Biochimica et Biophysica Acta 1798 (2010) 777–787

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m

Review

A nanometer scale optical view on the compartmentalization of cell membranes

Thomas S. van Zanten a, Alessandra Cambi b, Maria F. Garcia-Parajo a,c,⁎
a
BioNanoPhotonics Group, IBEC-Institute for Bioengineering of Catalonia and CIBER-bbn, Baldiri Reixac 15-21, 08028 Barcelona, Spain
b
Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands
c
ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain

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

Article history: For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of
Received 7 July 2009 conventional light microscopy. Major developments, especially in the past few years, have demonstrated
Received in revised form 13 September 2009 that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic
Accepted 20 September 2009
measurements, a wealth of new information is now emerging regarding the compartmentalization of cell
Available online 2 October 2009
membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of
Keywords:
the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed
Membrane nanodomain technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples
Lipid raft illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies,
Single molecule detection complementing the palette of biophysical techniques available nowadays.
Near-field scanning optical microscopy © 2009 Elsevier B.V. All rights reserved.
Super-resolution optical microscopy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
2. The cell membrane: more mosaic than fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
3. Probing the dynamic character of cell membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
4. Super-resolution optical microscopy beyond the diffraction limit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
4.1. Far-field optical nanoscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
4.2. Super-resolution near field scanning optical microscopy (NSOM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
4.3. Implementation of NSOM for quantitative bioimaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
5. Probing model and cell membrane architectures with near-field optical microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
5.1. Model membranes inspected by NSOM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
5.2. Cell membrane compartmentalization inspected by NSOM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
6. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

1. Introduction contrast with this hypothesis of simple homogeneous lipid mixing, an

ever increasing number of publications appear to confirm that cell
Our quest towards understanding the structure of biological membranes are heterogeneously arranged both in the plane of the
membranes gained considerable speed in 1971, when the fluid bilayer and across the two leaflets. This heterogeneity has been
mosaic model was published by Singer and Nicolson [1]. This model evidenced by the spatial and temporal confinement exhibited by
describes the plasma membrane as a lipid bilayer, forming a two- proteins and lipids in defined micro- and nanometric scale areas of the
dimensional fluid in which the molecules are randomly distributed. In membrane [2,3]. Dynamic events like change in mobility or temporal
association between lipids and proteins within these microdomains
can have direct impact on the biological function of these molecules
and therefore on cellular processes like cell activation, antigen
⁎ Corresponding author. BioNanoPhotonics Group, IBEC-Institute for Bioengineering
of Catalonia, Baldiri Reixac 15-21, 08028 Barcelona, Spain. Tel.: +34 93 4039615;
presentation and cell–cell interactions.
fax: +34 93 4020183. Although much effort has been directed towards studying the
E-mail address: [email protected] (M.F. Garcia-Parajo). dynamic character of cell membranes using biophysical approaches

0005-2736/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamem.2009.09.012
778 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787

such as fluorescence recovery after photobleaching (FRAP), fluores- residing proteins. The involvement of rafts in pathological conditions
cence correlation spectroscopy (FCS) and single particle tracking has further inspired many investigations aimed at unraveling the
(SPT) (all of them extensively reviewed by excellent specialists in the mechanisms behind the formation of lipid and protein domains, the
field) [4–7], much less is known about the real sizes and topological link between outer and inner leaflet rafts as well as the connection
architecture of these domains. The challenge in these studies is given with the cortical actin cytoskeleton.
by the dimensions involved, which are beyond the diffraction limit of An example of the dynamic role played by lipid rafts in the
light and the high packing density exhibited by lipids and proteins reorganization of receptor and signaling molecules is given by the
preventing their individual inspection using single molecule techni- formation of ceramide-enriched membrane domains upon cellular
ques. In this review we will focus on optical methods designed to activation. When stimuli such as CD95, CD40, FcγRII, LFA-1, infection
explore the nanoscale organization of the cell membrane, with a with Rhinovirus or UV light treatment occur, rafts are converted into
special focus on near-field optical microscopy (NSOM) to provide larger membrane platforms by the acid sphingomyelinase, an enzyme
optical super-resolution, not limited by the fluorescence properties of that hydrolyses the raft sphingomyelin into ceramide [20]. Ceramide
the probes neither by the excitation conditions used. However, one molecules spontaneously aggregate into larger platforms that allow
should be aware that recent super-resolution far-field approaches are the clustering of receptors and facilitate signal transduction by the
gaining increasing momentum at present and accordingly they also recruitment of intracellular signaling components. This indicates that
reviewed to some extent in here. Yet, before discussing the optical the plasma membrane lipid raft composition is subjected to
methodology, it is worthy to briefly summarize the importance of cell physiological alterations that subsequently affect cellular signaling
membrane compartmentalization for cellular function and the latest events.
consensus on the field. In virtue of their lipid anchor, glycosylphosphatidylinositol-
anchored proteins (GPI-APs) were among the first molecules to be
2. The cell membrane: more mosaic than fluid identified as “official” raft components [10]. Subsequent work from
Mayor's group demonstrated that GPI-APs are mainly organized in
Drawing an integral picture of the cell membrane that reflects its monomers with a fraction (20–40%) of nanoscale clusters [21].
spatial and dynamic complexity is just unrealistic. Nevertheless, one Interestingly, by using Förster resonance energy transfer (FRET) at
can summarize in a simplified manner, as the scheme shown in Fig. 1, the ensemble level, the same group recently demonstrated that the
the different domains and confined regions that have been identified GPI-AP nanocluster fraction tends to spatially concentrate in larger
so far. One should not be misled by this static picture since in fact large optically resolvable domains and that cortical actin activity affects the
stable domains have not been found in living cells [8]. Indeed, several formation, dynamics, and spatial organization of these nanoclusters
studies suggest that membrane microdomains are small and highly [22]. These studies showed the existence of a steady-state molecular
dynamic, constantly changing in size and composition [9–11]. complexation at the nanoscale that is regulated by the cortical actin,
One of the first structures found in the plasma membrane of implying that a link between outer and inner leaflets must exist.
eukaryotic cells were caveolae. These small (∼60 nm) flask-shaped Membrane compartments are indeed likely to exist both at the
membrane invaginations consist mainly of the caveolin protein, outer and at the inner leaflet of the plasma membrane. However, the
which binds cholesterol. Caveolae have been implicated in a range of mechanistic understanding of whether and how outer and inner
cellular functions, such as cholesterol transport, endocytosis and compartments are linked is still lacking. In signaling T cells, Lasserre
signal transduction [12]. and colleagues demonstrated that raft nanodomains are present at
A second main class of membrane inhomogeneities are lipid rafts. both leaflets and that, although sphingolipids and cholesterol are
Currently, lipid rafts (or membrane rafts) are defined as “small (10– particularly enriched at the outer leaflet, controlled depletion of both
200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid- these components also affected nanodomain formation at the inner
enriched domains that compartmentalize cellular processes. Small leaflet, suggesting again that a certain degree of leaflet coupling must
rafts can sometimes be stabilized to form larger platforms through occur [23]. On the other hand, Wu and coworkers showed large-scale
protein–protein and protein–lipid interactions [13]”. Lipid rafts have uncoupling of inner and outer leaflet rafts [24]. In fact, by allowing
been shown to play an important role in various biological mast cells to adhere onto micrometer-size functionalized patterned
phenomena, ranging from cell adhesion [14], to pathogen binding lipid bilayers, they did observe that FcɛRI clustering induces an actin-
[15], endocytosis [16] and immune cell signaling [17]. Also, the dependent co-redistribution of signaling proteins anchored to the
importance of lipid rafts in the pathogenesis of a variety of conditions, inner leaflet, whereas outer leaflet raft components, previously shown
such as virus infection [18], Alzheimer's and prion diseases as well as to redistribute with IgE-FcɛRI crosslinked with soluble ligands, did not
systemic lupus erythematosus (reviewed in ref. [19]) has been show any detectable colocalization with the surface patterned
elucidated. In particular, in these diseases lipid rafts have been features [24]. Considering the high heterogeneity of membrane
shown to promote altered signaling or allow abnormal folding of nanodomains and their dynamic nature, a possible explanation for
this discrepancy could lay in the different membrane receptors,
different cell types or different time scales analyzed in these studies.
Besides the lipid–lipid interactions that serve to target proteins to
lipid rafts, protein–lipid as well as protein–protein interactions are
also important for localizing some proteins to lipid rafts. This is
certainly the case for transmembrane proteins, whose association
with rafts is still under debate. The transmembrane region may serve
to target the proteins to the lipid domains simply based on the length
of the transmembrane segment itself [25]. Alternatively, raft-targeting
motifs such as palmitoylation can be present in the membrane
proximal cytoplasmic region [26,27]. Since palmitoylation can be a
transient event, targeting of proteins to rafts can be subject to tight
temporal regulation. This phenomenon is well known for the B cell
receptor signaling. Palmitoylation of the associated tetraspanin CD81
Fig. 1. Schematic and static representation of the various types of microdomains is required to recruit the B cell receptor to rafts, thus prolonging its
present in the cell membrane. signaling in B cells [28]. It should be noted that recent studies have
 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787 779

also demonstrated the formation of plasma membrane microdomains revealed that association with lipid rafts is not the dominant factor
in signaling T cells exclusively created by protein–protein networks governing lateral mobility, indicated by the absence of correlation
and not maintained by interactions with lipid rafts [29]. The between the diffusion coefficients and characterization as either raft
diffusional trapping through protein–protein interactions generates or non-raft marker. In SPT mobility has been investigated by tracking
microdomains that can recruit or reject specific cell surface proteins the movement of probes specifically bound to membrane components
during signal transduction. [40]. As a result of STP experiments, membrane compartmentalization
A third class of domains is characterized by the presence of has been widely recognized through the observation of TCZs, regions
tetraspanins, a family of integral proteins mainly localized at the in the membrane where a protein or lipid is confined much longer
plasma membrane and able to interact with one another and with than would be expected by simple Brownian motion [37]. TCZs as
numerous other transmembrane proteins, thereby assembling a described in the literature are typically 100–300 nm in size and have
network of molecular interactions also called the tetraspanin web lifetimes of hundreds of millisecond to seconds, depending on the
[30]. Based on a single-molecule analysis of the tetraspanin CD9, the experimental sampling rate [34,36,37]. A comprehensive review of
current view about these membrane domains is that small clusters of the literature on the dynamic aspects of cell membranes micro- and
tetraspanins, each specifically linked to an interacting molecule, nanodomains is beyond the scope of this paper, and the reader is
would move within the plasma membrane, often interacting with referred to excellent reviews in the field [4–8,37,40].
other domains, either tetraspanin-enriched or lipid rafts, and possibly
exchanging some of their components [31]. The association of 4. Super-resolution optical microscopy beyond the diffraction limit
tetraspanins with integrins is well documented [32,33], although
there is still little mechanistic insight into how tetraspanins facilitate Aside from the dynamic nature of membrane domains, revealing
integrin-mediated adhesion. their true size and composition requires high-resolution microscopy
The fourth type of organization discovered in membranes is techniques. In principle, Förster resonance energy transfer (FRET) is
so-called transient confinement zones (TCZ), which are supposedly capable of detecting proximity below the optical resolution (∼ λ/2,
formed by a membrane-associated actin mesh network [34]. Indeed, where λ is the wavelength of light) since the efficiency of the process
increasing evidence is clearly pointing towards an active role of depends directly on donor–acceptor distances, typically 1–10 nm [41].
cortical actin in the formation and dynamics of membrane nanodo- The basis for data interpretation is that clustering stabilizes
mains [35]. Interestingly, the diffusion rate of lipids in the plasma interparticle distances. FRET efficiencies that are independent of the
membrane is 5–100 times slower than in artificial bilayers, suggesting fluorophore densities have been interpreted as indicative for the
that long-range interactions between lipids and proteins or lipid and existence of clusters [10,41,42]. In addition, FRET experiments do not
the extracellular matrix may be responsible for this reduction. provide information on distances beyond 10 nm, and thus not able on
Kusumi's group has observed the movement of phospholipids at the its own to reveal the true size of domains. It should be noticed that in
single-molecule level with a temporal resolution of 25 μs and more recent years, time resolved FRET as combined with fluorescence
demonstrated that phospholipids undergo hop diffusion in compart- lifetime imaging (FLIM) and appropriate theoretical modeling is
mentalized plasma membranes, proposing the intriguing concept that providing more depth-inside in the size of nanodomains in both
transmembrane proteins anchored to the actin cytoskeleton mesh- model systems as well as in living cells, as recently reviewed by Loura
work would act as “rows of pickets” to temporarily confine diffusing et al. [43].
phospholipids [36]. This has shifted the original paradigm of the The increased spatial resolution of transmission electron micros-
plasma membrane as two-dimensional continuum fluid to the new copy (TEM) (0.1–10 nm) has been used extensively to visualize a wide
“partitioned fluid,” where proteins and lipids diffuse within TCZs [37]. variety of protein domains and lipid rafts on the cell membrane.
Lenne and colleagues have further substantiated these findings, Antibody gold particles of different sizes can be targeted to membrane
demonstrating that the cortical actin meshwork and the lipid-based components revealing their surface distribution [14,15,44–47]. For
domains are the two main compartmentalizing forces acting in the instance, nanoclustering of the pathogen recognition receptor DC-
plasma membrane [38]. More recently, the exact relationship SIGN on immature dendritic cells [15] and nanodomains of the
between protein dynamics and actin-defined compartments has leukocyte specific integrin LFA-1 at the cell surface of human
been directly visualized [39]. In this elegant paper, the authors not monocytes [14] have been revealed by TEM. Within the raft field,
only showed that the FcɛRI diffusion is confined within actin-poor probably the most intriguing observation has been the absence of co-
areas but also demonstrated that the size and location of these actin clustering between two putative raft markers (a GPI-anchored protein
barriers changed over time, indicating that the type of diffusion and GM1 lipids) [44]. Unfortunately, since TEM requires extensive
barriers formed by the cytoskeleton is time-dependent [39]. sample preparation it cannot be extended towards live cell imaging.
In summary, despite the increasing consensus regarding the fact High-resolution fluorescence microscopy is compatible with live
that biological membranes are compartmentalized both at the lipid cell imaging, provides excellent spectral contrast and in combination
and protein level, we still face major challenges in the investigation of with sensitive detectors allows the detection of individual molecules.
the plasma membrane structure. Given the dynamic nature and the Until only a few years ago, near-field scanning optical microscopy has
nanoscale dimensions of these membrane compartments, techniques been the only optical technique able to provide resolution beyond the
that monitor protein and lipid dynamics at high temporal and spatial diffraction limit of light. However, recently developed far-field
resolution are needed. Unfortunately, so far, no single technique can methods have also demonstrated optical resolution in the nanometer
combine high spatial resolution (to directly image nanoscale range, not only laterally but also in 3D. Each of the methods is briefly
domains) and fast image acquisition (to probe fast dynamics) in one discussed below in terms of their advantages but also limitations.
instrument. In here we will focus on techniques that provide optical Fig. 2 shows the principles of the four different methods developed so
resolution at the nanoscale and briefly mention the most relevant far, with a separation between far-field and near-field approaches.
findings obtained until now using high temporal resolution methods.
4.1. Far-field optical nanoscopy
3. Probing the dynamic character of cell membranes
Stimulated emission depletion (STED) microscopy was conceptu-
The dynamic nature of membrane domains can be investigated via ally introduced more than a decade ago by Hell and colleagues and
the lateral mobility of fluorescent probes in the cell membrane using successfully implemented recently [48–54]. STED creates a nano-
FRAP [6,9] or more locally via FCS [7]. In general, these studies metric optical region by first exciting fluorophores to an excited state
780 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787

Fig. 2. Different schemes for super-resolution imaging microscopy. (A) Stimulated emission depletion (STED) microscopy, as introduced by Hell's group (adapted from ref. [49]).
(B) Structured illumination concept as introduced by Gustafsson. Top: Circular observable region of radius k0 in frequency space observed by a conventional microscope. Bottom:
New set of information available in the form of moiré fringes (hatched circle) provided that the excitation light contains a spatial frequency k1. The new region has the same shape as
the normal observable region but it is centered at k1. The maximum spatial frequency that can be detected in this direction is k0 + k1 (adapted from ref. [54]). (C) Principle of PALM/
FPALM and STORM. PALM/FPALM are based on photoactivable autofluorescent proteins while STORM rely on on-off photo switchable organic fluorophores. The techniques use the
stochastic photoactivation of single molecules (set to the dark state at the beginning of the experiment as shown in the top panel) and their subsequent nanometric localization over
thousands of widefield image frames (series of small panels) to construct a super-resolution image (bottom panel). (D) NSOM uses a subwavelength aperture (∼50–100 nm) probe
to locally excite the sample surface and to generate point-by-point a super-resolution image related to the size of the probe. Only fluorophores at the cell surface are effectively
excited (red dots close to the near-field region) reducing the contribution of background fluorescence from other regions of the cell (dark dots in the interior of the cell).

over a diffraction-limited region using a pulsed laser. A second pulsed The two methods described above allow truly optical resolution at
laser illuminates the sample with a doughnut-shape like pattern in a the nanometer scale and can be readily extended to 3D imaging. The
wavelength that depletes the excited state of the fluorescent resolution in fluorescence microscopy can be increased even further
molecules back to the ground state. Fluorescence is effectively by allowing only a subset of fluorescent molecules to be photoactive at
detected only from the hole of the doughnut (Fig. 2A). The final a given time and ensuring that the nearest-neighbor distance between
spot size can be tuned to balance resolution against signal and active molecules is larger than the diffraction limit. Methods that
imaging speed by controlling the power of the depleting laser, and make use of this principle are photoactivatable localization micros-
indeed images with a resolution of ∼30 nm have been reported using copy (PALM/FPALM) [59,60] and stochastic optical reconstruction
this technique. However, because of its mere principle, STED requires microscopy (STORM) [61]. The basic premise of both techniques is to
precise control of the position, phase and amplitude of two laser fill the imaging area with many dark fluorophores that can be
beams (for single color fluorescence), and its best resolution is photoactivated into a fluorescing state by a flash of light. Because
restricted to certain dyes able to withstand repeated cycles of photoactivation is stochastic, only a few, well separated molecules
excitation and depletion at extremely high intensities. So far, the “turn on.” Then Gaussians are fit to their point spread functions (PSF)
technique has been mainly applied in neurobiology by the Hell's to high precision. After the few bright dots photobleach, another flash
group, delivering important information on the study of syntaxin of the photoactivating light activates random fluorophores again and
clusters and acetylcholine receptors on fixed cultured neurons, as well the PSFs are fit of these different well-spaced objects. This process is
as to the dynamics of synaptic vesicles over small fields of view at high repeated many times, building up an image molecule-by-molecule;
speeds [50-53]. More recently, STED has been also combined with FCS and because the molecules were localized at different times, the
to observe nanoscale dynamics of membrane lipids and GPI-anchored “resolution” of the final image can be much higher than that limited
proteins in living cells [54]. by diffraction (Fig. 2C). The main difference between PALM and
Saturated structured illumination microscopy (SSIM) is concep- STORM resides on the type of fluorophores used for photoactivation:
tually the opposite of STED (Fig. 2B). By using a structured light PALM relies on autofluorescent proteins, while STORM uses organic
illumination from two high-intensity power interference beams, most switchable dyes (from the cyanine family). The ascertainable
of the fluorescence molecules in the illuminating beams saturate, localization accuracy depends strongly on the total number of
leaving only small regions unsaturated at the shadows of the photons being detected. Especially PALM/FPALM can quantitatively
interference pattern: the higher the intensity, the smaller the regions map relative molecular densities with very high localization accuracy
[55–58]. The method can be implemented in a widefield (non- over wide fields and in living cells. As already mentioned, these forms
scanning) microscope and is capable of high frame rates over wide of nanoscale image reconstruction methodologies rely on photo-
fields of view. The practical resolving power is determined by the switchable fluorophores, and therefore imaging conditions are
signal-to-noise ratio, which is in turn limited by fluorescence consistent with single molecule detection and require so far long
photobleaching. In its linear form, SIM can work at lower intensities acquisition times. PALM/FPALM has been used to reconstruct images
reducing photobleaching but can provide only a two-fold resolution of various proteins in thin cellular sections and near the surfaces of
increase beyond the diffraction limit. The technique has been applied whole, fixed cells [59] to study the organization of different proteins
to study the organization of specific proteins at the neuromuscular within adhesion complexes [62] and to track large populations of
junction in Drosophila [58] and to elucidate the 3D structure of the single proteins molecules in the plasma membrane of living cells
nuclear periphery [56]. [63–65]. In particular Hess and colleagues exploited the technique to
 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787 781

follow the dynamic distribution of hemagglutinin proteins and surface induces changes in the far-field radiation, which is collected in
discriminate between different raft hypotheses [64]. They observed the far field by conventional optics and directed to highly sensitive
elongated clusters and irregular domain boundaries suggesting that detectors to provide an optical image [73–76]. An independent
line tension (and thus lipid fluid phase behavior) plays a limited role mechanism is used to control the distance separation between the tip
in domain shape and proposed that interactions between the and the sample and to simultaneously generate a topographic image
cytoskeleton and membrane proteins could produce such irregular [77,78]. In this way, a singular feature pertaining to NSOM is
cluster size distribution in accordance to previous work from Kusumi produced: correlative optical and topographical imaging with a
and Vale's groups [36,37,29]. spatial resolution determined by the probe configuration. Another
With the rapid and widespread implementation of different forms unique characteristic of near-field excitation is given by the finite size
of photoswitchable super-resolution optical microscopy, including of the probe itself: decreasing the area of illumination obviously
multicolor [62,66] and 3D capabilities [67–70], one word of caution reduces the interaction volume and background scatter, which is of
should be drawn to the biological community. In fact, several major importance in enhancing the sensitivity for spectroscopic
researchers in the field have already highlighted some of the caveats applications (fluorescence, Raman, etc.).
inherent to the approach [71,72]. They include cellular autofluores- Instead of using the probe to illuminate the sample, one can
cence or fluorescence from unactivated fluorophores that obscure the employ far field optics to illuminate the sample and use the probe to
signal from individual molecules, making sparsely labeled structures collect the evanescent field in close proximity to the sample surface.
difficult to image, and photodamage induced by the ultraviolet laser Although perfectly suitable for some photonic applications [79], its
used for photoswitching in PALM/FPALM that can limit its application use in fluorescence imaging is less appropriate since far field
in live cell imaging. Most importantly, there are also fluorescence illumination translates in unnecessary sample photobleaching. A
related problems such as over-expression, artifactual aggregation, different experimental strategy to NSOM is based on the use of
mistargeting or probe specificity that can complicate the interpreta- metallic tips, known in the literature as apertureless NSOM [80] when
tion of the images. Indeed, as stated by a recent review in the field: the tip is used as passive scatterer, or tip-enhanced NSOM when the
“One of the frustrations of super-resolution microscopy is that it is metallic tip is excited to enhance the electromagnetic field at the end
easy to get images, yet extremely difficult to get biologically of the tip apex [81]. In both cases, the sample is illuminated in the far-
meaningful ones. As the novelty of super-resolution microscopy field and a metal probe is placed in the tight focus of the illumination
wears off, and the focus shifts to its biological application, it will beam. The local interaction with the sample surface is subsequently
become increasingly important to adopt careful controls such as detected as a modulation in the scattered far field. Extreme sensitivity
correlative and/or simultaneous diffraction-limited imaging to insure is required to observe the weakly scattered light from the nanometer-
that the results are physiologically relevant [72]”. sized tip in the presence of the light scattered by the sample. When
combined with fluorescence, and the tip is properly excited with
4.2. Super-resolution near field scanning optical microscopy (NSOM) radial fields along the tip axis, optical resolutions in the order to 30 nm
can be achieved [82–84]. This method is however accompanied by a
In far-field optical microscopy, the diffraction limit implies that the large fluorescence background generated from far field illumination of
minimum distance Δx required to resolve independently two distinct the sample, requiring therefore modulation techniques to recover the
objects is dependent on the wavelength λ of the light used to observe high-resolution signal [85]. On the positive side of the balance, this
the specimen, and by the condenser and objective lens system, method is free from the associated practical difficulties of fabricating
through their refractive indices n and angle of acceptance α, such that circular apertures.
Δx = l/2n sinα. This implies that Δx exceeds 300 nm in the case of
visible light. When an object, such as microscopic specimen, is 4.3. Implementation of NSOM for quantitative bioimaging
illuminated with a monochromatic plane wave, the transmitted or
reflected light is collected by a lens and projected onto a detector to For biological applications, the most widely used configuration is
form the image. Usually, for convenience and practicality, the detector an aperture-type NSOM, incorporated into an inverted optical
is placed in the far-field, so that the far-field component of the light, microscope, with near-field excitation and far-field detection (see
which propagates in an unconfined way, is the only component used Fig. 3). This scheme preserves most of the conventional imaging
to generate the image. On the other hand, the interaction between the modes (confocal microscopy for instance), which remain available in
imaging light and the specimen also generates a near-field compo- combination with the near-field approach. Light that is emitted by the
nent, which consists of a non-propagating (evanescent) field existing aperture locally interacts with the sample. It may be absorbed, phase
only near the object at distances less than the wavelength of the light. shifted, or used to locally excite fluorescent markers, depending on
Because the near-field decays exponentially within a distance less the sample and the contrast mechanisms employed. In any case, light
than the wavelength, usually it cannot be collected by the lens, thus, it emerging from the imaging zone must be collected with the highest
is not detected. This effect leads to the well-known Abbé's diffraction possible efficiency. For this purpose, high NA (oil immersion)
limit. By detecting the near-field component before it undergoes microscope objectives are usually employed. The collected light is
diffraction, NSOM allows non-diffraction limited high-resolution directed to sensitive detectors, such as avalanche photo-diodes (APD)
optical imaging. This is achieved in NSOM by placing a probe tip in or photo-multiplier tubes (PMT), via suitable dichroic mirrors for
close proximity to the sample in order to illuminate and/or detect in spectral splitting or through a polarizing beam splitter cube for
the near-field. Thus, in NSOM microscopes, the resolution Δx no longer polarization detection. Filters are also commonly used to select the
depends on λ but instead on the aperture diameter of the probe spectral regions of interest removing unwanted spectral components,
(typically between 50 and 100 nm). In contrast to the previously and inverted optical microscopes are an advantageous solution for
described super-resolution techniques that are restricted to fluores- light collection, redistribution, and filtering.
cence and rely on the photophysical properties of the probes used, In Fig. 3, the excitation light from one or more laser sources is
NSOM can exploit many other optical contrast mechanisms (i.e., coupled into the optical fiber. The tip is maintained in the near-field of
absorption, polarization and spectroscopy) in addition to fluorescence. the specimen by the feedback system operating in close loop that
In its most commonly implemented mode, a subwavelength precisely controls the separation between the probe and the sample.
aperture probe is scanned in close proximity (b10 nm) to the In addition, a 3D scanner is employed to control the relative
specimen under study (Fig. 2D) to generate an image. Using the probe positioning of sample and probe. Depending upon design and
as a near-field excitation source, the interaction with the sample applications, in principle the scanner may either move the specimen
782 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787

Fig. 3. Schematics of our combined confocal/NSOM set-up. Two laser lines can be simultaneously coupled into the microscope using the confocal or NSOM excitation configuration
modes. Easy switching from one mode of excitation or the other is achieved by a flipable mirror (M2 in the scheme). Fluorescence light is collected using a high NA objective and
selected using appropriate filters. The fluorescence signal is then separated according to polarization (using a polarizing beam splitter) or spectral (using a dichroic mirror)
properties (S2 in the scheme) and sent to two sensitive detectors (APDs). The inset shows a 70-nm diameter NSOM probe. The aperture primarily determines the optical resolution of
the microscope.

or the probe. In the case of the scanner locked to the specimen, which is tip. Current developments using optical nanoantennas to concentrate
the most employed configuration for biological imaging, the sample is and enhance the electric field at the antenna end hold great promise
moved in a raster pattern. The image is generated from the signal for the use of these engineered probes in bioimaging [91].
arising from the tip–specimen interaction under the probe, which is
fixed and aligned confocally to the objective. The size of the area imaged 5. Probing model and cell membrane architectures with near-field
depends uniquely on the coarse of the scanner. During raster scanning, optical microscopy
data obtained both from the feedback system and from the optical
detectors are simultaneously stored by a computer point by point. 5.1. Model membranes inspected by NSOM
Finally, the PC compiles and renders the acquired data and furnishes
simultaneously topography and optical image of the specimen. Model membranes have been used for a long time to investigate
One of the major obstacles that have restricted the use of NSOM in the segregation behavior of lipids and different proteins in prede-
cell biology has been related to its difficulty to operate in liquid termined lipid mixtures, while reducing the complexity of the cell
conditions, a crucial step towards live cell imaging. Successful control membrane. The typical binary or ternary lipid mixtures used to mimic
of the tip–sample distance has been routinely achieved in air by using the lipid composition of cell membranes indeed phase-segregate into
tuning forks as sensing elements and driven at resonance [77,78]. liquid condensed (LC) and liquid expanded (LE) phases. By transfer-
However, this approach systematically failed once the tuning fork was ring monolayers of a lipid mixture on a substrate using standard
immersed in a liquid. Koopman et al have demonstrated that, in Langmuir-Blodgett techniques, Hwang et al. used NSOM to reveal
aqueous environments, sensitivity of the surface topography can be previously unresolved features of around 50 nm [92,93]. When a
regained by keeping the tuning fork dry in a “diving bell” enclosure higher pressure was used to form the monolayer, the domains of LC
just above the probe [86,87]. Alternatively Höppener and colleagues phase appeared to decrease in size and an increasingly complex fine
used the tuning fork with the tip placed perpendicular to the prongs of web structure of the LE phase emerged [92,93]. Cholesterol addition,
the fork and protruding about ∼ 2 mm below the fork. The typically enriching the LC phase, resulted in the formation of
configuration works thus as “tapping-mode” with the tip immerse elongated thin LC domains. From these morphology changes it was
in solution and the tuning fork kept dry above the liquid [88]. An concluded that cholesterol reduced the line tension between the
alternative method for position control is based on ion conductance. domains in regions of LC/LE coexistence. Likewise, the addition of the
The method relies on the use of sharp micropipettes. As the probe ganglioside GM1, again a LC constituent, affected the monolayer
approaches the sample, ion conduction is partially blocked and the morphology significantly. Moreover, GM1 induced a more pro-
change in conductivity is used as a measure of the tip–sample distance nounced segregation between the LC and LE phases. These results
[89]. This mechanism has been coupled to NSOM to obtain images in suggested the formation of genuine distinct domains, thus favoring
living cells [89]. the occurrence of a lipid raft type of phenomenon on model
Although NSOM provides nanometric optical resolution together membranes. The lipids typically enriching the LC phase are signifi-
with simultaneous topographic information using a multitude of cantly more saturated than lipids constituting the LE phase. Thus,
different optical contrast mechanisms, one should also be aware of the when all lipids pack in their subsequent phase, the LE phase will be
current limitations of the technique. For instance, NSOM is prone to lower in height. Indeed, by specifically labeling the LE phase a perfect
artifacts generated from the topographic signal used to control the correlation was found between topographical and fluorescence
separation between the tip and sample. Therefore relatively flat signals [94]. To extend these findings, Hollars and Dunn used
samples (with height differences below 1 μm) are preferred for tapping-mode feedback NSOM to additionally obtain compliance
imaging. As scanning technique, NSOM is inherently slow, and thus information of the lipid monolayer [95]. Because the carbohydrate
not suitable for recording fast dynamic processes in living cells. On the chains of the lipids from the LC phase are highly saturated they pack in
other hand, recent developments on NSOM combined with FCS might an ordered fashion as compared to the lipids from the LE phase. As
provide truly dynamic information at the nanometer scale [90]. expected, the LC phase was found less compliant than the LE phase
Finally, aperture probes have low throughput efficiencies (typically [95]. As such Hollars and Dunn demonstrated the strength of NSOM as
10- 4–10- 6), limiting the number of photons that can be forced out the compared to fluorescence or scanning probe techniques on their own.
 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787 783

In a more recent work supporting the formation of lipid rafts on

model membranes, small amounts of labeled GM1 revealed that GM1
is not homogeneously distributed throughout the LC phase. Instead,
they were seen to constitute their own 100–200 nm sized domains
[96]. In fact, upon closer examination the labeled GM1 distribution
appeared to be more complex. To better characterize the GM1
behavior, GM1 lipids were labeled with Bodipy. This fluorophore
displays a redshift in the emission spectra when present in higher
concentrations due to excimer formation, thus being able to probe the
local lipid density [97]. Due to the strong tendency of GM1 to partition
in gel or liquid-ordered phases high concentration GM1 was found in
the LC phase, showing the redshifted emission, even while using low
deposition pressures [98]. Nevertheless, a rather large fraction of
single Bodipy-GM1 was still found randomly distributed in the LE
phase. Upon increasing the deposition pressure towards expected cell
membrane pressures, the LC domain phases became smaller and the
labeled GM1 appeared to preferentially partition into the LC phase
[98].
The use of NSOM to investigate monolayers has been extended
towards bilayers [99] and protein containing lipid layers [88,100–102].
The addition of proteins to such lipid phase-segregated model
systems will be an important step in understanding how lipid based
interaction can influence protein distribution. Subsequently, moni-
toring the dynamics would then provide a more complete spatio-
temporal map of proteins and lipids in a lipid bilayer. Work in this
direction has been performed using atomic force microscopy in
combination with FCS [103]. The recent proof-of-principle indication
that dynamical studies can be also performed with NSOM [90] opens
up an exciting field that combines high-resolution imaging with
ultrafast dynamics. Indeed, the advantage of performing FCS on
confined volumes has been recently demonstrated on living cells
[54,104]. The incorporation of this approach in NSOM would provide
in addition to surface sensitivity, topography and resolution, also
temporal information.

5.2. Cell membrane compartmentalization inspected by NSOM Fig. 4. Super-resolution image of a dendritic cell expressing the pathogen recognition
receptor DC-SIGN. (A) Simultaneously obtained topography (gray) and NSOM
fluorescence (color spots) on a small region of the dendritic cell surface. The color-
Within cell membrane quantitative imaging, NSOM has been coding reflects the emission dipole moment of individual molecules on the cell
mainly used to investigate the degree of clustering of different membrane, with red and green spots corresponding to different molecular in-plane
receptors on the cell membrane. In some cases, the association of orientations. Most of the spots are yellow and have different physical sizes, a clear
multiple components has been also investigated using dual color signature for clustering of DC-SIGN. (B) Intensity brightness distribution over 1200
different spots from multiple NSOM images. The long tail of the distribution reflects the
NSOM. In the context of receptor clustering our group has used NSOM
diverse clustering exhibited by DC-SIGN. The inset shows the expanded intensity axis
to image pathogen recognition receptors with high spatial resolution and superposed to it, the brightness distribution of individual Cy5 molecules (red bars).
on cells of the immune system, providing insight into the mechanisms
exploited by the cell to ensure high performance of these receptors
[87,105–107] (Fig. 4). By labeling the pathogen recognition receptor significant higher levels of cytokines [108]. By means of these high-
DC-SIGN with a specific monoclonal antibody, we found that as much resolution NSOM experiments it was shown that the TCR reorgani-
as 80% of DC-SIGN is clustered on the cell membrane of immature zation plays a significant role in antigen recognition and cytokine
dendritic cells [107]. These domains were randomly distributed over production.
the plasma membrane with a size distribution centered ∼185 nm. In the case of members of the epidermal growth factor (EGF)
Interestingly, we discovered a remarkable heterogeneity of the DC- receptor tyrosine kinase family, clustering is thought to have a
SIGN packing density within the clusters. This suggests that the large negative effect. Some EGFs, like the erbB2 receptor, are found to be
spread in DC-SIGN density per cluster likely serves to maximize the over-expressed in breast cancerous cells. It is thought that this over-
chances of DC-SIGN binding to a large variety of viruses and expression leads to cluster formation causing the highly oncogenic
pathogens having different binding affinities [107]. Indeed, the activation of very potent kinase activity. Indeed, by applying NSOM
organization of DC-SIGN in nanodomains appeared crucial for efficient the clustering behavior of EGF receptors was found to be associated
binding and internalization of pathogens [15]. with the activation state of the cell [109]. Additionally, it was found
Recently, Chen et al. used NSOM in combination with quantum that EGF cluster sizes increased if the quiescent cells were treated
dots to label the T cell receptor (TCR) of T cells in live animals before with EGF activators to the same extend as cells over-expressing these
and after cell stimulation [108]. In the resting state, the TCR EGFs [109]. Since activation of the EGF signaling pathways requires
complexes were found monomerically organized on the T cell extensive interaction between individual members of the EGF family,
membrane. Upon T cell stimulation, the TCR complexes reorganized it is likely that concentrating one of these EGF receptors in clusters
and formed 270–390 nm sized domains. Interestingly, these small- increases the likelihood of co-clustering of other EGF members. This
sized domains were not only formed but also sustained for days. co-clustering would then subsequently increase the EGF signaling
Additional experiments showed that although unstimulated cells efficiency. In other words, a higher local concentration will decrease
could produce an immune response, stimulated cells produced the lag time for direct inter-receptor contact.
784 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787

Cell signaling events commonly involve a multitude of spatially As such, NSOM is capable of bridging the gap between 10 to 300 nm
segregated proteins and lipids. As such, standard confocal microscopy providing valuable information at these important spatial scales.
studies in biology usually involve multiple colors corresponding to Fig. 5 shows as example the powerfulness of dual color excitation
multiple specifically labeled proteins. However, inherent to all lens- and detection NSOM when imaging two different components of the
based techniques are chromatic aberrations that cause multiple cell membrane. The high packing density of both components on the
wavelengths to never perfectly overlap. In contrast, NSOM guarantees cell surface combined with the limited resolution of confocal
a perfect overlay between multiple excitation wavelengths, an microscopy suggests colocalization between GM1 nanoclusters and
essential requirement to resolve the true nanoscale landscape of cell the receptor DC71. However, high-resolution NSOM shows clearly
membranes. Already in 1997, Enderle et al. used for the first time that these receptors do not compartmentalize in the same regions of
dual-color NSOM to directly measure the association of a host protein the cell membrane.
(protein4.1) and parasite proteins (MESA and PfHRP1) in malaria More recently, NSOM has been also used to spatially relate
(Plasmodium falciparum) infected erythrocytes [110]. As the parasitic topographical features to two different lipid species [113]. Both
proteins interact with the host proteins, 100 nm sized knob-like GM1 and GM3 were seen to cluster in 40–360 nm domains that
topographical features appear on the membrane of the host cell. To distributed randomly on the plasma membrane of epithelial cells.
investigate the direct interaction of host and parasite proteins, the However, upon closer examination it appeared that the GM3 clusters
proteins were specifically labeled and subsequently imaged with were localized on the peaks of microvillus-like structures [113]. In
NSOM. As expected, the fluorescence from the two labeled parasitic contrast, the majority of the GM1 lipid clusters were found in the
proteins and the labeled host protein were found on the knob like valley or slopes of these topographical protrusions [113]. These results
structures. However, this did not necessarily involve colocalization of highlight the importance of correlating topography and optical
host and parasite proteins [110]. information uniquely afforded by NSOM. Along these lines, it is
The increased co-localization of individual components on the cell worthy to mention that several groups have also implemented AFM in
membrane has been actually demonstrated on two members of the combination with confocal microscopy in order to correlate topogra-
interleukin family by combining dual-color excitation and single phy with fluorescence information, albeit at lower optical resolution
molecule detection NSOM [111]. IL2R and IL15R did not interact if (diffraction-limited). On the other hand, a combination of AFM and
their organization was monomeric. However, in their clustered form, confocal FCS can also provide complementary information on the
both receptors were found to co-localize significantly suggesting that dynamics of different nanoenviroments on membranes and correlate
clustering of both receptors takes place in the same nanocompart- it with topographic information as afforded by AFM [103].
ments [111]. Interestingly, IL2R and IL15R clusters were found to have
a constant packing density albeit forming domains of different sizes 6. Summary and outlook
[111]. Although the receptors were found to pack at different
densities, the linear increase in number of receptors with domain In summary, the past few years have witnessed tremendous
size suggested a general building block type of assembly for these technical advances in super-resolution optical microscopy using both
receptors [111]. far and near-field methods. This has in turn further increased our
Ianoul et al. have also used dual-color NSOM to investigate the understanding on the compartmentalization of the cell membrane
association of β-adrenergic receptors (βAR) and caveolae of the and its implications in cellular function and diseases. However, a
surface of cardiac myocytes [112]. The study showed that ∼15–20% significant number of questions are still open and awaiting for
β2ARs colocalize in caveolae. The lack of complete colocalization of techniques that combine high spatial and temporal resolution in one
β2AR with the caveolae suggested that the diverse functional and the same instrument. Far-field super-resolution methods have
properties of the β2AR could arise from its association with multi- already demonstrated the possibility of following the dynamics of
protein complexes of different compositions that may not be caveolar slowly moving receptors on the cell membrane on small fields of
in nature. Interestingly, the fraction of β2ARs not colocalizing with views [53,63–65] or in combination with a FCS approach [54]. Further
caveolae appeared proximal to it, indicating β2AR complexes are pre- developments of probes and instrumentation will certainly lead to
assembled in, or near caveolae. More conventionally used techniques improvement of these techniques.
such as FRET are unable to report on such a proximity effect at spatial Within the context of near-field super-resolution, first demon-
scales N10 nm. On the other extreme, diffraction limited techniques strations of NSOM measurements on living cells have been reported
such as confocal microscopy will not be able to reveal a lack of co- [114–116] although high-resolution dynamics on the membrane of
localization if multiple components are located at distances b300 nm. living cells is yet to be demonstrated. Obviously, if the scanning speed

Fig. 5. Dual color super-resolution NSOM in aqueous conditions of different membrane components. (A) Representative confocal fluorescence image taken at the focal plane of the
glass surface of a monocytic cell showing the merging of the lipid GM1 (labeled with CTxB, red) and the receptor CD71 (labeled with specific antibodies, green) (40 × 40 μm2). An
area of interest, as indicated by the white box is further inspected by confocal microscopy in (B) (10 × 10 μm2). Multiple yellow patches in the confocal image suggest colocalization
between GM1 and CD71. (C) NSOM image of the highlighted area in B, obtained after excitation using a probe of ∼100 nm in diameter (5 × 5 μm2). The apparent colocalization
observed in confocal disappears upon high-resolution inspection afforded by NSOM (physically separated red and green spots) indicating that CD71 does not colocalize with GM1,
entirely consistent with its assignment as non-raft marker.
 T.S. van Zanten et al. / Biochimica et Biophysica Acta 1798 (2010) 777–787 785

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