Abberior Instruments Guides Applications

STED and virology

Super resolution microscopy is a widely used methods, such as STimulated Emission Depletion
research method for studying biological (STED) microscopy or single-molecule localization
features beyond the diffraction limit, such as methods, which offer resolution capabilities of about
nuclear pores, clathrin coated vesicles, the 20  nm. Numerous studies using super resolution
periodicity of the actin skeleton in neurons, imaging techniques have already expanded the
and viruses. In this white paper we will focus scientific knowledge of the Human Immunodeficiency
on the advantages of STED microscopy and Virus (HIV-1) [1, 2], Influenza A virus [3, 4], Herpes
its contributions to the virology field. We will Simplex Virus (HSV) [5], Respiratory Syncytial Virus
provide insight into the advances in virus (RSV) [6], Vaccinia Virus [7], Adeno-Associated Virus
research made possible by STED microscopy (AAV) [8], Nipah Virus (Niv) [9], Adenovirus [10] and
and give a hint to were the journey might go. many more.
© Abberior Instruments GmbH 2020 | Rev. 1.0 20200505 | Subject to change without notice.

Virus particles vary in size across different virus STED microscopy for virus research
species. One of the smallest viruses, the parvovirus,
has a particle size of approx. 20 nm while the largest The gold standard technique for imaging virus
known virus particles of the Mimivirus family possess particles was and still is electron microscopy (EM).
a capsid size of up to 500 nm, which already compares The main advantage of EM is undoubtable the
to the size of a bacterium (Fig. 1). Therefore, due to angstrom-range resolution it provides, which exceeds
their size, virus particles and their substructures can the resolution capabilities of all other available
be studied in detail using super-resolution microscopy imaging techniques. The extremely good resolution,
however, comes at the cost of highly fixed specimens
with low contrast. Furthermore, labeling and detection
of specific proteins inside large assemblies and
• Super-resolution STED microscopy is a powerful cells is challenging with EM. On the other hand,
technique to observe the finest structural details light microscopy methods, and especially confocal
in cells and viruses fluorescence microscopy, generate high-contrast
• It covers the full resolution spectrum required to images, with specific labels for the proteins of interest.
study viruses even in a live environment and in However, the resolution of these techniques is limited
interactions with the host cells to about half the wavelength of light (~200 nm). The
• MINFLUX will open completely new paths of Nobel Prize winning methods of super-resolution
virus imaging microscopy, specifically STED microscopy and
single molecule localization microscopy techniques,
overcome this resolution barrier by exploiting the on-

1/5
 STED and virology

Fig. 1: Comparison of different virus particle sizes with the resolution of different imaging techniques.

off switching of fluorescent dyes. These techniques light on, for example, virus-host interactions. In
improve confocal resolution by roughly a factor of conventional microscopy, however, our understanding
10, achieving resolution capabilities closer to those of the protein position is blurred by diffraction. Super
offered by EM (see Fig. 1). Furthermore, the nanoscale resolution microscopy techniques overcome this
resolution of STED microscopy combined with the blurring effect and thus lend a much higher confidence
less destructive sample preparation and labelling to the results of colocalization experiments. STED
methods of fluorescence make it possible to study microscopy is especially well-suited for these studies
the structural information of native (unfixed) virus since resolution can be improved not only laterally, but
particles in the 100 nm-range [2]. An example of how axially as well, giving rise to isotropic resolution of up
STED microscopy has contributed to HIV-1 research to 60 nm without any post-processing. Additionally,
is detailed in the box on page 3. if two spectrally different dyes are used which can
both be depleted by the same STED laser doughnut
Protein-specific labeling coupled with the high- (e.g. STAR ORANGE and STAR RED, or Alexa594 and
contrast imaging of light microscopy enables ATTO647N), there is no chromatic shift between the
colocalization studies of different proteins to shed two channels. Because the central zero of the STED
doughnut defines the point from which photons are
emitted, if two channels share the same depletion
doughnut, colocalization is intrinsically given. Various
studies have used STED microscopy to visualize the
HSV genome inside infected cells [11] or to study the
virus-host interaction of the Influenza A virus [3, 4].

Fig. 2: Env protein distribution (red/yellow) on single HIV-1

particles (green). Vpr.eGFP was used as a marker for HIV-
1 particles. Env clusters on the virus particle surface were
labelled by Fab fragments coupled to STAR635P. Only with
super-resolution microscopy can single Env clusters be
revealed (compare middle column (STED) and right column
(confocal)). Images by Jakub Chojnacki (used with permission).

2/5
 STED and virology

The possibility of live-cell imaging

and the tracking of dynamic events A glimpse into HIV-1 studied by STED microscopy
over time is a great advantage
of fluorescence microscopy, Human Immunodeficiency Virus 1 (HIV-1) maturation occurs concomitantly
and STED microscopy uniquely with cleavage from the plasma membrane of its host cell. During maturation, the
combines this advantage with protease is active, and cleaves the polyprotein Gag into its components, leading to a
nanoscale resolution. Live-cell rearrangement of the virus proteins. Mature HIV particles enter cells more efficiently.
STED microscopy has proven However, there is no apparent change of the surface protein (envelope protein, Env)
valuable in many studies [12- structure or composition. Compared to other virus particles, only a few envelop
14]. A major concern in live-cell proteins are incorporated on the HIV surface (7-14 Env proteins per virus particle). The
STED microscopy is bleaching question remains of how the internal morphological change influences the Env protein
and phototoxicity. While on the particle surface, and what the surface effect is that increases entry efficiency
bleaching can be overcome into new host cells.
by using photostable dyes or
exchangeable fluorophores [18], In 2012, the research team of Hans-Georg Kräusslich answered these questions
minimizing phototoxicity is less using STED microscopy, when the method was still in its infancy. In a collaboration
straightforward. Nevertheless, with Stefan Hell’s group, they showed that Env proteins on single virus particles form
with STED microscopy, it is often clusters after virus maturation, and that this clustering is induced by maturation
overlooked how resolution and through an interaction between the Env tail and the internal virus proteins. Since
laser power are interdependent. virus particles (diameter ~120-150 nm) are much smaller than the diffraction limit of
Due to the square-root light, only direct observation by super-resolution microscopy was able to resolve this
dependence of resolution on clustering (see Fig. 2) [1].
STED power, high STED laser
power is only needed to squeeze In a follow-up study at the University of Oxford, researchers were able to determine
out the last few nanometers of the mobility of the Env trimers on a single virus particle. This study showed that the
resolution. In STED microscopy, HIV-1 lipid envelope is a low mobility environment due to its high lipid order. The
the achieved resolution is tunable standard technique to image mobility is Fluorescence Correlation Spectroscopy
via the STED laser power and this (FCS). However, since the virus particles are smaller than a conventional focused
can be increased only as much spot, intensity fluctuations due to particle movement can only be observed by a
as required to answer a given combination of STED and FCS, to reduce the focal volume [23].
scientific question. Furthermore,
novel technologies such as pulsed The high lipid order observed led to a collaboration between labs in Spain, France,
STED combined with adaptive Germany and Australia to study the lipid environment of budding HIV particles in cells
illumination methods (e.g. by STED-FCS. The study showed that around an HIV-1 budding site, the virus seems
MINFIELD, DyMIN and RESCue) to generate its own lipid environment, which is likely due to the recruitment of specific
[15-17], allow for prolonged lipids through the main structural polyprotein Gag [24].
imaging at high resolution with
low light levels and thus less In summary, major contributions to basic research on HIV-1 were achieved using STED
phototoxic effects. An example microscopy. Also in the future, STED and super-resolution microscopy in general will
on HIV-1 imaging is shown in be instrumental in the research on small structures such as viruses.
Fig. 3. Here, it is also important
to note that since a virus particle
contains only a small number of
proteins and hence labels, gentle adaptive illumination in confocal mode with a low magnification objective
methods help minimize photobleaching, thus allowing lens to easily identify rare events. After finding the
one to image with higher resolution, or for a longer event, a region of interest can be chosen to target the
time in a live experiment. event, and the STED laser can be turned on to acquire
a super-resolution image. The imaging of capsid
Especially sparse events are difficult to image in EM positive objects after HIV infection of cells close to
and can usually only be found by correlative imaging the nuclear pore [19] is one example of how STED
of light microscopy and EM. Identification of sparse microscopy can be used to study rare events.
events, such as cell infection or virus particles binding
to enter a cell, or nucleus, are the perfect target for Scientific research involving virus imaging requires
STED microscopy. Since every STED microscope a large range of imaging scales, from a single virus
is inherently a confocal microscope (with the STED particle on the coverslip [2], to whole infected cells [19]
laser turned off), it is possible to scan the sample or even intact living mice [21] to, for example, observe

3/5
 STED and virology

the immune response in lymph nodes. Thus, the ideal

super-resolution microscope needs to be flexible to
perform all given imaging tasks. STED microscopes Got questions?
reliably meet this need and can be adapted to a
variety of imaging experiments. Objective lenses Please contact
and mounting media can be chosen as needed for info@abberior-instruments.com
the experiment. Adaptive optics allows STED and abberior-instruments.com
confocal imaging deep inside the sample by using a
deformable mirror to correct for aberrations induced
by the sample. With these tools at hand, the STED
microscope is able to image across scales and highly [22] offers isotropic resolutions up to 2 nm – the
adapts to the experimentalist’s needs. size of a single fluorescent molecule. Already STED
microscopy was able to change the way we look at
virus particles, so the impact MINFLUX will have
Conclusion and Outlook on scientific research cannot be underestimated.
In this article, we provide a brief overview of super This new level of resolution is expected to have a
resolution microscopy, focusing on STED microscopy tremendous impact on virology research. MINFLUX
and its contributions to virus imaging and virus tracking can be performed at extremely high speeds
research. Developments in STED microscopy with true molecular precision, making it possible to
instrumentation are rapid and, while the technique has observe rearrangements inside a single protein over
already seen widespread use in scientific research, we time, or to follow single virus particles over a long time
are confident that it’s potential can only increase in the with unprecedented precision and speed.
future. One should not forget that the first ever images
on a home-built STED microscope were acquired only
less than two decades ago. Since then, tremendous
amounts of engineering and innovation have made References
STED microscopy what it is today: a reliable, easy-to- [1] Maturation-Dependent HIV-1 Surface Protein Redistribution Re-
use super resolution imaging technique that can be vealed by Fluorescence Nanoscopy, Chojnacki, J. et. al, SCIENCE,
operated as intuitively as a confocal microscope, by 26 OCT 2012: 524-528
everyone.
[2] Stimulated Emission Depletion Nanoscopy Reveals Time-
Course of Human Immunodeficiency Virus Proteolytic Maturation,
The potential of super resolution microscopy extends Hanne, J., et al, ACS Nano 2016 10 (9), 8215-8222, DOI:10.1021/
far beyond STED. The novel technique MINFLUX acsnano.6b03850

A B C
Confocal MINFIELD

160 nm

Fig. 3: MINFIELD STED presented on human immunodeficiency virus type 1 (HIV-1 labelled with SNAP-tag between matrix and
capsid in the Gag protein and stained with Siliconrhodamine). (A) Confocal (left) and MINFIELD STED (right) images with a field
size of 160 nm. (B) Scheme of the immature HIV-1 particle with labels indicated in red. (C) Imaging examples of single HIV-1
particles.

4/5
 STED and virology

[3] IFITM3 Clusters on Virus Containing Endosomes and Lyso- [21] In vivo mouse and live cell STED microscopy of neuronal actin
somes Early in the Influenza A Infection of Human Airway Epitheli- plasticity using far-red emitting fluorescent proteins. Wegner, W. et
al Cells, S. Kummer et al., Viruses 2019, 11(6), 548 al., Sci Rep 7, 11781 (2017). https://doi.org/10.1038/s41598-017-
11827-4
[4] Visualization of early influenza A virus trafficking in human
dendritic cells using STED microscopy, F. Baharom et al., PLoS [22] Nanometer resolution imaging and tracking of fluorescent
ONE 12(6): e0177920. molecules with minimal photon fluxes, Balzarotti, F. et al., SCI-
ENCE, 10 FEB 2017: 606-612
[5] Structural analysis of herpes simplex virus by optical super-res-
olution imaging, Laine, R., et al., Nat Commun 6, 5980 (2015). [23] Envelope glycoprotein mobility on HIV-1 particles depends on
https://doi.org/10.1038/ncomms6980 the virus maturation state. Chojnacki, J., et al., Nat Commun 8, 545
(2017). https://doi.org/10.1038/s41467-017-00515-6
[6] Multicolor Stimulated Emission Depletion (STED) Microscopy
to Generate High-resolution Images of Respiratory Syncytial Virus [24] HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol
Particles and Infected Cells, M. Mehedi et al., Bio Protoc. 2017 Sep mobility in living cells creating a nanodomain platform for virus
5; 7(17): e2543. assembly. Favard, C., et al., Science advances, 5(10), 2019,
eaaw8651. https://doi.org/10.1126/sciadv.aaw8651
[7] Gray, Robert & Albrecht, David. (2019). Super-resolution Micros-
copy of Vaccinia Virus Particles. 10.1007/978-1-4939-9593-6_16.

[8] Super-resolution imaging of nuclear import of adeno-associ-

ated virus in live cells, Kelich, Joseph M et al., Molecular Therapy
- Methods & Clinical Development, Volume 2, 15047

[9] A stochastic assembly model for Nipah virus revealed by

super-resolution microscopy, Liu, Q. et al., Nat Commun 9, 3050
(2018). https://doi.org/10.1038/s41467-018-05480-2

[10] Tracking Viral Genomes in Host Cells at Single-Molecule

Resolution, Wang, I-Hsuan et al., Cell Host & Microbe, Volume 14,
Issue 4, 468 - 480

[11] Visualizing the replicating HSV-1 virus using STED super-res-

olution microscopy. Li, Z. et al., Virol J 13, 65 (2016). https://doi.
org/10.1186/s12985-016-0521-7

[12] Live-cell imaging of dendritic spines by STED microscopy, Nä-

gerl, V. U. et al., Proceedings of the National Academy of Sciences
Dec 2008, 105 (48), 18982-18987; DOI: 10.1073/pnas.0810028105

[13] Live-Cell Superresolution Imaging by Pulsed STED Two-Pho-

ton Excitation Microscopy, Takasaki, K. T. et al., Biophysical Jour-
nal, Volume 104, Issue 4, 770 - 777

[14] Two-colour live-cell nanoscale imaging of intracellular targets,

Bottanelli, F. et al., Nat Commun 7, 10778 (2016). https://doi.
org/10.1038/ncomms10778

[15] Adaptive-illumination STED nanoscopy, Heine, J. et al, Pro-

ceedings of the National Academy of Sciences Sep 2017, 114 (37)
9797-9802; DOI: 10.1073/pnas.1708304114

[16] MINFIELD STED microscopy, Göttfert, F. et al, Proceedings of

the National Academy of Sciences Feb 2017, 114 (9) 2125-2130;
DOI: 10.1073/pnas.1621495114

[17] Far-field optical nanoscopy with reduced number of state tran-

sition cycles, Staudt, T. et al., Opt. Express 19, 5644-5657 (2011)

[18] Whole-Cell, 3D, and Multicolor STED Imaging with Exchange-

able Fluorophores, Spahn C. et al., Nano Letters 2019 19 (1), 500-
505, DOI: 10.1021/acs.nanolett.8b04385

[19] Analysis of CA Content and CPSF6 Dependence of Early HIV-1

Replication Complexes in SupT1-R5 Cells, Zila, V. et al., mBio Nov
2019, 10 (6) e02501-19; DOI: 10.1128/mBio.02501-19

[20] Resolution scaling in STED microscopy, Harke, B. et al., Opt.

Express 16, 4154-4162 (2008)

5/5