Fphy 06 00157
Fphy 06 00157
Fphy 06 00157
Department of Physics, Randall Centre for Cell and Molecular Biophysics, King’s College London, London, United Kingdom
Bridging the gap between traditional immunology and nanoscale biophysics has
proved more difficult than originally thought. For cell biology applications however,
super-resolution microscopy has already facilitated considerable advances. From
neuronal segmentation to nuclear pores and 3D focal adhesion structure—nanoscopy
has begun to illuminate links between nanoscale organization and function. With
immunology, the explanation must go further, relating nanoscale biophysical phenomena
to the manifestation of specific diseases, or the altered activity of specific immune
cell types in a bodily compartment. What follows is a summary of how nanoscopy
has elucidated single cell immunological function, and what might be achieved in the
future to link quantifiable, nanoscale, biophysical phenomena with cell and whole tissue
functionality. We explore where the gaps in our understanding occur, and how they might
be addressed.
Keywords: T cells, immunology, SMLM, nanoscopy, super-resolution
Edited by:
Jorge Bernardino de La Serna, The so called immunological synapse has proved fertile ground for the intersection of nanoscopy
United Kingdom Research and
with immunology [1]. The word synapse originates from Greek sun–together and hapsis–joining.
Innovation, United Kingdom
Like a neuronal synapse, the immune synapse (IS) describes a communication zone at the interface
Reviewed by:
between two cells. In the case of the T cell synapse, a T lymphocyte and an antigen presenting
Florian Baumgart,
cell (APC) communicate through an intricately arranged array of receptors. Archetypal immune
Technische Universität Wien, Austria
Susanne Fenz, synapses can be recapitulated through use of supported lipid bilayers (SLBs) loaded with peptide
Universität Würzburg, Germany bound major histocompatibility complex (p-MHC) [2], or more simply through the use of
Ana Mafalda Santos, activating antibodies bound to glass, directed at the T cell receptor [3, 4]. In the past few years,
University of Oxford, United Kingdom several proteins in the T cell membrane have been found to be organized on nano-length scales,
*Correspondence: and links to cell function have begun to emerge.
Dylan M. Owen In the fields of super-resolution and biophysics, the IS has had a dual function–as an area for cell
dylan.owen@kcl.ac.uk biological insight in itself, and as a proving ground for super-resolution technique development.
Cell biology has benefitted from the system as headway has been made in understanding the role of
Specialty section: nanoscale molecular organization of the TCR [5–7], LFA-1 [8, 9], LAT [10, 11] and the nanoscale
This article was submitted to meshworks formed from fibers like actin [12]. Elsewhere, super-resolution imaging has been used to
Biomedical Physics,
study nanoscale structures such as the nuclear pore complex, mechanisms of segmentation within
a section of the journal
Frontiers in Physics
organelles such as the mitochondria, or the axon and dendrites of neurons [13, 14]. In these cases,
the use of diverse nanoscopic methodologies helped independent researchers report and verify the
Received: 24 September 2018
same structures.
Accepted: 18 December 2018
Published: 24 January 2019
The organization of the TCR has been subject to rather intense investigation over the last few
years, in part because of controversy over its nanoscale organization, and in part due to artifacts
Citation:
Shannon M and Owen DM (2019)
emanating from the imaging. It therefore provides a useful case study of many researchers being
Bridging the Nanoscopy-Immunology involved in the elucidation of a phenomenon using new technology.
Gap. Front. Phys. 6:157. Bjorn Lillemeier’s group were among the first to show that T cell receptor forms nanoclusters
doi: 10.3389/fphy.2018.00157 in activated cells, which group together into islands to amplify a signal, allowing T cells to
surpass a threshold for activation [6]. In a noisy, live nanoscale. Molecular clustering is necessary for the structured
environment, resolution was reduced, but new information formation of adhesions in T cells [24], and new nanoscopy
afforded by this technique showed that the TCR and LAT indeed techniques have allowed us to show that there are levels of
organize into nanoscale islands upon activation by a single organization beyond microscale adhesion clusters. The dynamics
pMHC-TCR interaction in vivo.This, the authors posit, is a of such clusters likely occur across many timescales, and while
nanoscale spatial mechanism for signal amplification—one that live cell PALM in the T cell synapse has provided important
negates the need for 10 or more antigen peptides to be presented insights into nanocluster dynamics with temporal resolution of
for full T cell activation [15]. 1–2 s [10, 11, 25–27], fast dynamics will rely on the development
However, the existence of TCR clusters in antigen experienced of much faster localization imaging.
“resting” cells is less clear, and new analysis suggests that pre- Nanoscale T cell adhesions are short lived, small and distinct
clustered TCR may be an artifactual symptom of molecular from conventional focal adhesions observed in other cell types.
overcounting [8, 16]. Alternatively, such clusters may represent Recent super-resolution microscopy work has shown that T
ligand-independent triggering of the TCR that happens when cell adhesions based around LFA-1 integrin are very small
T cells come into contact with various surfaces commonly throughout the cell—on the length scale of nascent adhesions
used for in vitro assays, including poly-l-lysine [17]. Molecular at around 100 nm in diameter [9]. Those adhesions that are
overcounting occurs when multi-blinking fluorescent emitters anchored to ligand on the outside of the cell, and to actin on the
are detected more than once, in slightly different places, resulting inside remain engaged from the leading edge through the lamella,
in a single molecule looking like a cluster in the output data. but have a total lifespan of <1 min. As the field progresses, further
Varying the label density is a possible way to rule out false clusters nanoscopic investigation may help us to answer questions about
in super resolution localization data [8]. When this technique was the precise composition and compartmentalization of nanoscale
used, no TCR clusters were found in resting T cells. Biologically, T cell adhesions. One possibility is that actin and the membrane
new data suggests that the T cell receptor would seem to benefit work together to compartmentalize nano-adhesions, a concept
from being a single entity in that this would speed the scanning pioneered by Kusumi [28, 29] and suggested for T cells by
phase in resting T cells: if TCRs are non-clustering, they have Lillemeier [6].
a higher probability of coming into contact with pMHC [18]. Recent work has linked the modulation of such nanoscale
Recent work by Cai et al. supports this. Here, high resolution adhesions with altered migration due to the loss of function
lattice lightsheet microscopy, along with quantification using the mutation of a phosphatase associated with autoimmune disease:
displacement of quantum dots showed how microvilli are used by PTPN22 [9]. Upon loss of this phosphatase, effector CD8+
T cells to speed the scanning process [19]. Jung et al. used variable and CD4+ cells migrate considerably faster and alter their
angle TIRF and localization of fluorescently labeled TCR and nano-adhesion structure as well as their link to the actin
Zap70 (Syk family kinase involved in TCR activation) to observe cytoskeleton. In humans suffering from rheumatoid arthritis,
nano-clustering at the tips of the microvilli [20]. The precise PTPN22 mutation or deficiency is linked to an accumulation
characteristics of TCR and Zap70 clusters, and the function of the of such effector T cells in the joints [30]. Therefore, it is
clustering observed at the dwell sites of microvilli requires further possible that phosphatases like PTPN22 act as nanoscale control
investigation. The Zap70 catch and release model [21] appears switches for integrin based adhesions, and that the lack of this
to match well with microvillar scanning, which might provide control causes a default fast migration phenotype, contributing
the amplification needed for further signaling and full activation to the mis-localization of cells and development of autoimmune
in the presence of a strong, albeit rare signal. Together, these disease conditions. The effects on the base migration phenotype
works provide evidence that the nanoscale spatial localization of and accompanying nano-adhesion architecture caused by this
multiple signaling molecules like this are likely to be involved in mutation warrant more investigation. This might be done in
the regulation and amplification of an initial TCR-pMHC signal. other fast moving cell types affected by phosphatase mutation
[31], as well as in specific T cell subtypes such as T effector
memory cells that migrate in the periphery or central memory
NANOSCALE T CELL MIGRATION T cells that migrate mainly in the secondary lymph nodes [32].
speed [35]. Along blood vessels, migration and diapedesis relies molecule numbers, such that enough information is collected in
on actin “treadmilling” and integrin based adhesions to anchor only a few raw frames to extract statistically relevant nanoscale
themselves and translate actin flow into cell movement [35]. information about cluster dynamics. Through advances in such
In the diverse environment of the periphery, it is likely that quantitation techniques, live cell microscopy techniques and
these modes of migration are mixed and complimentary and are reversibly switchable or replenishable fluorophore engineering
probably true for other exploratory immune cells. Interestingly, [41–43], it is likely that the dynamics of individual protein
it is also clear that subtypes of T cells develop and maintain set clusters of signaling molecules will be investigable in live T cells.
migratory patterns as they differentiate. Some predominantly Point pattern analysis at physiologically relevant spatio-temporal
move around the peripheral tissues (e.g., stem memory T cells), resolution will provide highly quantifiable data, potentially
or remain resident there (T resident memory), while others allowing sensitive processes to be statistically analyzed.
reside largely in the secondary lymph nodes, such as the central
memory T cells [32, 36].
INCREASING THE SUPER-RESOLUTION
Insight into the nanoscale behavior of adhesion receptors
and effectors may provide immunological understanding as to SAMPLE SIZE
why these cells maintain such behaviors. One question might be
One concern within circles of immunologists is the relevance of
whether cells coded for certain kinds of migration also maintain
tiny changes in the properties of nanoscale signaling domains
“cluster memory,” where adhesion clusters adopt specific
to immune system function. That changes within nanoscale
clustering formations in the membrane based on preformed
signaling domains are very small might seem like truism, but
intracellular clusters. Such preformed clusters of integrins and
there is a valid point here. While researchers might use T
associated intermediates/effectors have been observed in vesicles
cells sourced from several different people and observe high
in other cells [37]. If such vesicles are preformed and stored
replicability across many samples, the nanoscale change may be
in pools, they could be used to quickly enact specific kinds
tiny: cluster size changes of a few nanometers for example. Several
of prepackaged migration, by using spatial platforms for fast
functional changes have been linked to nanoscale alterations: the
signaling in a migratory niche. The precise arrangement of
increased clustering of LAT derived from sub-cellular vesicles
molecules within vesicles, and their mechanism of formation is
that correlates with T cell receptor activation and synapse
unknown, but may soon be accessible with the combination of
formation [10], the co-clustering of Zap70 and TCR with LAT
new optical and analytical tools. This hypothesis of nanoscale
[25]as well as Lck [7, 8, 27] upon T cell activation. Recently, links
spatial memory might extend to vesicular adhesion receptors
to mechanism have been posited during the activation of B cells,
and effectors—importantly, integrins can be pre-activated within
where actin and tetraspanin reorganizes B cell receptors [44], in
such vesicles [37].
macrophages, where Src family kinases cause actin to rearrange
The optical tools to analyze such phenomena exist. Lattice
FcγR1 nanoclusters [45], and in the regulation and formation of
light sheet microscopy (LLSM) already allows for high
the natural killer cell lytic synapse [46].
spatiotemporal resolution at low laser power. Whole 3D
Such studies provide important insight into phenomena in
volumes can be acquired very quickly, enabling imaging of single
single cells. However, the low n-numbers lend themselves to
cell dynamics with high temporal resolution. Low phototoxicity
study of the average changes within a heterogeneous dataset
and fast 3D imaging with high signal to noise has allowed the
composed of diverse clusters, and do not allow access to
confirmation and measurement of dynamics of the well-studied
population data when looking at multiple cell types in the same
immunological synapse in a 3D matrix between a T cell and a
experiment as is common in immunology labs. This denies
dendritic cell [38], as well as microvilli [19]. Recent advances
researchers access to possible minority phenomena–nanoscale
in adaptive optics for microscopes has increased the reach of
machinations that start small, residing at the tail ends of data
the lattice light-sheet technique, achieving sub 100 nm lateral
distributions, and result in whole cell change, as well as the effect
and axial resolution even in highly scattering tissues [39]. The
of the change on the rest of the immune cell population in a given
technique has also been used to get single molecule localization
niche.
information, and has provided impressive PAINT images in
fixed cells, where the narrow and homogenous Bessel beam
lightsheet allowed for superior optical sectioning [38]. HIGH THROUGHPUT TRANS-SCALE
While LLSM has the advantage of high speed and low MICROSCOPY
dose, allowing access to fast processes that occur in immune
cells, it has not yet been used for quantification of single While the average super-resolution experiment relies on data
molecules in live cells. Extending this technique to identify from a few tens of cells, the jewel in the crown of immunology,
single molecule involved in nanoclusters in live cells might be flow cytometry, is able to analyze tens of thousands of cells
achievable through its combination with new live cell cluster in a matter of minutes. This allows for imaging and analysis
quantification techniques [40]. Instead of focusing on optics or of a diverse population, often giving a snapshot of immune
fluorescent protein development, the algorithm relies on statistics cell populations in a given bodily niche. If super-resolution
to increase the temporal resolution, by calculating the fewest microscopy is to be used for population based imaging,
points required to reliably state the relative size and density of the number of cells imaged, and their diversity must be
an under-sampled nanocluster. The algorithm works with low increased. To achieve this, researchers have been making efforts
FIGURE 1 | Nanoscale imaging with multiple metrics and high throughput microscopy. Nanoscale readouts, combined with high throughput analysis may link single
cell, multi cell and in vivo behavior to separate T cell subtypes and disease phenotypes. Combining 3D nanoscale live-cell imaging of many targets with other
nanoscale measures such as tension, temperature, conformation will help us to make causal links to single cell behavior. The use of machine learning and automation
to image 1,000 s of cells and billions of molecules will help us to create true trans-scale experiments. Diagram created using biorender.io.
toward increasing the throughput of an average super-resolution of a mixed population of T cells must be imaged all at once,
experiment by automating the process [47, 48], and by using flat identified separately and characterized on the nanoscale by super-
field illumination to increase the number of cells collectable in resolution.
each image [49, 50]. Automated super-resolution imaging and single particle
Flat field super-resolution microscopy attempts to increase tracking has been performed by Masato Yasui of the Ueda lab in
the illumination area to match the capabilities of scientific Osaka [47]. His system uses fast focusing based on pixel intensity
complementary metal oxide semiconductor (sCMOS) cameras, at the iris to select the correct focal plane, then machine learning
which offer a huge field of view [49]. sCMOS cameras used to find and image cells with a given morphology and fluorescent
for localization microscopy also have lower readout noise and signal. Combined with automated addition of chemicals or
rapid readout rates, but are not as sensitive as EMCCD cameras. desired treatments using robotics, the system has the capacity to
The latter have been the equipment of choice for most single image several 100 cells per day. Single particle tracking or super-
molecule localization microscopy applications. However, while resolution localization microscopy can then be carried out in
EM amplifies the signal it also amplifies the camera noise. sCMOS multiple conditions, resulting in hundreds of cells per condition,
cameras do not use electron multiplication but have extremely and millions of analyzable molecules. In conjunction are servers
low camera noise. Together, this means that contrast is higher that can handle the localization of huge numbers of molecules,
and therefore single molecules can be localized more precisely and localization and analysis algorithms that make most efficient
by the latest sCMOS cameras than by EMCCD [38]. Combined use of the CPU or GPU of the systems involved. As an automated
with the faster readout rate and large field of view, as well as process, new ways to validate the quality of super-resolution data
potential for better quantum yields in the future, sCMOS will [51, 52], could be built into the process of screening before image
likely supersede EMCCD cameras for localization microscopy. processing.
Most super-resolution microscopes illuminate the sample
such that there is a relatively narrow Gaussian distribution of MINORITY BIOLOGY
intensity from the center of the field of view, which is made
narrower in some cases by the use of a condenser lens to Increasing the n number from an average of 10 cells to
increase excitation intensity. Fluorophores near the edge of this 1,000 cells may also allow us access to new biological
illumination zone are less strongly excited than those in the phenomena, undetectable when comparing the means of two
center, and as a result are less precisely localized. To address this, conditions. Heterogeneity of clustering in cells is clear—
Suliana Manley’s group set up a flat field microscope, which relies usually the distributions display a wide range of cluster
on an inexpensive microlens array to image 100 µm2 regions shapes, densities and colocalizations with other clustered/non-
with super-resolution, more than quadrupling the current limits clustered molecules. Data comprised of many cells and many
[49]. This could well be applied to immunology where samples thousands more single molecule detections has the potential to
be mined in various ways to reveal minority or rare nanoscale DNA tension sensors represent another way single molecule
phenomena. data could be enriched, and have successfully been used
Minority phenomena represent putative events that have a lot to investigate the force applied by TCR when it binds to
of power for change, but occur at the fringes of distributions. One pMHC [58]. Here, the authors describe a positive relationship
might imagine a molecular cluster exhibiting divergent behavior. between the pulling force of TCR upon peptide-MHC and
Such a cluster might be made of precisely organized layers of full T cell activation. LFA-1 binding to its ligand ICAM-1
kinases, phosphatases and mechanical intermediates [such as augmented TCR/pMHC tension, implying crosstalk between
a focal adhesion [53]] which at a given quorum of molecular the two pathways. Such an interaction represents a catch
participants [molecular conformations and phosphorylation bond—one that becomes stronger as the force across it is
states considered] initiates a signal cascade affecting neighboring increased. Other molecules that undergo catch bond behavior
clusters. This seed cluster could build up a level of signaling that have been investigated by optical traps, such as vinculin
then reorganizes actin [through the local action of Rho-GTPases [59]: an intracellular actin/integrin linker that reveals cryptic
for example [54]] or the strength of an adhesion [through binding sites as it is stretched. Many techniques can now
modulation of vinculin binding proteins [55]] to change the be utilized on a single cell or single molecule level to map
direction of cell migration or to initiate the formation of a full forces in cells [59]. If such techniques can be multiplexed
synapse after initial microvillus based scanning. with super resolution microscopy, this may allow for matching
Such a cluster would certainly be overlooked by conventional tension with the spatio-temporal arrangement of molecules on
comparison of mean values between two separate distributions the nanoscale. Adding layers of information to localization
representing the characteristics of thousands of clusters. The data could provide a route to discover whether specific
kind of N-numbers available by increasing the throughput of structures or cluster types (with regards molecular content,
super-resolution microscopy may allow for statistical analysis of density, size, and location) link to the mechanical tension
segregated minority events at the tail ends of the population they exert on their surroundings. This might also be used
distribution on the nanoscale but also at single cell or cell to find out whether clusters of molecules such as integrins
population level. The transition from “scanning” to “swarming” are homogenous in their mechanical tension within single
mode of neutrophils in the lymph node is a good example of a clusters. By imaging cells live, we may be able to discern
single cell instigating a change in the behavior of the population whether these characteristics play a role in the basis of many
[56]. High throughput, trans-scale type experiments may aid us immune cell functions, such as the formation of microvilli, of
to investigate such phenomena, allowing elucidation of the link migration in response to chemokine, or of immune synapse
between initial nanoscale behavior and cell to population wide formation. Temperature sensors represent another avenue of
functional outcomes. development which may be combined with super-resolution
imaging. Recently, it was reported that mitochondria are
maintained at a temperature of 50 degrees celcius, far above that
FUNCTIONAL SUPER-RESOLUTION of the cytoplasm [60] using a mito-targeted organic probe [61].
IMAGING ERthermAC, a thermosensitive organic dye, has been used to
detect large temperature changes in the endoplasmic reticulum
Nanoscale colocalization of transmembrane proteins with of adipocytes [62]. It should not be discounted therefore that
intracellular signaling intermediates with well-characterized these temperature changes and others might occur in immune
functions can provide some level of insight into functional cells.
output. Such “proxy” measures are likely to lead to important Finally, the imaging of ions at super-resolution would be
discoveries, and with sub 10 nm resolution can complement useful for many areas related to immune cell behavior. Changes
FRET techniques. However, clear links between nanoscale in ion flow through single channels are good candidates for
molecular arrangements and their mechanisms will require minority events that may cause cell wide nanoscale changes that
the combination of super-resolution microscopy with direct translate to function. Calcium ions are highly relevant to T cell
functional readouts, which together might provide clearer activation, delivered through store operated calcium channels
answers about the importance of nanoscale events and how they (SOC) [63] but also T-type channels [64] upon activation of the
relate to cell function, cell population function and immune T cell receptor. Potassium ions are shown to be inhibitory to T
system function (Figure 1). cells, particular in the tumor microenvironment [65]. In addition,
One example of linking function to super resolved coordinate potassium efflux may be involved in chronic maintenance
data is Travis Moore’s work linking LFA-1 integrin conformation of TEM cells, but several different potassium channels have
with its affinity for ligand [57]. By labeling the head group of the compensatory roles, and the dynamics of ion flow at the level
integrin and the inner leaflet of the plasma membrane, Moore of single receptors is unknown [65]. The investigation of ion
and the AIC at Janelia research institute used interferometric flux at super-resolution is therefore an area ripe for study in
PALM to directly detect conformational change in the protein by the context of peripheral T cell migration, interaction with
measuring the distance between the two [57]. The structure of target cells and during a failed response to cancer. Imaging
LFA-1 integrin relates to its affinity, therefore it is important that ions, their respective channels/pumps and intermediates on
the researchers confirmed that the integrin structures predicted the nanoscale has the potential to reveal more about how T
from x ray crystallography matched those in cells. cells respond quickly to different environments, and may be
achievable soon through the development of a raft of ion sensing to immunologists, and the right combinations of techniques
probes [61]. will soon help us link that nanoscale world to holistic, causal
outcomes.
CONCLUSION
AUTHOR CONTRIBUTIONS
Through the combination of cell friendly single molecule
imaging, artifact controls, high throughput microscopy and All authors listed have made a substantial, direct and
multiple metrics on single molecules, trans-scale experiments intellectual contribution to the work, and approved it for
can be undertaken. Such techniques may hold the key to publication.
many immunological phenomena that start on the nanoscale,
representing the natural next step for immune function basic FUNDING
research. They may help us to understand how immune cells
can be used for cancer immunotherapy [66], how the immune This work was supported by ERC Starter Grant #337187. MS was
system co-develops with the body’s bacterial population [67], and supported by the King’s Bioscience Institute and the Guy’s and
how immune cells malfunction and attack our own cells, causing St Thomas’ Charity Prize Ph.D. Programme in Biomedical and
autoimmune disease. The nano-scale is now truly accessible Translational Science.
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66. Spitzer MH, Carmi Y, Reticker-Flynn NE, Kwek SS, Madhireddy D, Martins Copyright © 2019 Shannon and Owen. This is an open-access article distributed
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