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Illuminated up close: near-field optical microscopy of cell surfaces
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 119 – 125
Perspective
nanomedjournal.com
Illuminated up close: near-field optical microscopy of cell surfaces Daniel M. Czajkowsky, PhD a , Jielin Sun, PhD b , Zhifeng Shao, PhD a,⁎ a
Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China b Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China Received 16 June 2014; accepted 10 August 2014
Abstract Invented in the 1990s, near-field optical microscopy (NSOM) was the first optical microscopy method to hold the promise of finally breaking the diffraction barrier in studies of biological samples. This promise, though, failed to materialize at that time, largely owing to the inability to image soft samples, such as cell surfaces, without damage. However, steady technical improvements have now produced NSOM devices that can routinely achieve images of cell surfaces with sub-100 nm resolution in aqueous solution. Further, beyond just optical information, these instruments can also provide simultaneous topographic, mechanical, and/or chemical details of the sample, an ability not yet matched by any other optics-based methodology. With the long recognized important roles of many biological processes at cell surfaces in human health and disease, near-field probing of cell surfaces is indeed now well poised to directly illume in biomedicine what has, until recently, been unknowable with classic light microscopy. From the Clinical Editor: This paper presents a novel and important near-field microscopy-based method directly enabling the imaging of cell surfaces with sub-100nm resolution. Unlike other optics-based methods, the presented technique can also provide topographic, mechanical and chemical details of the samples. © 2015 Elsevier Inc. All rights reserved. Key words: Super resolution; Cell membrane; Nanodomains
Since its invention nearly four hundred years ago, 1 optical microscopy has played an irreplaceable role in unravelling the structural underpinnings of biological systems, from the initial discovery of individual cells by van Leeuwenhoek to the organization of cells in complex tissues to the delineation of chromosomal territories within individual nuclei. 1-3 However, up until recently, what has been possible to resolve with conventional light microscopy has remained stubbornly limited by diffraction to about 250 nm laterally and several times worse axially. 4 With this limitation, structural features that are in closer proximity to each other than these distances cannot be spatially distinguished. As many subcellular structures are themselves smaller than 250 nm, there has thus been a long-standing interest in biology in techniques or technologies that can supersede this limitation and resolve what is unknowable using classical light microscopy.
This work was supported by NSFC (91129000, 11374207, 31370750, and 21273148), the Shanghai Science Commission (10PJ1405100), and the K.C. Wong Education Foundation (H.K.). ⁎Corresponding author. E-mail address: [email protected] (Z. Shao).
In the last few decades, we have indeed witnessed unprecedented developments in this regard: there are now approaches that, although still limited by diffraction, can nonetheless attain a resolution of ~ 100 nm in all three directions and others that overcome the diffraction barrier altogether, yielding resolutions down to ~ 20 nm laterally and ~ 50 nm axially. 5-7 These approaches fundamentally differ from classical microscopy by using either novel illumination or detection schemes. The most successful of these methods include structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). 5-7 SIM works through patterned illumination that encodes the higher resolution spatial details of the sample in multiple images that can then be recovered with extensive post-exposure analysis. STED also works with “patterned” illumination but here the pattern is produced by a pair of beams designed to drastically reduce the emission volume to nanoscopic dimensions, which is then used to scan the sample to obtain the image. By contrast, PALM and STORM take advantage of a property of many fluorescent molecules to photo-switch: the molecules are first induced into a non-emitting,
http://dx.doi.org/10.1016/j.nano.2014.08.002 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Czajkowsky DM, et al, Illuminated up close: near-field optical microscopy of cell surfaces. Nanomedicine: NBM 2015;11:119-125, http://dx.doi.org/10.1016/j.nano.2014.08.002
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dark state and then a very low fraction of these molecules are stochastically photo-activated to emit. The individual fluorophores are then localized, which can be achieved down to less than 10 nm, as this is not limited by diffraction but instead by the number of detected photons. 8 Iterative accumulation of sufficient localizations allows a reconstruction of the full image at a much higher resolution than conventional diffraction-limited microscopy. Although such technologies continue to evolve, their power is already evident with several seminal contributions to our understanding of biological super-structures, including the discovery of a linearly periodic arrangement of actin-spectrin structures along the axons of neurons 9 and the delineation of the multilayer molecular architecture of the focal adhesion core. 10 These techniques have thus justly attracted a great deal of attention from a broad spectrum of biologists. In light of this great interest, we note that another super-resolution method, near-field optical microscopy (NSOM), 11-16 should be of especial interest to the biomedical community, owing to its unique capabilities to image biological processes at the cell membrane, the site of many medically important events. Like STED, this method breaks the diffraction barrier by reducing the size of the region of molecules that fluoresce. However unlike STED, this is achieved in NSOM by simply bringing a very small aperture at the end of a tapered glass fiber in extremely close proximity (~ 10 nm) to the sample surface (Figure 1). In this near-field regime, the illumination intensity is limited laterally as well as axially, thus bypassing the diffraction limit in all three dimensions, and bringing the lateral resolution to below 20 nm in raster-scanned images. This technique in fact significantly pre-dates the development of the aforementioned super-resolution methods, being developed on the heels of the transformative scanning tunneling microscope. 17,18 The validity of the NSOM concept was well demonstrated at that time, at least for imaging in air or vacuum, and the potential of such a technique in biomedicine was also appreciated almost immediately. 18,19 However, initial applications in biological research turned out to be rather disappointing, owing to the difficulty of maintaining the probe at a fixed distance from the sample in solution (see below). 15 Because of these difficulties, early optimism faded and enthusiasm for this methodology quickly waned. Still, this technology continued to evolve owing to the persistent efforts of a few labs. It is the purpose of this Perspective to bring attention to recent breakthroughs in this methodology and to provide a succinct account of the nanoscopic information that can presently be achieved with these devices.
Improving the original NSOM design: effective shear-force feedback in solution The most commonly used method in NSOM is rather straightforward. Optical fibers are pulled to form tapered probes between 20 and 100 nm in diameter, and then coated with an optically opaque metal (typically aluminum) to establish a transmitting aperture at the apex. As the tapered end has dimensions much smaller than the illuminating wavelength, these probes can exhibit low light throughput. In particular, as the size of the fiber diameter first approaches and then becomes much smaller than the
Figure 1. Schematic diagram of NSOM. A tapered optical fiber with a metalcoated aperture is brought within nanometers of the sample, which severely limits sample illumination. Images are obtained by raster-scanning the fiber at a constant probe-sample distance.
wavelength, light propagation becomes significantly perturbed by the small dimensions of the conduit: the number of propagating modes decreases as the fiber diameter reduces until the fiber is too small to support even a single mode, at which point the field decays exponentially. 13,20 Those light modes that do not propagate are either reflected back into the fiber or are absorbed by the aluminum, which can lead to severe heating when the power is excessive. 13 Despite these heating effects and this low efficiency of light transmission, these probes have indeed proven reasonably effective for many applications. 13,18,19,21 However, these probes are also extremely delicate and any forceful contact with the sample surface causes irreparable damage to the probe or the sample. This is indeed what led to the early disappointments when attempting to image biological samples in solution, a requirement for any technique engaged to resolve most biologically relevant features. In the original setup, the probesample distance was maintained by the “shear-force” detection scheme 22,23 In this approach, a piezoelectric device drives the optical fiber into lateral oscillation, and shear forces between the tip and sample lead to a reduction in the amplitude, which is monitored to maintain a constant probe-sample distance. In air or vacuum, where there are high-quality factor (Q) resonances, very small shear
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forces can lead to measurable changes in the amplitude, and this method is indeed highly effective for samples in which the molecules are adsorbed to a flat substrate, enabling, for example, the detection of the orientation and spectral properties of single molecules. 19 Ideally, these shear forces would be owing to effects when the tip is not touching the sample. Instead, however, evidence suggested that the oscillating probe tip makes intermittent contact with the surface, causing a reduction in the amplitude. 15,24 When the probe is submerged in aqueous solution, the amplitude is drastically reduced owing to the viscosity of the solution, and much greater shear forces are needed to produce measurable changes in the amplitude. Such greater shear forces, resulting from contact with the sample, led to the significant difficulties using this method to study biological samples in solution. 15 Although it was recognized that reducing the amount by which the probe is submerged in solution would lead to higher Q oscillations, early attempts in this regard failed to produce satisfactory results. 25 Recently though, Garcia-Parajo, van Hulst and colleagues have developed a device that successfully yields Q values of greater than 1000 in solution, 26,27 roughly 20-fold greater than earlier attempts. 25 As the probe-sample damping force scales inversely with Q 28, this device is expected to require a likewise 20-fold smaller probe-sample force than its predecessor, perhaps as low as 100 pN. 26 In this device, the optical fiber is attached to one of the prongs of a small piezoelectric tuning fork such that it projects out only a small distance (500 μm) from the end of the fork (Figure 2, A). With such a small projection, the motion of the fiber essentially mirrors that of the tuning fork, and so is thus determined by the inherently larger Q oscillation of the tuning fork. The distance by which the fiber is inserted in the solution is maintained relatively small (200 μm) as a consequence of the “diving bell” architecture of the device so as to minimize the reduction in Q. 26 This instrument has been applied to study a range of biological processes within the cell membrane at sub-diffraction resolution27,2932 . Of particular note is the direct observation of coalesced lipid rafts smaller than 120 nm laterally containing the ganglioside, GM1, upon complexation with its multivalent ligand, the pentameric cholera toxin B-subunit (CTB) (Figure 2, B). Interestingly, this coalescence occurred with an enhanced clustering of other raftassociated proteins in close proximity to, but not actually intermixing with, these CTB-GM1 nanodomains (Figure 2, B). 32
Height regulation using ion-conductive micropipettes In parallel with the shear force based probe control, an entirely different approach was developed that took advantage of the well advanced patch clamp technology. Here, the probe consists of an optical fiber inserted into a tapered glass micropipette, the end of which is coated with aluminum, similar to the shear force design (Figure 3, A). The tip-sample distance in this device is controlled by monitoring the ion current through the micropipette, as is done in scanning ion conductance microscopy (SICM). 33 When the micropipette is brought into close proximity to the sample surface (roughly the size of the aperture), the current decreases as a result of simple occlusion. Adjusting the pipette-sample height to maintain a constant reduced
Figure 2. NSOM with an improved shear-force detection scheme. (A) By attaching the optical fiber to a high-Q piezoelectric tuning fork and housing the device in this “diving bell” construction, fiber oscillations with Q N 1000 in solution were achieved.26 (B) Nanodomains of typical raft constituents, ganglioside GM1 complexed with CTB (red) and the GPI-linked protein CD55 (green), are found, surprisingly, only in close proximity to each other but not actually overlapping.32 Detecting such a distinction is only possible with sub100 nm resolution imaging capabilities. Scale bar: 1 μm. Reprinted with permission from Ref. [32]. Copyright 2010 National Academy of Sciences.
current while scanning the sample maintains a constant pipettesample distance and enables the generation of a topographic profile of the sample. In SICM, the attainable lateral resolution depends on the size of the aperture, and although pore sizes down to 6 nm have been achieved, 34 more common inner tube diameters are ~ 50 nm, roughly the best lateral resolution attainable by this technique. 35-37
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Figure 3. NSOM with an ion current tip-sample height regulation. (A) Schematic diagram of the device. (B) Simply raster-scanning the pipette across a sample that exhibits large changes in height can result in collisions producing an increased current. The system would then respond by decreasing the probe-sample distance, which further damages the probe and/or sample. (C) Images of IEC9 cells obtained with AC-mode SICM with a vibrating amplitude of 70 nm. At a larger scale (top), the expected cell shape of this layer of confluent cells is apparent. At smaller scales (bottom), higher resolution surface features, less than 100 nm in depth, are readily resolved. Scale bars: top: 9 μm lateral; 4 μm vertical; bottom: 2 μm lateral; 100 nm vertical.
While this NSOM/SICM technique indeed proved effective for relatively flat samples, studies of cells were too complicated for SICM. This difficulty stemmed from the relative insensitivity of the ionic current to abrupt changes in the nearby topography (Figure 3, B). As a result, for practical reasons, imaging of cell surfaces with this method had to be performed far (~250 nm) from the sample, which significantly reduced the attainable resolution. 38 To overcome this limitation, the concept of an alternatingcurrent (AC) imaging mode was proposed. 39-41 In this mode, in analogy with tapping mode AFM (atomic force microscopy), the probe is vertically oscillated while scanning to reduce the duration of probe-sample contact and likewise the extent of lateral forces between the probe and sample. The current alternates in phase with the height of the probe from the sample surface and the AC-component of the fluctuating current at this frequency is monitored during imaging. It was in fact shown,
theoretically, that the AC-component changes more sharply with vertical distance from the sample than the DC component. 40 Thus, this mode should be more sensitive than simply monitoring the DC-component. This device, used as a height monitor in NSOM or simply in SICM, was indeed shown to be more effective than its earlier “contact-mode” counterpart (Figure 3, C). 39-41 Since, for practical reasons, the amplitude of oscillation in the AC mode must be within several tens of nanometers, the AC mode is most effective for samples that do not vary in height by much more than this distance. Imaging entire cells can require greater height ranges, and often proved challenging to be well imaged with this mode. In AFM, to overcome a similar problem 20 years ago, researchers devised a so-called pulsed force or force-volume mode in where the cantilever is vertically scanned over distances of microns from the sample surface. 42-44 By scanning over such greater distances, the tip can thereby sense
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Figure 4. Imaging with hopping-mode SICM. (A) Principle of the method. (B) A spectacular hopping mode SICM image of the stereocilia of auditory hair cells. The arrow points to the kinocilium. Reprinted with permission from Ref. [45]. Copyright 2009 Nature Publishing Group.
the sample surface during a vertical approach, rather than a lateral one. Lateral scanning is only performed when the probe is far from the sample. This concept has now been successfully implemented in an SICM by Korchev, Klenerman and colleagues. 45 The pipette initially starts far from the sample and then is brought closer to the sample until the DC current is reduced by a predetermined amount, usually ~1% (Figure 4, A). This is recorded as the height of the sample and the pipette is withdrawn to a position far away from the sample surface, at which point it is moved laterally to the next imaging point. The typical drift problems with DC measurements are overcome in this method by re-setting the current measurement at the furthermost point from the sample surface. This method has proven exceptionally effective at imaging cell surfaces, particularly those with very large surface protrusions, such as stereocilia of auditory hair cells 45 (Figure 4, B). It is straightforward to incorporate NSOM capabilities into such a setup. Using a micropipette also affords additional sample characterizing options that are not possible with optical fibers or other super resolution techniques. For example, ligands to specific receptors can be administered locally through the imaging pipette, which together with the monitoring of a cellular response, enables a labelfree detection of functional receptor distribution within the cell membrane. This was utilized in a recent characterization of the differences in the cell membrane distribution of β1- and β2-adrenergic receptors between healthy and diseased rat cardiomyocytes. 46 Applying ligands specific for one or the other receptor, and monitoring (with conventional microscopy) binding using a fluorescent cytosolic sensor for cAMP, which increases upon receptor activation, the authors identified a markedly heterogeneous distribution of β2ARs in the healthy cells that was strikingly absent in the diseased cells. This suggested that changes in the cell membrane compartmentalization may contribute to the disease phenotype. Other examples of additional information other than topography that can be obtained with this instrument include measurements of the local elasticity of the cell, 37,47 direct measurement of ion channel activity on small cellular structures by directly patch-clamping the cells following imaging, 48 and chemical measurements of locally
secreted compounds when combined with an electrochemically sensitive electrode. 49
Looking forward Recent developments, although incremental in some sense, are in fact significant advances in terms of what they now enable for practical applications. Both approaches are suitable for labs as a do-it-yourself type of instrumentation, though the micropipette-based approach has a slight advantage in that, with the wide availability of scanning probe system controllers in commercial AFM systems, simple adaptations might be sufficient for most applications. However, as discussed above, pulled micropipettes may never achieve the same spatial resolution as that of a pulled fiber, since, as the size of the bore is reduced, it becomes increasingly difficult to fill with electrolytes and the ionic current also drops to values comparable to noise. Still, this deficiency may be compensated for by the wide range of additional information that may be simultaneously acquired. Future improvements to fiber-based approaches are likely to come from improved strategies to deliver greater, more highly localized illumination intensities such as with the use of nano-antennas directly attached to the probe aperture. 50,51 Such devices have already demonstrated a resolving capacity of 10 nm, imaging Cy3-labeled DNA molecules, 52 and of 30 nm in images of proteins within cell membranes. 50 Higher resolution might be expected with novel antenna designs or materials. 14 However, the forces applied with this device on the cell surface are, in practice, still somewhat significant (~ 350pN 26), which may damage local structures. 37 By contrast, SICM is inherently non-contact and has been used to resolve delicate extra-cellular or membranous structures. 45,53 Higher resolution features will require pipettes with smaller inner diameters that nonetheless allow sufficient current to pass. 37 One intriguing possibility is to embed a conductive nanowire within the coated micropipette: the nanowire would improve electrical conduction
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and the glass wall of the pipette would enable delivery of the near field optical illumination. Given the rapid advances in present-day nanofabrication capabilities, it may not be farfetched to envision that such hybrid probes could indeed be constructed. If so, one may expect 20–30 nm NSOM, in the same league as fiber probes or STORM and STED, but with the SICM combined. Such a system could provide a means to routinely resolve complexes consisting of only a few molecules within the membrane, enabling an even finer distinction of the differences between membrane-associated complexes. The NSOM devices described here are now capable members in the toolbox of biomedicine – with the next step in improved resolution, they could become one of the most powerful. Acknowledgments We wish to thank Drs. Thomas van Zanten and Maria Garcia-Parajo for Figure 2, B and Drs. Pavel Novak and David Klenerman for Figure 4. This work was supported by NSFC (91129000, 11374207, 31370750, and 21273148), the Shanghai Science Commission (10PJ1405100), and the K.C. Wong Education Foundation (H.K.).
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Perspective
nanomedjournal.com
Illuminated up close: near-field optical microscopy of cell surfaces Daniel M. Czajkowsky, PhD a , Jielin Sun, PhD b , Zhifeng Shao, PhD a,⁎ a
Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China b Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China Received 16 June 2014; accepted 10 August 2014
Abstract Invented in the 1990s, near-field optical microscopy (NSOM) was the first optical microscopy method to hold the promise of finally breaking the diffraction barrier in studies of biological samples. This promise, though, failed to materialize at that time, largely owing to the inability to image soft samples, such as cell surfaces, without damage. However, steady technical improvements have now produced NSOM devices that can routinely achieve images of cell surfaces with sub-100 nm resolution in aqueous solution. Further, beyond just optical information, these instruments can also provide simultaneous topographic, mechanical, and/or chemical details of the sample, an ability not yet matched by any other optics-based methodology. With the long recognized important roles of many biological processes at cell surfaces in human health and disease, near-field probing of cell surfaces is indeed now well poised to directly illume in biomedicine what has, until recently, been unknowable with classic light microscopy. From the Clinical Editor: This paper presents a novel and important near-field microscopy-based method directly enabling the imaging of cell surfaces with sub-100nm resolution. Unlike other optics-based methods, the presented technique can also provide topographic, mechanical and chemical details of the samples. © 2015 Elsevier Inc. All rights reserved. Key words: Super resolution; Cell membrane; Nanodomains
Since its invention nearly four hundred years ago, 1 optical microscopy has played an irreplaceable role in unravelling the structural underpinnings of biological systems, from the initial discovery of individual cells by van Leeuwenhoek to the organization of cells in complex tissues to the delineation of chromosomal territories within individual nuclei. 1-3 However, up until recently, what has been possible to resolve with conventional light microscopy has remained stubbornly limited by diffraction to about 250 nm laterally and several times worse axially. 4 With this limitation, structural features that are in closer proximity to each other than these distances cannot be spatially distinguished. As many subcellular structures are themselves smaller than 250 nm, there has thus been a long-standing interest in biology in techniques or technologies that can supersede this limitation and resolve what is unknowable using classical light microscopy.
This work was supported by NSFC (91129000, 11374207, 31370750, and 21273148), the Shanghai Science Commission (10PJ1405100), and the K.C. Wong Education Foundation (H.K.). ⁎Corresponding author. E-mail address: [email protected] (Z. Shao).
In the last few decades, we have indeed witnessed unprecedented developments in this regard: there are now approaches that, although still limited by diffraction, can nonetheless attain a resolution of ~ 100 nm in all three directions and others that overcome the diffraction barrier altogether, yielding resolutions down to ~ 20 nm laterally and ~ 50 nm axially. 5-7 These approaches fundamentally differ from classical microscopy by using either novel illumination or detection schemes. The most successful of these methods include structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). 5-7 SIM works through patterned illumination that encodes the higher resolution spatial details of the sample in multiple images that can then be recovered with extensive post-exposure analysis. STED also works with “patterned” illumination but here the pattern is produced by a pair of beams designed to drastically reduce the emission volume to nanoscopic dimensions, which is then used to scan the sample to obtain the image. By contrast, PALM and STORM take advantage of a property of many fluorescent molecules to photo-switch: the molecules are first induced into a non-emitting,
http://dx.doi.org/10.1016/j.nano.2014.08.002 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Czajkowsky DM, et al, Illuminated up close: near-field optical microscopy of cell surfaces. Nanomedicine: NBM 2015;11:119-125, http://dx.doi.org/10.1016/j.nano.2014.08.002
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dark state and then a very low fraction of these molecules are stochastically photo-activated to emit. The individual fluorophores are then localized, which can be achieved down to less than 10 nm, as this is not limited by diffraction but instead by the number of detected photons. 8 Iterative accumulation of sufficient localizations allows a reconstruction of the full image at a much higher resolution than conventional diffraction-limited microscopy. Although such technologies continue to evolve, their power is already evident with several seminal contributions to our understanding of biological super-structures, including the discovery of a linearly periodic arrangement of actin-spectrin structures along the axons of neurons 9 and the delineation of the multilayer molecular architecture of the focal adhesion core. 10 These techniques have thus justly attracted a great deal of attention from a broad spectrum of biologists. In light of this great interest, we note that another super-resolution method, near-field optical microscopy (NSOM), 11-16 should be of especial interest to the biomedical community, owing to its unique capabilities to image biological processes at the cell membrane, the site of many medically important events. Like STED, this method breaks the diffraction barrier by reducing the size of the region of molecules that fluoresce. However unlike STED, this is achieved in NSOM by simply bringing a very small aperture at the end of a tapered glass fiber in extremely close proximity (~ 10 nm) to the sample surface (Figure 1). In this near-field regime, the illumination intensity is limited laterally as well as axially, thus bypassing the diffraction limit in all three dimensions, and bringing the lateral resolution to below 20 nm in raster-scanned images. This technique in fact significantly pre-dates the development of the aforementioned super-resolution methods, being developed on the heels of the transformative scanning tunneling microscope. 17,18 The validity of the NSOM concept was well demonstrated at that time, at least for imaging in air or vacuum, and the potential of such a technique in biomedicine was also appreciated almost immediately. 18,19 However, initial applications in biological research turned out to be rather disappointing, owing to the difficulty of maintaining the probe at a fixed distance from the sample in solution (see below). 15 Because of these difficulties, early optimism faded and enthusiasm for this methodology quickly waned. Still, this technology continued to evolve owing to the persistent efforts of a few labs. It is the purpose of this Perspective to bring attention to recent breakthroughs in this methodology and to provide a succinct account of the nanoscopic information that can presently be achieved with these devices.
Improving the original NSOM design: effective shear-force feedback in solution The most commonly used method in NSOM is rather straightforward. Optical fibers are pulled to form tapered probes between 20 and 100 nm in diameter, and then coated with an optically opaque metal (typically aluminum) to establish a transmitting aperture at the apex. As the tapered end has dimensions much smaller than the illuminating wavelength, these probes can exhibit low light throughput. In particular, as the size of the fiber diameter first approaches and then becomes much smaller than the
Figure 1. Schematic diagram of NSOM. A tapered optical fiber with a metalcoated aperture is brought within nanometers of the sample, which severely limits sample illumination. Images are obtained by raster-scanning the fiber at a constant probe-sample distance.
wavelength, light propagation becomes significantly perturbed by the small dimensions of the conduit: the number of propagating modes decreases as the fiber diameter reduces until the fiber is too small to support even a single mode, at which point the field decays exponentially. 13,20 Those light modes that do not propagate are either reflected back into the fiber or are absorbed by the aluminum, which can lead to severe heating when the power is excessive. 13 Despite these heating effects and this low efficiency of light transmission, these probes have indeed proven reasonably effective for many applications. 13,18,19,21 However, these probes are also extremely delicate and any forceful contact with the sample surface causes irreparable damage to the probe or the sample. This is indeed what led to the early disappointments when attempting to image biological samples in solution, a requirement for any technique engaged to resolve most biologically relevant features. In the original setup, the probesample distance was maintained by the “shear-force” detection scheme 22,23 In this approach, a piezoelectric device drives the optical fiber into lateral oscillation, and shear forces between the tip and sample lead to a reduction in the amplitude, which is monitored to maintain a constant probe-sample distance. In air or vacuum, where there are high-quality factor (Q) resonances, very small shear
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forces can lead to measurable changes in the amplitude, and this method is indeed highly effective for samples in which the molecules are adsorbed to a flat substrate, enabling, for example, the detection of the orientation and spectral properties of single molecules. 19 Ideally, these shear forces would be owing to effects when the tip is not touching the sample. Instead, however, evidence suggested that the oscillating probe tip makes intermittent contact with the surface, causing a reduction in the amplitude. 15,24 When the probe is submerged in aqueous solution, the amplitude is drastically reduced owing to the viscosity of the solution, and much greater shear forces are needed to produce measurable changes in the amplitude. Such greater shear forces, resulting from contact with the sample, led to the significant difficulties using this method to study biological samples in solution. 15 Although it was recognized that reducing the amount by which the probe is submerged in solution would lead to higher Q oscillations, early attempts in this regard failed to produce satisfactory results. 25 Recently though, Garcia-Parajo, van Hulst and colleagues have developed a device that successfully yields Q values of greater than 1000 in solution, 26,27 roughly 20-fold greater than earlier attempts. 25 As the probe-sample damping force scales inversely with Q 28, this device is expected to require a likewise 20-fold smaller probe-sample force than its predecessor, perhaps as low as 100 pN. 26 In this device, the optical fiber is attached to one of the prongs of a small piezoelectric tuning fork such that it projects out only a small distance (500 μm) from the end of the fork (Figure 2, A). With such a small projection, the motion of the fiber essentially mirrors that of the tuning fork, and so is thus determined by the inherently larger Q oscillation of the tuning fork. The distance by which the fiber is inserted in the solution is maintained relatively small (200 μm) as a consequence of the “diving bell” architecture of the device so as to minimize the reduction in Q. 26 This instrument has been applied to study a range of biological processes within the cell membrane at sub-diffraction resolution27,2932 . Of particular note is the direct observation of coalesced lipid rafts smaller than 120 nm laterally containing the ganglioside, GM1, upon complexation with its multivalent ligand, the pentameric cholera toxin B-subunit (CTB) (Figure 2, B). Interestingly, this coalescence occurred with an enhanced clustering of other raftassociated proteins in close proximity to, but not actually intermixing with, these CTB-GM1 nanodomains (Figure 2, B). 32
Height regulation using ion-conductive micropipettes In parallel with the shear force based probe control, an entirely different approach was developed that took advantage of the well advanced patch clamp technology. Here, the probe consists of an optical fiber inserted into a tapered glass micropipette, the end of which is coated with aluminum, similar to the shear force design (Figure 3, A). The tip-sample distance in this device is controlled by monitoring the ion current through the micropipette, as is done in scanning ion conductance microscopy (SICM). 33 When the micropipette is brought into close proximity to the sample surface (roughly the size of the aperture), the current decreases as a result of simple occlusion. Adjusting the pipette-sample height to maintain a constant reduced
Figure 2. NSOM with an improved shear-force detection scheme. (A) By attaching the optical fiber to a high-Q piezoelectric tuning fork and housing the device in this “diving bell” construction, fiber oscillations with Q N 1000 in solution were achieved.26 (B) Nanodomains of typical raft constituents, ganglioside GM1 complexed with CTB (red) and the GPI-linked protein CD55 (green), are found, surprisingly, only in close proximity to each other but not actually overlapping.32 Detecting such a distinction is only possible with sub100 nm resolution imaging capabilities. Scale bar: 1 μm. Reprinted with permission from Ref. [32]. Copyright 2010 National Academy of Sciences.
current while scanning the sample maintains a constant pipettesample distance and enables the generation of a topographic profile of the sample. In SICM, the attainable lateral resolution depends on the size of the aperture, and although pore sizes down to 6 nm have been achieved, 34 more common inner tube diameters are ~ 50 nm, roughly the best lateral resolution attainable by this technique. 35-37
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Figure 3. NSOM with an ion current tip-sample height regulation. (A) Schematic diagram of the device. (B) Simply raster-scanning the pipette across a sample that exhibits large changes in height can result in collisions producing an increased current. The system would then respond by decreasing the probe-sample distance, which further damages the probe and/or sample. (C) Images of IEC9 cells obtained with AC-mode SICM with a vibrating amplitude of 70 nm. At a larger scale (top), the expected cell shape of this layer of confluent cells is apparent. At smaller scales (bottom), higher resolution surface features, less than 100 nm in depth, are readily resolved. Scale bars: top: 9 μm lateral; 4 μm vertical; bottom: 2 μm lateral; 100 nm vertical.
While this NSOM/SICM technique indeed proved effective for relatively flat samples, studies of cells were too complicated for SICM. This difficulty stemmed from the relative insensitivity of the ionic current to abrupt changes in the nearby topography (Figure 3, B). As a result, for practical reasons, imaging of cell surfaces with this method had to be performed far (~250 nm) from the sample, which significantly reduced the attainable resolution. 38 To overcome this limitation, the concept of an alternatingcurrent (AC) imaging mode was proposed. 39-41 In this mode, in analogy with tapping mode AFM (atomic force microscopy), the probe is vertically oscillated while scanning to reduce the duration of probe-sample contact and likewise the extent of lateral forces between the probe and sample. The current alternates in phase with the height of the probe from the sample surface and the AC-component of the fluctuating current at this frequency is monitored during imaging. It was in fact shown,
theoretically, that the AC-component changes more sharply with vertical distance from the sample than the DC component. 40 Thus, this mode should be more sensitive than simply monitoring the DC-component. This device, used as a height monitor in NSOM or simply in SICM, was indeed shown to be more effective than its earlier “contact-mode” counterpart (Figure 3, C). 39-41 Since, for practical reasons, the amplitude of oscillation in the AC mode must be within several tens of nanometers, the AC mode is most effective for samples that do not vary in height by much more than this distance. Imaging entire cells can require greater height ranges, and often proved challenging to be well imaged with this mode. In AFM, to overcome a similar problem 20 years ago, researchers devised a so-called pulsed force or force-volume mode in where the cantilever is vertically scanned over distances of microns from the sample surface. 42-44 By scanning over such greater distances, the tip can thereby sense
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Figure 4. Imaging with hopping-mode SICM. (A) Principle of the method. (B) A spectacular hopping mode SICM image of the stereocilia of auditory hair cells. The arrow points to the kinocilium. Reprinted with permission from Ref. [45]. Copyright 2009 Nature Publishing Group.
the sample surface during a vertical approach, rather than a lateral one. Lateral scanning is only performed when the probe is far from the sample. This concept has now been successfully implemented in an SICM by Korchev, Klenerman and colleagues. 45 The pipette initially starts far from the sample and then is brought closer to the sample until the DC current is reduced by a predetermined amount, usually ~1% (Figure 4, A). This is recorded as the height of the sample and the pipette is withdrawn to a position far away from the sample surface, at which point it is moved laterally to the next imaging point. The typical drift problems with DC measurements are overcome in this method by re-setting the current measurement at the furthermost point from the sample surface. This method has proven exceptionally effective at imaging cell surfaces, particularly those with very large surface protrusions, such as stereocilia of auditory hair cells 45 (Figure 4, B). It is straightforward to incorporate NSOM capabilities into such a setup. Using a micropipette also affords additional sample characterizing options that are not possible with optical fibers or other super resolution techniques. For example, ligands to specific receptors can be administered locally through the imaging pipette, which together with the monitoring of a cellular response, enables a labelfree detection of functional receptor distribution within the cell membrane. This was utilized in a recent characterization of the differences in the cell membrane distribution of β1- and β2-adrenergic receptors between healthy and diseased rat cardiomyocytes. 46 Applying ligands specific for one or the other receptor, and monitoring (with conventional microscopy) binding using a fluorescent cytosolic sensor for cAMP, which increases upon receptor activation, the authors identified a markedly heterogeneous distribution of β2ARs in the healthy cells that was strikingly absent in the diseased cells. This suggested that changes in the cell membrane compartmentalization may contribute to the disease phenotype. Other examples of additional information other than topography that can be obtained with this instrument include measurements of the local elasticity of the cell, 37,47 direct measurement of ion channel activity on small cellular structures by directly patch-clamping the cells following imaging, 48 and chemical measurements of locally
secreted compounds when combined with an electrochemically sensitive electrode. 49
Looking forward Recent developments, although incremental in some sense, are in fact significant advances in terms of what they now enable for practical applications. Both approaches are suitable for labs as a do-it-yourself type of instrumentation, though the micropipette-based approach has a slight advantage in that, with the wide availability of scanning probe system controllers in commercial AFM systems, simple adaptations might be sufficient for most applications. However, as discussed above, pulled micropipettes may never achieve the same spatial resolution as that of a pulled fiber, since, as the size of the bore is reduced, it becomes increasingly difficult to fill with electrolytes and the ionic current also drops to values comparable to noise. Still, this deficiency may be compensated for by the wide range of additional information that may be simultaneously acquired. Future improvements to fiber-based approaches are likely to come from improved strategies to deliver greater, more highly localized illumination intensities such as with the use of nano-antennas directly attached to the probe aperture. 50,51 Such devices have already demonstrated a resolving capacity of 10 nm, imaging Cy3-labeled DNA molecules, 52 and of 30 nm in images of proteins within cell membranes. 50 Higher resolution might be expected with novel antenna designs or materials. 14 However, the forces applied with this device on the cell surface are, in practice, still somewhat significant (~ 350pN 26), which may damage local structures. 37 By contrast, SICM is inherently non-contact and has been used to resolve delicate extra-cellular or membranous structures. 45,53 Higher resolution features will require pipettes with smaller inner diameters that nonetheless allow sufficient current to pass. 37 One intriguing possibility is to embed a conductive nanowire within the coated micropipette: the nanowire would improve electrical conduction
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and the glass wall of the pipette would enable delivery of the near field optical illumination. Given the rapid advances in present-day nanofabrication capabilities, it may not be farfetched to envision that such hybrid probes could indeed be constructed. If so, one may expect 20–30 nm NSOM, in the same league as fiber probes or STORM and STED, but with the SICM combined. Such a system could provide a means to routinely resolve complexes consisting of only a few molecules within the membrane, enabling an even finer distinction of the differences between membrane-associated complexes. The NSOM devices described here are now capable members in the toolbox of biomedicine – with the next step in improved resolution, they could become one of the most powerful. Acknowledgments We wish to thank Drs. Thomas van Zanten and Maria Garcia-Parajo for Figure 2, B and Drs. Pavel Novak and David Klenerman for Figure 4. This work was supported by NSFC (91129000, 11374207, 31370750, and 21273148), the Shanghai Science Commission (10PJ1405100), and the K.C. Wong Education Foundation (H.K.).
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