, excited with a laser with wavelength of 532
nm and by 1.𝑁 4𝑛𝜆
𝐴2
~ 570 nm along the axial direction (in-depth) of a
medium of refractive index n=1.5 [14]. The actual resolution in the
non-ideal experimental conditions is often even lower [15]. This type of
resolution is not enough to image a virus without obtaining a blurred
image.
Conventional wide-field and laser scanning confocal fluorescence
microscopes principles have been developed by introducing some struc-
tural optical modification to improve their spatial resolution. Structured
illumination (SIM) [16,17] achieved improvement in axial resolution in
the range of 100–150 nm. These approaches are quite simple and as
such still offer the advantages of direct applicability, using convention-
ally prepared fluorescent probes and samples. However, due to their still
limited optical resolution, their use is not wide-spread in virus research.
3
Chemical Physics Impact 2 (2021) 100013
FSR principles and techniques
FSR microscopy techniques recently established [14,18], com-
prise among the most notable, single-molecule switching or localiza-
tion microscopy (SMSM) divided into Photo-Activation Localization
Microscopy (PALM) [19–21] and Stochastic Optical Reconstruction
Microscopy (STORM) [22,23], and stimulated emission depletion mi-
croscopy (STED) [24]. As a common feature, these methods permit to
achieve spatial resolution below 100 nm, enabling the imaging of viruses
for the examination of their architectural details and their interactions
with the cell constituents throughout virus replication, spread, and in-
fection. These methods have permitted to resolve images better than 20
nm and achieve 10 nm localization accuracy. While high resolution can
be achieved with these methods, they suffer from slow acquisition time
and/or post-processing of images, as well in some cases of photo-toxicity
or photo-bleaching of fluorescent tags that make volumetric time elapse
imaging of living samples impractical.
Principles of the most prominent FSR microscopy methods with their
typical achievable resolution are provided in Fig. 2.A, where the inten-
sity distribution of 550 nm laser beam through a commonly used oil im-
mersion objective (Obj) with numerical aperture NA = 1.4, is displayed
in the focal point. The beam intensity distribution at the focal point
in the lateral and axial directions are ~250 nm and ~600 nm, respec-
tively, in a confocal or wide-field microscope with schematics shown in
Fig. 2.B. While scanning confocal fluorescence microscopy using point
detectors and a pinhole for read-out to improve spatial resolution and
reduce the contribution from the sample background out of focus, wide-
field microscopy does not require scanning and has generally a larger
field of view.
By using SIM and similar techniques based on illumination with
structured multifocal points, or Airyscan detection mode [25], it is pos-
sible to achieve a spatial resolution of 100 nm and 300 nm in the lat-
eral and axial directions, respectively, accounting for an improvement
of a factor of 2 versus conventional confocal or wide-field microscopes.
These approaches use the variation of light patterns in the detection
channel to reconstruct the image (Fig. 2.C). A series of images from the
CCD camera is collected by turning the illumination structure and from
these images analysis, it is possible to recreate the final image with high-
resolution information [14]. In ref. [26] 3D-SIM permitted a two-fold
increase in resolution in all three dimensions of the Hendra virus (HeV)
in fixed infected primary bovine, porcine aortic endothelial cells, and
bat-derived cell lines. Here the HeV M and G proteins structures were
confirmed as being around HeV virion and SR imaging demonstrated
for the first time sub-virion imaging of paramyxovirus proteins and the
respective localisation of HeV G, M and N proteins within the viruses.
Recently using SIM and image reconstructing algorithm combined with
SEM, Zika virus main stages of its assembly from viroplasm formation to
the viral release in mammalian cells were studied [27]. 3D-SIM has been
used to study vaccinia virus replication resolving the virus morphologies
at the scale of single virus particles [28].
sists of the formation of an induced evanescent wave at the interface
of two media having different refractive indices, generally in a limited
sample region close to the interface area between the sample and a
glass coverslip or tissue culture container. In this case, the excitation
beam is reflected totally at this interface, reducing the energy at a sam-
ple region of interest with thus minimal background. This can reduce
the excitation power in axial z-direction thus reducing background and
permitting to achieve ~ 100 nm resolution in the axial direction [30].
TIRF combined with SIM has been implemented and can provide a good
spatio-temporal resolution, remaining a facile method, retaining versa-
tility, and permitting to achieve 3D live-cell imaging capability by using
conventional microscopy fluorophores. Therefore, it is considered the
best method for studies requiring at the most 100 nm resolution, while
not enough in areas such as virus research, requiring less than 100 nm
resolution.
S. Castelletto and A. Boretti
Interestingly by combining TIRF microscope with a Bayesian analysis
[31] of the stochastic blinking and bleaching (3B SR) of Alexa Fluor
488 dye, a recent study [32] shows an achieved resolution of 50 nm
in imaging the viroplasms (VPs), cytoplasmic electron-dense inclusion
where the Rotavirus genome replication and assembly occur (Fig. 3).
This showed the structural organization of seven viral proteins within
and around the VPs organized in five concentric layers.
Sub-diffraction resolution below 100 nm attained in fluorescence mi-
croscopy relies on fluorescent labels being switched “on” and “off” be-
tween so-called “bright” and “dark” states either deterministically or
stochastically. This switching mechanism permits to fluoresce to only
a small subset of all labels in the focal spot, thus, to be individually
distinguished and localized. Their imaging and localization contribute
to a reconstructed image defeating the light diffraction limit [34]. The
underlining difference between techniques relies on the type of switch-
ing mechanism or localization, of either deterministic or stochastic ori-
gin. In a deterministic approach the fluorophore from its excited state
is forced into the dark state by using a dual excitation beam as in STED
microscopy [24] and in the Reversible Saturable Optical (Fluorescence)
Transition (RESOLFT) microscopy [35,36]. In the first method, the dark
state is a ground state while in the second is a long-lived metastable
state or intersystem crossing state.
To date, only a few demonstrations of the application of the above
FSR methods to study the bio-functionalities of viruses even in fixed
cells can be accounted for, with the principal focus been the human im-
munodeficiency (HIV) virus [37]. FSR microscopy studies of HIV and
4
Chemical Physics Impact 2 (2021) 100013
Fig. 2. A. Lateral or in-plane (x-y) and ax-
ial (z-direction, depth) resolution of various
imaging schemes. Schematics of B. Confocal
and wide-field combined, C SIM, D STED, and
E 3D STORM [33]. EMCCD is an electron-
multiplied charge-coupled device used to im-
age low brightness fluorophores for higher
imaging resolution.
Fig. 3. Rotavirus RRV-infected MA104 cells
were fixed and processed for transmission
electron microscopy or immunofluorescence
microscopy. (A) Transmission electron mi-
croscopy of a VP (identified by the dotted white
ellipse). (B) Diffraction-limited image of VPs
(white arrows). (C) 3B-SR image reconstructed
from B. (D–E) 3B-SR images of individual VPs
labeled with different antibodies. Image from
ref. [32]. Creative Commons CC BY license. cre-
ativecommons.org/licenses/by/4.0/.
their insight have been recently reviewed in ref. [14,38,39] and a re-
cent perspective on the use of FSR microscopy for HIV can be found
in ref. [2]. These studies together with examples applied to Rotavirus
[32,40], Hendra virus [26], Vaccinia Virus [28], VSV-like particles [41],
Influenza A virus (IAV) [42-44], ZIKA virus [27], Herpes simplex virus
[45], Ebola virus [46], while in still limited demonstrations, illustrate
the journey of SR imaging to bring a better understanding of cell-virus
biological processes and their dynamics with nanoscale resolution.
Details on the techniques applied for the specific viruses and major
insights on the virus interaction with the cells will be discussed in the
following sections, while we summarise the main or more recent ad-
ditional virus studies, methods, and finding using FSR or single virus
tracking in Table 1.
We discuss some common fluorescent tags used in viral imaging and
other solid-state tags. We then focus on the application of these tech-
niques for viral particle imaging, while reminding the reader of more
specific details of the techniques to other reviews [14,61]. For a more
extensive summary of the many other techniques principle of operation
and schematics see for example ref. [62].
Fluorescent tags in viral particle imaging
One of the key issues of fluorescence microscopy-based methods ap-
plied to the virus is the labeling or fluorescence tags. In general, with
all the above FSR approaches significant attention must be taken in the
labeling of the virus by fluorescent markers or tags if compared with
S. Castelletto and A. Boretti Chemical Physics Impact 2 (2021) 100013

Table 1
Summary of specific referenced virus studies, FSR or single virus tracking methods used, and key findings.

Virus type Methods Key Findings Refs.

HIV-1 STED Dynamics imaging of Gag protein and Env during virus [47,48]
maturation.
HIV-1 STED-FCS Study of the mobility of the proteins on the surface of [49]
individual HIV-1 particles in live-cells context.
HIV-1 dSTORM-TIRF 2D molecular distribution of the major structural proteins of [50-53]
the HIV-1 after infection. Colocalization (Env and Gag) at the
HeLa cell membrane for the virus assembly
SERINC5 and anti-retroviral factor mechanism study.
HIV-1 PALM and TIRF HIV assembly and budding studies at the plasma membrane [54]
of CD4+ T cells
HSV-1 dSTORM Localized the protein layers on the virus in infected cells [55]
HSV-1 STED Identify proteins for processes of cell attachment and [56]
membrane fusion.
Nipah Virus dSTORM Reveal virus assembly model [57]
Rotavirus TIRF combined with Study of replication and assembly [32]
Image reconstructing
algorithms
Influenza Virus A Single virus tracking IAV virions encapsulating quantum dots are tracked in [43,58]
real-time, dynamic of the uncoating is revealed in real-time.
Influenza Virus A dSTORM Virus matrix protein localization [44]
Influenza Virus A STED Virus events trafficking in human dendritic cells after [59]
internalization
Ebola Virus Single Virus Tracking Virus internalization [46]
Zika Virus 3D-SIM plus image Main stages of virus assembly and release in mammalian [27]
reconstructing cells
algorithm and SEM
Human respiratory STED High-resolution images of the virus [60]
syncytial virus

confocal microscopy. The size of the fluorescent tags is of paramount im-
portance to directly target the biological structures of interest and track
cellular processes in specific locations without introducing artifacts and
interfere with the virus and cells’ functionality. Besides, they must have
photo-physics properties that are compatible with the microscopy and
FSR principles, and ideally, they should not photo-bleach to provide ac-
cess to an extended time for tracking. For FSR tagging each virus compo-
nent with high molecular specificity and efficiency is essential to achieve
relevant information in their dynamics during the virus lifecycle. Fur-
thermore, depending on the process associated to each viral component
to be imaged, fluorophores with distinct fluorescence properties, such as
brightness, fluorescence lifetime, photostability, emission wavelength,
photo-switching capabilities have to be specifically tailored. For exam-
ple, some virus components such as proteins and the lipid envelope can
be labeled using synthetic fluorophores as described also in ref. [63].
Specifically, several tags have been used for fluorescence microscopy
of viruses such as covalent labeling, intercalating and lipophilic dyes,
for single virus imaging, fluorescent proteins, fluorescent nanoparticles
such as quantum dots (QDs), and metal nanoparticles, as discussed in
detail in ref. [7]. So far, the virions have been conjugated mostly with
organic fluorophores or genetically combined to a green fluorescent pro-
tein (GFP). The fluorescence can be induced on the virion by the so-
called split-GFP technology consisting of an engineered particle with a
GFP segment bond to a protein of interest associated with another GFP
segment [64]. A recent comprehensive review of the type of fluorescent-
labeled viruses and fluorescence microscopy methods to image them so
far is reported in ref. [6].
Antibodies are considered large fluorescent tags whose size can bias
the super-resolved image and therefore introduce errors in the localiza-
tion of the tagged molecules. Smaller tags such as nano-bodies or click
chemistry are therefore essential for FSR microscopy applications [65].
Live-cell FSR microscopy studies of viruses appear to be even more sus-
ceptible to issues with labeling with stricter requirements of minimal
interaction with the virus structure and functionalities. Antibodies or
nano-bodies for fluorescence tagging have limited possibility in live-cell
imaging because they can only be applied to study external surfaces of
the viruses and cells.
Fluorescence proteins have been advanced for virus labeling appli-
cations using conventional microscopy and they can be implemented for
live-cell FSR imaging. Other organic-dye tagging such as tetracysteine
(TC) tag, CLIP-tag, SNAP-tag, or artificial amino acids and click chem-
istry are viable labeling methods to accomplish FSR imaging studies of
virus replication cycle in living-cell [66].
Standard commercially available Alexa Fluor and ATTO dyes [67],
which cover the visible wavelength range, are switched reversibly be-
tween an “on” and an “off” state with the addition of thiol‐containing
reducing agents which are used to efficiently induce a relatively stable
non-fluorescent state [67]. These small organic fluorophores are rele-
vant for super‐resolution imaging in living cells.
Solid-state nanoparticles such as QDs has been more recently applied
for SVT [7,43,68,69] also in combination with other labeling methods
[70], suggesting that solid-state labeling is the latest trend in the single
virus tracking [43,58].
Other solid-state nanoprobes have been proved efficient for FSR
imaging. For example, nanodiamonds (NDs) also can bind to viruses,
such as hepatitis B or C, from blood plasma isolated from infected pa-
tients [71]. As NDs contain a nitrogen-vacancy (NV) color centers that
can be used as a fluorescent tag in the near-infrared without photo-
bleaching, they can be bonded to viruses and used are FSR solid-
state probes. FSR imaging using NDs has been already demonstrated
in several examples with 20 nm resolution using SMSM [72,73], spin-
dependent fluorescence [74], STED [75], SPIN-RESOLFT [76] as well
as the paramagnetic properties of NV centers can be used to perform
spin nanoscopy [73], magnetic imaging combined with FSR [77], and
spin-STORM [78]. NDs containing NV is also successfully applied to
two-photon microscopy with adaptive optics [79]. STED microscopy
has achieved the best resolution using NV in diamond [80], and SMSM
techniques benefit from NDs as labels due to lower photo-toxicity and
better photo-stability not requiring near UV excitation to induce photo-
switching mechanism. The current limitation to use NDs in FSR imag-
ing of biological systems such as virus is the relatively large and variable
size of NDs (~35-50nm), while other applications as fluorescent labeling
of the biological sample have been established [81]. Other fluorescent
probes based on solid states nanomaterials have been recently applied

5
S. Castelletto and A. Boretti Chemical Physics Impact 2 (2021) 100013

to SMSM such as silicon carbide nanoparticles [82] and boron nitride

flakes [83], the first having the possibility to achieve dual-color photo-
switching using 640 nm beam, thus reducing further photo-toxicity and
photo-bleaching. Both materials can be fabricated in < 8nm size, while
still maintaining their fluorescence brightness. However, none of the
latest proposed novel fluorescent solid-state labels for single-molecule
imaging, have been applied to viral imaging as they are at a very early
stage of development.
The tagging of the viral genome of the virus appears much more
difficult and so far studied mostly for HIV by fusing a fluorescent
protein to an RNA-binding protein or by using fluorescence in situ
hybridization [2].

STED microscopy and fluorescence correlation spectroscopy in

viral imaging

STED microscopy [84] (Fig. 2D) relies on using generally a green

laser excitation to induce spontaneous decay of the fluorophores from
their excited state to the ground state by emitting fluorescence photons.
A red-shifted laser excitation (known as STED laser) is applied to the
excited fluorophore to induce stimulated emission to the ground state
occurring without photon emission in the spectral region of interest. The
STED beam is engineered via a phase modulator to achieve in the focal
point a “donut-shaped” intensity distribution, corresponding to a cen-
tral intensity minimum, where the spontaneous fluorescence is depleted
everywhere except at the center. This permits localization of the fluo-
rophore with a point spread function much smaller than the focal spot of
the excitation beam, which is otherwise diffraction-limited. The resolu-
tion is given by the efficiency of the fluorescence depletion depending on
the intensity of the STED laser. The resolution can be tuned by adjusting
the STED laser shape and intensity, achieving a resolution < 50–60 nm in
the plane direction in cellular imaging [85,86] and < 100 nm resolution
in the axial direction in biological samples [87]. The advantage of STED
microscopy is the direct creation of the image in real-time with a simple
acquisition process, without the need for image frame post-processing,
which may induce potential post-processing image artifacts. However,
achieving high resolution and high speed comes at the expense of photo-
toxicity. When exposing samples with high laser intensities in the ~ GW
cm−2 as with the STED-beam, radicals or singlet oxygen can be gener-
ated causing photo-bleaching and photo-toxicity in living systems with
subsequent cell death. Living cell imaging with STED has been how-
ever achieved [88-90] by optimising the sample preparation protocols
and using fast beam-scanning methods. Nevertheless, STED microscopy
even if it can be used for fast live and fixed cells studies, due to the in-
evitable still too high laser power required, it is not suitable for a long
Fig. 4. a Live confocal imaging of HIV-1 virus in a 2 × 2 μm2 imaging frame
period of live-cell imaging. using the GFP labeling the viral protein Vpr, GFP-Vpr (green). The scale bar
High resolution can be combined also with high imaging speed in is 200 nm. In red the image scanning line locating the position of the virus.
STED microscopy. As STED is based on point by point laser beam scan- b, c The fluorescence intensity for GFP-Vpr (green) in confocal operation and
ning, the imaged field of view impacts its imaging speed with smaller Env (orange) in STED operation on mature HIV-1 particle. The fluorescence
image implying a faster acquisition time. The maximum temporal reso- intensity is scanned along with the red line position (x-axis) of the virus and
lution of STED-microscopy of 5–10 ms was achieved in the investigation versus time (y-axis), respectively. Scale bars are x-axis (white) = 200 nm, y-axis
of HIV-1 (the most virulent of the two HIV strains) acceptance into HeLa (grey) = 4.4 ms. d Mapping of the virus protein Env position and time as acquired
cells. Such high temporal resolution was achieved by reducing the ac- in c, in terms of the correlation function, showing the possibility to track the
intensity correlation of the proteins at the virus position with super-resolution.
quisition time by employing ultrafast laser-scanners on small areas of
e Normalised autocorrelation curves G(𝜏) of Env diffusion (grey and black lines)
interest, namely a reduced field of view [91]. Here STED was modified
for a specific position on the scanned line for the mature (red), immature (blue),
to be able to image the cellular uptake of viral particles with 5- to 10-ms and fixed HIV-1 particles (purple). Figure from ref. [95]. Creative Commons CC
temporal resolution. BY license. creativecommons.org/licenses/by/4.0/.
In general, for all FSR methods included STED, to achieve sub-
diffraction resolution on a given field of view a long acquisition time is
needed. The field of view is however important in imaging virus interac- lease of individual proteins. The structural polyproteins Gag of HIV-1
tions with cells and it cannot be sacrificed. Currently, STED provides the are essential players for the assembly, release, and maturation of virus
best imaging speed of the fluorescence-based SR techniques however at particles and are considered for anti-viral therapy [92]. The incomplete
the expense of a limited field of view. Several studies using STED were Gag lattice of immature virions was distinguishable from the condensed
performed in HIV-1. distribution of mature protein subunits using STED, as compared to elec-
For HIV-1 to achieve a morphologically mature fully infectious virus tron tomography [93]. The maturation is a critical step to understand
particle status, it undergoes a process called maturation with the re- the HIV-1 replication cycle and to develop anti-HIV-1 therapy. The HIV-

6
S. Castelletto and A. Boretti
1 maturation and how the virus evolves into a lipid environment were
studied using STED. The study permitted visualization of the protein
on the surface of individual HIV-1 particles. The first visualization of
the viral envelope (Env, the trimeric viral envelope surface glycopro-
tein) glycoprotein distribution on the surface- with features of 40nm-
of individual HIV-1 particles was achieved with STED using anti-gp120
antibodies as reporters [47,48]. This study revealed that the maturation
was induced by clustering on the virus surface of the Env proteins that
depended on the Gag-interacting Env tail [47]. STED was able to visual-
ize these proteins on the virus’s surface in different stages as it was not
possible with confocal fluorescent microscopy [47]. Previous live-cell
and STED studies on HIV-1 were limited by requiring antibody labeling
for Env detection. In [94] a bio-orthogonal amino acid is combined with
click chemistry for site-specific and minimally invasive labeling.
STED was combined with another method called Fluorescence Corre-
lation Spectroscopy (FCS) [95], based on the correlation analysis of the
fluorescence intensity fluctuations of the tagged molecules over time
while they diffuse. From the correlation of their intensity fluctuations,
it is possible to determine the molecular transit times and their diffu-
sion coefficient. FCS measurements can also enable the determination
of physical parameters governing cellular mechanisms and dynamics, es-
pecially when high sensitivity and fast dynamic resolution are required
[96]. FCS when combined with STED (known as STED–FCS) can achieve
the determination of the molecular diffusion with high spatial localiza-
tion at the level of an individual molecule. STED-FCS also improve tem-
poral resolution [97] to make it more applicable to virus study.
STED-FCS permitted to study the lipid envelope surrounding some
viruses in detail for example, in a live-cell context. Specifically, STED-
FCS has been utilized on HIV-1 and permitted to study how HIV-1
evolves into a lipid environment, allowing the virus to assemble in the
plasma membranes [95]. The study permitted to determine the mobility
of the protein on the surface of individual HIV-1 particles [95]. Specif-
ically, it was confirmed that during its assembly, HIV-1 incorporates
copies of Env1, 2 (the trimeric viral envelope surface glycoprotein), and
the main structural protein Gag3 (a polyprotein initially accumulating
into immature virus particles) [98]. To achieve a morphologically ma-
ture fully infectious virus particle, maturation with the release of indi-
vidual proteins occurs.
In Fig. 4 STED-FCS is applied to measure the autocorrelation curves
G(𝜏) of Env diffusion on HIV-1 particles for different virus phases, named
mature, immature, and mature fixed HIV-1 virus conditions. It was
found that Env undergoes an increase in mobility due to maturation.
This technique has provided appreciable information on the protein’s
assembly dynamics and fusion at virus surfaces in vivo.
The membrane-proximal external region (MPER) of the HIV-1 Env
has been identified as a potential vaccine target [99]. The MPER acces-
sibility is limited making its native structure poorly understood, as such
different and exclusive models are explaining the molecular mechanism
of MPER used by broadly neutralizing antibodies (bnAbs) to recognize
it. Ref. [100] studies the accessibility of antibodies to the native Env
MPER on single HIV virions using STED.
Broadly neutralizing antibodies 10E8 and 4E10 Fabs were conju-
gated with the fluorescent probe Abberior STAR RED (also known as
KK114) to analyze MPER accessibility through STED microscopy in the
native Env complex.
STED imaging of fluorescently labeled Fabs reveals a common pat-
tern of native Env recognition for HIV-1 antibodies targeting MPER or
the surface subunit gp120. Fig. 5 (a) presents the model of HIV viri-
ons, and Fig. 5 (b,c) the visualization of these HIV virions by STED mi-
croscopy super-resolution.
The transport, acidification, and fusion of single influenza virus A
(IVA) in living cells were studied by using fluorescence microscopy [3],
which permitted to dissect individual stages of the viral entry pathway
[101]. More recently three-color STED microscopy was applied to IVA to
visualize early IAV events trafficking in human dendritic cells following
the internalization of virus particles [59]. In particular, it was visualized
7
Chemical Physics Impact 2 (2021) 100013
the input IAV nucleoprotein, early and late endosomal compartments,
and dendritic cells membrane/cytosol.
Human respiratory syncytial virus (RSV) forms pleomorphic virus
particles made of long filaments about 100 nm in diameter and up to
about 10 𝜇m in length. RSV affects human lung epithelial cells induc-
ing filopodia, cellular protrusions that extend to neighboring uninfected
cells. SR microscopy techniques are needed to visualize the interactions
between RSV infected cells and virus particles. In ref. [60], STED mi-
croscopy combined with software providing image deconvolution to cor-
rect for optical distortion has revealed filamentous RSV particles along
these filopodia, providing clues that filopodia facilitates RSV spreading
from cell to cell. Specific dyes were used to prepare fixated cells with
RVS and STED permitted to discern and enumerate the RVS particles
attached to filopodia.
For the Herpes Simplex Virus 1 (HSV-1) the processes of cell attach-
ment and membrane fusion involve many different envelope glycopro-
teins (called gB, gC, gD, gH, and gL) which bind to cellular receptors.
Using STED it has been shown that most of these proteins are distributed
evenly round purified virions, while gD localizes essentially as clusters
that are distinct from gB and gH/gL while it localizes with other gly-
coproteins on cell-bound particles. This redistribution of gD upon cell
attachment could be responsible for the activation of membrane fusion
[56].
Single-molecule switching microscopy
Other FSR techniques use a stochastic intensity switching, resulting
in fluorescence intermittency of fluorescent molecules or other probes,
in the entire field of view of a wide-field microscope. Using imag-
ing reconstruction algorithms, the precise localization of the probes is
achieved. Single-Molecule Switching Microscopy (SMSM) is the general
term used to group all the techniques based on this approach, which
includes PALM [21,41] and STORM [22,33] as well as some variations
of them such as Super-resolution Optical Fluctuation Imaging (SOFI)
[102].
SMSM-methods are usually based on wide-field microscopy with
read-out achieved by EMCC detection (Fig. 2 B). Here a sequence of
100 up to 10,000 individual camera frames are taken, imaging only a
limited number and different subsets of individual isolated fluorescent
labels each time, as they are stochastically switched-on for each subse-
quent camera frame. The sub-100 nm resolution image is reconstructed
from these many camera frames by determining the spatial positions of
the individual fluorescent molecules from their intensity “on” and “off”
states temporal dynamics. The various methods differ owing to the use
of different fluorophores photo-physics to achieve their stochastic on-off
switching. PALM employs the fluorescence activation, using specific ex-
citation lasers, of so-called “photo-activable” fluorescent labels, brought
to an “on” state by the laser excitation; their emission then undergoes a
subsequent photo-bleaching. Specifically, PALM uses photo-activatable
GFP (PA‐GFP) or mEOS as labels, which are genetically engineered fluo-
rescent proteins. PA‐GFP is initially non-fluorescent, while it is activated
in a fluorescent state using a pulse of 405‐nm laser light. By regulating
the laser intensity, only subsets of PA‐GFP molecules become activated
and are imaged and resolved using 561 nm laser, while the other re-
mains dark. STORM instead originally utilized stochastic fluorescence
transitions of organic dye [22]. In STORM the switchable fluorophore
is a Cy3–Cy5 dye pair attached to DNA or protein. As opposite to PALM
where photo-bleaching occurs, in the STORM method, the fluorescence
is brighter and each fluorescent molecule can be cycled hundreds of
times before the irreversible bleaching occurs [22]. Later, studies found
that conventional dye fluorophores could be photo-switched without
the need for an activator fluorophore [23,103] enabling the application
of existing antibody and nucleic acid labels in SMSM. Direct STORM
(d-STORM) relies on the intrinsic cycling of fluorophores between “on”
and “off” states without the need for a secondary activator dye as in the
original STORM. In a more recently developed direct STORM (dSTORM)
S. Castelletto and A. Boretti Chemical Physics Impact 2 (2021) 100013

Fig. 5. (a) Model for MPER accessibility within native Env complexes based on Cryo-EM reconstructions. In the Top Contours showing the exposure of the MPER
helix in native Env (green) to 10E8 compared to no 10E8. Bottom cartoons showing the different Env components, and positions of the neutralizing epitopes for
bnAbs used PGT145 (V1/V2), VRC01, and b12 (CD4bs) and 10E8/4E10 (MPER). Image from [100]. This open-access article is distributed under Creative Commons
Attribution License 4.0(CC BY). (b) MPER Env site visualized by the signal of 10E8-KK114 bound to HIV-1 virions acquired in confocal (top) or STED modes (bottom).
(c) STED images of 2G12, 10E8-KK114, 4E10-KK114, and Annexin V-ATTO 647 bound to HIV-1 virions (top) and corresponding pixel areas (bottom). Image from
[100]. This open-access article is distributed under Creative Commons Attribution License 4.0(CC BY).

[104], conventional organic fluorophores, such as Cy5 and Alexa 647
[23] are used for reversible photo-switching without requiring an acti-
vator molecule, this further improves the quality of signals.
SMSM provides a higher resolution of 10-20 nm localization preci-
sions compared to STED. SMSM is also commonly paired with TIRF.
SMSM-based imaging has been further improved by optimization of the
fluorophores photo-switching methods [23,105,106] by using as exam-
ple multicolor imaging [107,108,109] and by introducing various 3D
SMSM approaches [33,110].
SMSM makes use of laser intensities in the ~kW cm−2 , much lower
compared to STED, however, for photo-switching, it is often required
UV laser irradiation which can cause pronounced photo-toxicity.
Currently, SMSM approaches encompass higher image resolution
and lower photo-toxicity if it is compared to the other methods, how-
ever, the downfall is the requirement for acquisition of many camera
frames and the use of post-processing algorithms to create a final highly
resolved image which limits the temporal speed. SMSM techniques in
general feature a very high spatial resolution at the expense of reduced
imaging speed due to the acquisition of thousands of photo-switching
cycles necessary to build up the final image.
The long acquisition can introduce bias such as drift, which requires
fiducial markers and correction to avoid image artifact. Long acquisition
times more importantly introduce an upper limit to the time resolution
and speed when it is needed to resolve live-cell dynamics or to achieve
real-time imaging of viruses infecting the cell as an example. Optimized
image acquisition and processing protocols [111,112] have been devel-
oped to mitigate these issues. Due to a several seconds typical acquisi-
tion time associated with SMLM, the technique appears less suitable for
live-cell imaging while it is better applicable to fixed samples requiring
the highest possible, molecular level resolution.
SMLM time resolution has been improved to reach 0.5 s in using sin-
gle excitation in a 2D imaging in cell environment [111], even if at a
reduced spatial resolution of 25 nm; while a 3D spatial resolution of ~30
nm in the lateral directions and ~50 nm in the axial direction could be
achieved with a time resolution of 1-2 s. This temporal speed is not still
enough to capture live imaging of virus-cell molecular interactions as-
sociated with molecular diffusion and clustering dynamics, which occur
within a time scale of milliseconds at nanometer distances. dSTORM us-
ing small organic fluorophores with high photostability permits longer
acquisition time thus can achieve a lateral resolution of 15–20 nm [67].
In ref. [55], dSTORM was used to determine the position of pro-
tein layers within individual Herpes simplex virus type-1 (HSV-1) par-
ticles. The envelope-anchored glycoproteins were discerned from tegu-
ment proteins, both in purified virions and in virions present in infected
cells.
Several studies using dSTORM and PALM as well as Single Particle
Tracking PALM were performed on the HIV-1 virus revealing its proteins
component, assembly, maturation, and replication cycles, whose details
are summarized in ref. [38,98].
In ref. [50], dSTORM is combined with standard immunocytochem-
istry and TIRF microscopy to quantify the 2D molecular distribution of
the major structural proteins of the HIV-1. HIV-1 is imaged before and
after the infection of lymphoid cells, after fixing the infected cells. It was
found that after HIV-1 infection, the HIV-1 capsid proteins undergo re-
arrangement. HIV-1 virus contained the GFP-viral protein (GFP-Vpr), to
allow visualizing particles with conventional fluorescence microscopy
[50]. Subsequently, dSTORM and TIRF using multicolor tags also re-
vealed information on protein colocalization (Env and Gag) at the HeLa
cell membrane for the virus assembly [52] as well as recruitment of
tetherin clusters to Gag assembly sites at the membrane of HeLa cells
[51].
Recently, 2D and 3D dSTORM [53] revealed the mechanism by
which Serine incorporator protein 5 (SERINC5), an anti-retroviral fac-
tor, inhibits the Env mediated HIV-1 virus fusion with target cells by
imaging the distribution of Env on a single virus. It was first confirmed
that the Env glycoproteins form clusters on the surface of mature virions
[47] and the SERINC5 disrupted Env clusters without affecting the over-
all Env content, suggesting an indirect effect of SERINC5 on Env cluster
formation. Recently dSTORM (based on commercial Nikon N-STORM)
has been used to study a matrix protein localization at the base of IAV
filaments, emerging from the membrane of infected stained cells [44].
Correlative fluorescence light microscopy permits a combination of
SEM and FSR and it has been applied to the influenza virus [42], where
3D STORM and SEM are combined (Fig. 6).
PALM microscopy has also demonstrated the ability to observe the
virus assembly at the host cells with nanoscale resolution. In particular,
it has been applied to immune system living host CD4+ T cells where
HIV assembly and budding at the plasma membrane of the cells was
studied [54,98]. It has been implemented so-called live PALM, consist-
ing of combining PALM and TIRF-microscopy, with analysis methods

8
S. Castelletto and A. Boretti
based on hidden Markov statistical analyses and Bayesian inference.
This approach has permitted to achieve a description of the virus for-
mation at the plasma membrane of individual cells, with 50 nm spatial
resolution and millisecond temporal resolution. HIV-1 particle assembly
at the host cell plasma membrane occurs via thousands of viral Gag pro-
teins forming particles with a diameter of 100–130 nm. By introducing
into the HIV-1 Gag precursor and derivatives, a photoactive fluorescent
9
Chemical Physics Impact 2 (2021) 100013
Fig. 6. Correlated 3D STORM and SEM images
of individual filamentous influenza viruses (A)
Process to achieve correlative images. (B) Cor-
related images of STORM, overlaid image, and
SEM image of the virus. (C) Magnified images
of the selected areas in (B). (D) Transverse lo-
calisations of the selected area in (B). (E) Nor-
malized photon numbers per switching event.
(F) Cross-correlation between STORM and SEM
images. (G) Correlative two-color STORM and
SEM images of virus filaments with two differ-
ent immuno-labeling STORM image, Overlaid
image, SEM image. Scale bars are 500 nm De-
tails are reported in ref. [42]. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and re-
production in any medium, provided the origi-
nal author and source are credited.
Fig. 7. PALM and TIRF Imaging of mem-
brane Gag cluster sizes and densities at the
T-cell surface of fixed Jurkat T cells that ex-
press wild type (WT) Gag, NL4.3ΔPolΔEnv
Gag, WM, MACASP1, or Δp6. Purified virus-
like particles are on the left upper panel from
WT Gag productive cells used as a size ref-
erence. The major image with a 2 𝜇m scale
has a zoom of a selected area with a 1 𝜇m
scale bar. Image adapted from ref. [54]. Cre-
ative Commons CC BY license. creativecom-
mons.org/licenses/by/4.0/.
protein mEOS2 to form Gag(i)mEOS2, PALM imaging was achieved. In
Fig. 7 images using Live PALM show HIV-1 with various Gag proteins
at the plasma membrane of living host CD4+ T cells.
The modality of assembly for the Nipah virus was for the first time
revealed by using an SMSM similar to a dSTORM combined with TIRF,
showing the virus assembly model previously undetermined using bio-
chemical analyses and electron microscopy [57].
S. Castelletto and A. Boretti
Depth imaging
Another relevant criterion for FSR methods to be applied to biologi-
cal samples imaging and virus imaging and tracking is the sample depth
penetration of the excitation beam.
Refractive index mismatch between air and tissue is responsible for
optical aberrations and light scattering, limiting factors to achieve res-
olution in sample depth by using fluorescence microscopy. The limited
sample depth penetration is due to poor image resolution and limited
contrast due to increased noise levels due to scattering. These effects
can be mitigated by using two-photon-based excitation at a longer laser
wavelength which reduces scattering [113], by the use of objective
lenses with fluid better matching the sample’s refractive index such as
glycerol-immersion microscope [114], or by the use of adaptive optics
which can limit effects of optical aberrations [115].
Light-sheet fluorescence microscopy (LSDM) [116] is another
method that permits superior sample depth penetration allowing 3D
imaging, while it does not improve spatial resolution, and it is advanta-
geous to combine it with FSR approaches for better 3D imaging of live
cells [117,118]. This approach has been used for virus imaging com-
bined with SIM and RESOLFT for herpesvirus [45,119]. The method
permitted to image and track the herpesvirus capsids transport occur-
ring by diffusion.
LSDM is also used successfully for Single Virus tracking, together
with other approaches described above when SR is needed.
Single virus or particle tracking
Single virus or particle tracking (SVT) is another method to image
viruses [7]. While FSR microscopy can provide nanometric resolution
in imaging viruses, their trajectory tracking can be achieved without
the need for FSR methods by simply using wide-field microscopy, TIRF
microscopy, confocal microscopy, and spectroscopy. These methods al-
low for the imaging of virions labeled with photo-stable organic fluores-
cent dyes or fluorescent proteins and permit tracking their position with
highly accurate particle localization in the nanometer range better than
the resolution of microscopes images [7]. For example in ref. [46], Ebola
virus dynamic entry and internalization into live cells were visualized
by fluorescent virus labeling using SVT at the single virus level. This for
the first time permitted to observe that Ebola virus internalization in
cells occurs through lipid rafts.
A label-free method has been used to track HIV such as interfero-
metric scattering microscopy (iSCAT) [120]. iSCAT permits visualiza-
tion of the position of the virus using the interference of light owing to
reflection from the surface of the coverslip and from the virus itself with
high temporal and spatial resolution virus tracking. iSCAT in conjunc-
tion with SMSM was used to investigate the diffusion of single Simian
Virus 40 particles labeled with QDs. Due to the achieved 8 ms temporal
resolution and 9 nm spatial resolution, it was possible to determine the
orientation of the virus on supported lipid bilayers [68].
Wide-field microscopy allows SVT in a large volume due to the large
depth of focus associated with a low signal loss in depth. It is better
suited when there are low auto-fluorescence and low concentration and
distribution of fluorescent viruses. It has been used to study the trans-
port, acidification, and fusion of single influenza viruses in living cells
and it was possible to separate distinct phases of the viral internaliza-
tion within cells [101]. However, wide-field microscopy with samples
exhibiting a high background results in out-of-focus probes with low-
contrast images, thus lower resolution as it was revealed in HIV assem-
bly tracking by using fluorescence-labeled Gag protein [121].
SVT has been combined with FSR microscopy to increase the tempo-
ral resolution of PALM and STORM. Combining Single-particle tracking
with PALM (known as SPT-PALM) enabled monitoring of the molecular
diffusion of single-molecule Gag and tsO45 proteins from the vesicular
stomatitis virus G (VSVG) [41].
10
Chemical Physics Impact 2 (2021) 100013
Discussion and conclusions
FSR methods have largely progressed from the time of their incep-
tion, at a current stage of development and deployment which has pro-
vided many commercial systems, that are progressively becoming of
common use in biomedical imaging laboratories. However, for virus
imaging, there is not yet one perfect approach, as the above-described
techniques have a unique set of advantages and disadvantages. These
approaches are evolving to combine more than one imaging method
to mitigate disadvantages and leverage on the respective advantages or
simplicity of use. Current virology studies employing FSR imaging have
contributed to many novel insights even if still achieved mostly in fixed
cells environment and in vitro cell cultures. As such their potentials to
study the dynamic behavior of viruses’ components and their interac-
tions with living cells or tissues environment, is not yet accomplished.
New development of FSR methods has recently seen the combination of
STED and STORM (MINFLUX), merging the advantages of both meth-
ods, thus permitting to discern molecules with 1 nm precision at 9 nm
distance and 100 times better temporal resolution in single molecules
tracking. Due to MINFLUX recent application in 3D imaging in fixed
and live cells [122], it could be further applied in observing the virus
components dynamics in living cells [123].
However, one of the major problems for expanding the use of FSR
imaging in live-cells virology is the need for biosafety containment level
3 or 4 (BSL-3 and BSL-4 laboratories), which are not very common. In
this regard, the development of commercial nano imagers could mit-
igate safety requirements as well the use of pseudovirus that can be
handled in biosafety level 2 laboratories in place of wild‐type viruses.
Pseudovirus [124], deprived of the certain virulent genome, can only
replicate once, and are obtained by combining proteins from the surfaces
of other viruses, as an example for SARS-COV-2 virus with replication-
defective HIV or another retrovirus [125], could advance the in vivo
FSR microscopy application to a virus.
Based on the so far few demonstrations and on the HIV-1 viruses
studies, where the key mechanisms of the viruses and the cell interaction
and internalization were unveiled, the current methods can be applied to
other viruses for example SARS-CoV2, towards the development of drugs
that specifically prevent these processes. There is in fact a clear need
to complement existing viral imaging techniques with super-resolution
methods for a better understanding of the SARS-CoV2 replication cycle
in presence of chemotherapeutic and chemopreventive agents [126].
It can be finally concluded that the above-mentioned techniques
could be implemented not only to advance virions’ imaging and mo-
tion analyses to provide information about biological mechanisms, as it
has been shown now in some examples, but also to facilitate the devel-
opment of antiviral therapeutics and vaccines. This seems to be possible
at the current stage of development and even better when their full po-
tential could be reached, which is in vivo imaging and single-particle
tracking could be implemented in cultured cells with different thera-
peutics.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
The authors received no funding.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.chphi.2021.100013.
S. Castelletto and A. Boretti
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