Nanomedicine: Nanotechnology, B

Diagnostic

Developing scanning probe–based nanodevices—

stepping out of the laboratory into the clinic

Martin Stolz, PhD, Ueli Aebi, PhD4 Daniel Stoffler, PhDM.E. Muller Institute for Structural Biology, Biozentrum, University of Basel, Switzerland

Received 25 April 2006; accepted 19 July 2006

www.nanomedjournal.com

Abstract This report focuses on nanotools based on the scanning force microscope (SFM) for imaging,

1549-9634/$ – see fro

doi:10.1016/j.nano.20

No conflict of inte

4 Corresponding

University of Basel, B

E-mail address: u

measuring, and manipulating biological matter at the sub-micron scale. Because pathophysiological

processes often occur at the (sub-) cellular scale, the SFM has opened the exciting possibility to spot

diseases at a stage before they become symptomatic and cause functional impairments in the affected

part of the body. Such presymptomatic detection will be key to developing effective therapies to slow

or halt disease progression.

D 2007 Elsevier Inc. All rights reserved.

Key words: Multifunctional SFM probes; Articular cartilage; Osteoarthritis; Coronary arteries; Arteriosclerosis; Molecular

motors; AFM; Clinical diagnostics; Nanotechnology; Tissue engineering; Actin; Asymmetric unit membrane;

Nuclear pore complex; Nanomedicine; IT SFM; Advanced health care

Introduction

Living matter is composed of a large number of

biological bnanomachinesQ, so it seems hardly surprising

that many diseases have their onset at the nanometer scale.

In fact, the idea of using bnanotoolsQ for detecting diseases

and repairing the human body was first put forward in the

late 1950s by the renowned American physicist and Nobel

laureate Richard Feynman during his famous speech,

bThere’s Plenty of Room at the BottomQ [1]. One such

nanotool is the scanning force microscope (SFM), a

variation of the scanning tunneling microscope for which

Gerd Binnig and Heinrich Rohrer were awarded the Physics

Nobel Prize in 1986 [2,3]. The SFM uses a fine tip at the

end of a cantilever, which scans over a specimen surface and

thereby maps its elevations and depressions with great

accuracy. The SFM provides not only the beyesQ for imaging

biological matter at scales from nanometers up to milli-

meters, but also the bfingersQ for measuring and manipulat-

ing biological matter at the level of single molecules,

nt matter D 2007 Elsevier Inc. All rights reserved.

07.01.001

rest was reported by the authors of this paper.

author. M.E. Mqller Institute for Structural Biology,

asel, Switzerland.

eli.aebi@unibas.ch (U. Aebi).

organelles, up to entire cells and tissues. For example, by

varying the force by which the tip of the SFM presses onto

the specimen surface while monitoring its deformation at a

given point—called indentation-type (IT) SFM—the stiff-

ness of the specimen may be determined at this position.

Moreover, the SFM tip may be bfunctionalizedQ to serve as asensor for a broad range of molecular targets. It is expected

that in the near future novel biochemical and biophysical

tests will emerge that, in turn, will greatly improve the

sensitivity and speed of medical diagnostics. Evidently, the

prospects of this unique nanosensor and nanoactuator in

biomedical research and clinical applications are only

limited by our imagination.

Imaging: Visualizing dynamic processes at the molecular

scale, from bsnapshotsQ to movies

Transmission electron cryomicroscopy (cryo-EM) [4-6]

is a powerful method for studying the architecture and

assembly of macromolecules and their interactions with

substrates in a close-to-native environment [7]. However,

major drawbacks regarding applications in biology are the

high investment for a state-of-the-art microscope and the

ability of this technique to provide only bsnapshotsQ of

iology, and Medicine 3 (2007) 53–62

Fig 1. SFM imaging of the mammalian urothelial plaques and plaque particles. A, SFM imaging provides absolute height measurements of the asymmetric unit

membrane (AUM) on stable immobilized urothelial plaques at a mica surface in a close-to-native buffer environment: The overall height of the entire urothelial

plaque was measured to ~12.5 nm; the height of the lipid membrane is ~5.5 nm (A, substrate level, i.e., mica surface; step size between A and B; B, lipid

bilayer (~5.5 nm) plus cytoplasmic face (~0.5 nm); C, overall height 12.5 F 0.7 nm); the leaflet above the lipid bilayer at the luminal side raised by ~6.5 nm

(step size between B and C) above the luminal membrane surface and the donut-shaped protrusions of the cytoplasmic side is ~0.5 nm above the lipid

membrane. (B–E) Surface topography of the 16-nm particle by SFM. B, Low-magnification view of the luminal face of several interconnected urothelial

plaques. C, High-magnification view of the luminal surface of an urothelial plaque. D, Low-magnification view of the cytoplasmic face of several urothelial

plaques. E, High-magnification view of the cytoplasmic face of several urothelial plaques. F, The averaged images of the luminal (upper left half in orange)

and cytoplasmic (lower right half in blue) surfaces of urothelial plaques. (A–E, Scale bars = 100 nm; F, center-to-center distance = 16 nm).

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–6254

the biological specimen (being immobilized in a thick layer

of vitrified ice). Also, cryo-EM does not allow measuring of

the functional properties of tissue.

Over the past decade, the SFM has opened completely

new vistas for analyzing the surface topography of biological

matter in its aqueous environment and at a resolution

comparable to that achieved by EM [8-18]. The most recent

generation of high-speed SFMs now permits the recording

of dynamic biological processes at close-to-video speed

[19-23]. A dynamic process may involve lateral [24-26] or

rotational motions [27,28], as well as bspatially stationaryQprocesses, those that do not exhibit any lateral or rotational

movement such as, for example, the opening and closing of

gates and channels [29-32].

Imaging the asymmetric unit membrane

The distinct nanostructure of the urothelial plaque, also

termed basymmetric unit membraneQ (AUM), offers an ideal

system for membrane visualization using the SFM. Accord-

ingly, the apical surface of the mammalian bladder

urothelium is covered almost entirely by rigid-looking,

concave membrane plaques consisting of 16-nm protein

particles that naturally form two-dimensional crystalline

arrays (Figure 1, A, B, and D) [33-36]. These plaques play

important roles in urothelial function and diseases, including

(1) the permeability barrier function to protect the underly-

ing tissues from the potentially harmful components of the

urine; (2) the reversible adjustment of the apical urothelial

surface area during the urination cycle; and (3) the receptor

function for the uropathogenic type 1-piliated bacteria,

which cause the majority of urinary tract infections, one of

the most common bacterial infections [37,38].

Urothelial plaques are composed of distinct 16-nm

diameter particles (Figure 1, C). Figure 1, F depicts the

averaged SFM images of the luminal and cytoplasmic

surfaces of urothelial plaques. In their native environment,

the urothelial plaque particles are hexagonally packed and,

based on their bfolded-ribbonQ topology [33], are predes-

tined to cooperatively rearrange during bladder distention.

Possibly the plaque particles undergo conformational

changes induced by effector molecules contained in the

urine. Experiments are now needed to track the formation of

plaques by time-lapse SFM and to eventually investigate

different conformations of individual plaque particles [36].

Imaging actin dynamics and assembly

Actin’s oligomerization, polymerization, and polymor-

phism have been investigated over the past decade at atomic

detail by combining x-ray and EM data. Figure 2, A depicts

negatively stained actin filaments recorded by transmission

electron microscopy (TEM). Actin exhibits a myriad of

diverse functions, most of which ultimately depend on its

intrinsic ability to rapidly assemble and disassemble

filamentous structures [39-41]. Actin controls the mechan-

ical stability and dynamics of the cytoskeleton and thereby

the overall shape, motility, and plasticity of cells. Actin is

also directly involved in muscle contraction where actin

filaments (F-actin) are serving as bmolecular tracksQ for

myosin motors to step along.

The bulk of structural information portrays actin in one

specific functional state. Hence, imaging F-actin under

native conditions by SFM, such as shown in Figure 2, B, is

useful not only to compare the native structure of the

filament with its atomic model, but also to eventually

directly monitor structural changes induced by chemical or

mechanical effectors [20,21,42]. For example, the new

generation of high-speed SFMs promises to directly

monitor muscle contraction at work. Here, the SFM tip

Fig 2. Imaging of actin filaments by transmission electron microscopy (TEM) and by SFM. A, Negative-stain TEM image of actin filaments. B, Native actin

filaments grown on the positively charged surface of a lipid film and transferred onto a hydrophobic support suitable for SFM and kept in buffer solution. SFM

imaging done in tapping mode of native actin filaments revealed filament dimensions (36-nm crossover repeat of the long-pitch helix crossover strand) similar

to those obtained by x-ray diffraction and electron microscopy. The width represents ~40 to 60 nm. (Scale bars: A, 100 nm, 20 nm (inset); B, 100 nm.

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–62 55

would be employed to track myosin heads in their different

conformational states during their cyclic interaction with

F-actin filaments.

Consequently, our knowledge about actin filaments has

evolved from a rigid bbeads-on-a-stringQ model to that of a

complex, highly dynamic protein polymer. To this end, the

SFM is the sole microscope providing us with high-

resolution images revealing the dynamics of actin in its

various functional states. Thus, time-lapse SFM should

further our present understanding of actin filament structure,

polymerization, and dynamics, as well as the interaction of

actin with myosin motors during muscle contraction and

cell locomotion.

Imaging nuclear pore complex architecture and dynamics

Nuclear pore complexes (NPCs; Figure 3, A) are large

macromolecular assemblies embedded in the double mem-

brane of the nuclear envelope (NE). They are the major

gateways mediating transport of ions, small molecules,

proteins, RNAs, and ribonucleoprotein particles in and out

of the nucleus in interphase cells (see Fahrenkrog and Aebi

[43]). Delineating the three-dimensional (3D) structure and

molecular architecture of the NPC is a prerequisite to

mechanistically understand its role in mediating nucleocy-

toplasmic transport. Over the past several years significant

advances have been made in the use of electron microscopy

techniques to elucidate the 3D structure and architectural

design of the NPC [44]. These studies indicated that NPCs

are rather dynamic supramolecular assemblies exhibiting a

high degree of structural plasticity, where significant

structural changes can be induced by cellular signals such

as calcium or nucleotides [44].

Whereas EM provides only snapshots of distinct func-

tional states, time-lapse SFM allows the imaging of the

same specimen in various stages of activity. Hence, many

laboratories began imaging NPCs by SFM (listed in [29]).

However, in most cases the specimen was chemically fixed,

exposed to detergents, or dehydrated and rehydrated at some

stage during its preparation for SFM. Thus, it remained

unclear whether the responses of NPCs to effector mole-

cules depicted in some of these studies did indeed represent

bona fide structural changes or distinct functional states

in response to adding effectors or ligands, or whether

they were merely due to specimen preparation effects,

for example, differential extraction of labile or weakly

bound NPC constituents or plastic deformations caused by

surface tension.

To overcome these limitations, it was important to

develop an isolation-preparation protocol for Xenopus

oocyte NEs that avoids exposure to detergents, chemical

fixation, or dehydration-rehydration steps. Based on such a

protocol [29], we established SFM imaging conditions

allowing the repeated scanning of these native NE samples

in physiological buffer environment, so that one and the

same NPC can be watched in response to chemical effectors.

As depicted in Figure 3, B and C, the NE appears highly

asymmetric with regard to the cytoplasmic and nuclear

surface topography of its NPCs, similar to metal-shadowed

specimens imaged by EM [45]. In areas devoid of NPCs,

bremnantsQ of the nuclear lamina meshwork [46] could

readily be depicted (Figure 3, C). With this preparation

protocol at hand, we performed time-lapse SFM experiments

to monitor the nuclear surface topography of completely

unfixed NEs. These experiments revealed the repeated

Fig 3. Imaging native nuclear pore complexes (NPCs) by SFM. A, A new structural consensus model of the NPC. The cytoplasmic filaments (blue) and the

struts of the nuclear basket (orange), absent in the cryo-EM tomographic 3D reconstruction because of their inherent flexibility, were modeled into the 3D

reconstruction based on thin-section images (where the filaments are visible) and height measurements of the nuclear basket (see D and E) by SFM.

Corresponding SFM images of native NPCs kept functional in near-physiological buffer reveal a distinct morphology for the cytoplasmic (B) and the nuclear

face (C) of spread Xenopus nuclear envelopes. The insets in B depict a high-magnification view of the rotational symmetry of individual NPCs (top) and NPCs

with clearly visible bplugsQ (bottom). The inset in C depicts a high-magnification view of the nuclear baskets. C and D were tilted 80 degrees using the scanner

software to improve the 3D appearance. D and E reveal reversible calcium-mediated structural changes of the nuclear baskets (i.e., the distal rings) by time-

lapse SFM of the same individual NPCs. In absence of calcium, the distal rings are bclosedQ (D). Adding 100 MM Ca2+ to the buffer medium opens the distal

rings (E). This process is reversible (e.g., by adding 1 mM EGTA, which selectively chelates calcium). Calcium affects only the opening of the distal ring of the

nuclear basket, not its overall height, as shown in the averaged radial height profiles in C and D. The 3D models in C and D depict the tentative interpretation

of the opening and closing of the distal ring upon changing the calcium concentration. In this model, the distal ring might act as an iris-like diaphragm. For

better comparison, the same individual NPCs were marked with blue arrowheads. (Scale bars, 100 nm.)

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–6256

opening and closing of the nuclear baskets of the NPCs at

their distal end in response to adding or removing calcium

(Figure 3, D and E). More specifically, the observed

structural changes were such that 20- to 30-nm-diameter

openings at the distal end of the baskets occurred upon

addition of micromolar amounts of calcium (Figure 3, E)

without, however, affecting the overall height of the baskets.

This reversible structural change of the nuclear baskets

may be interpreted in terms of their distal ring acting as an

biris-likeQ diaphragm, as suggested previously by Pante and

Aebi [47], which is mostly closed in the absence of calcium

(Figure 3, D) and opens upon the addition of calcium

(Figure 3, E). Therefore, it may be assumed that most likely

the distal ring of the nuclear basket undergoes similar struc-

tural changes while mediating nucleocytoplasmic transport.

Measuring: nanotechnology-based clinical

diagnostic tools

In addition to visualizing dynamic processes, the SFM

also allows us to explore the mechanical properties of

proteins, their supramolecular assemblies, organelles, cells,

and tissue in vitro and, ultimately, in situ by IT SFM [48].

Evaluating the material properties of a specimen at all

relevant length scales bears the prospect of developing a

new generation of precision instruments that in combination

with automated inspection protocols may allow, for exam-

ple, the doctor to perform a much more quantitative and

objective inspection of the patient. The following two

examples focus on how the SFM may be used in diagnosing

two frequent and devastating diseases, arteriosclerosis and

osteoarthritis. Arteriosclerosis is the major cause of disabil-

ity and death in the First World nations, occurring mostly in

the form of a myocardial infarction or stroke. Similarly,

arthritis has been diagnosed in over 50 million Americans,

with another 20 million Americans reporting chronic joint

symptoms. Cost-of-illness estimates include both direct

medical costs and indirect costs associated with lost

productivity. The total costs of osteoarthritis and chronic

joint symptoms in the United States in 1997 was $85 billion,

including $50 billion in direct costs and $35 billion in

indirect costs, approximately 1% of the US gross domestic

product. These costs will probably continue to rise with the

aging population, with the number of people aged 65 and

older with arthritis or chronic joint symptoms expected to

nearly double from 20 million in 2001 to 40 million by 2030

(Centers for Disease Control and Prevention).

Exploring the vulnerability of coronary artery plaques

Arteriosclerosis is a progressing disease leading to

bplaqueQ formation in arteries supplying critical organs with

blood such as the heart or the brain. Two types of plaques

Fig 4. Imaging and measuring native coronary artery tissue. A, SFM imaging reveals the intact endothelial cell layer lining the lumen of a porcine artery in

physiological buffer. Individual cells can clearly be delineated, as can their segregating cell junctions. B, The surface endothelial layer of a wild-type mouse

coronary artery was mechanically removed by surgical tools for inspection in physiological buffer. The SFM image reveals the subsurface layer that is

supporting the endothelium. The inset documents the 67-nm axial repeat of the collagen fibrils. C, SFM imaging of rat-2 cells as imaged under culture

conditions. Different cells and their cytoskeletal actin network are visible. (Scale bars: A, 5 Mm; B, 2 Mm, inset 500 nm; C, 25 lm).

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–62 57

exist: fibrous, stable plaques that are rarely life-threatening

[49-53], and vulnerable, lipid-rich plaques prone to me-

chanical rupture thus leading to myocardial infarction and

stroke. Conventionally, discrimination of the two types of

plaques is performed histologically during an autopsy [54].

In our attempt to investigate plaques at different stages of

their development by SFM, we recently established two new

preparation protocols that not only take into account the

fragility of the soft coronary arteries by maintaining its bona

fide structure in a nondestructive sample treatment, but also

permit the investigation of cultured cell lines under well-

defined culture conditions [55]. As a first result, we

succeeded in improving the imaging resolution of those

soft biological specimens as documented in Figure 4, A to C.

Figure 4, A and B, depict SFM images of native coronary

artery endothelium in physiological buffer taken from a pig

or a wild-type mouse, respectively. For imaging the

underlying vascular extracellular matrix that consists mainly

of collagen fibrils, we mechanically removed the endothelial

cell layer (Figure 4, B). In addition to the preparation of

pieces of native coronary arteries, cultured vascular endo-

thelial cells may be studied as an in vitro model system. As

a first step toward this latter goal, Figure 4, C displays an

example of imaging living rat fibroblasts by SFM. The cells

were imaged under optimal culture conditions, that is, by

continuously exchanging the culture medium at 378C to

assure viability of the cells in the SFM over extended

periods of time. Such in vitro model systems may eventually

be used for testing drugs and pharmaceutical agents

targeted, for example, at cytoskeletal elements to affect

their 3D structure and spatial organization, and hence their

mechanical properties.

The ability to inspect the bona fide structure and assess

the local biomechanical properties of soft biological tissue is

paramount, because the investigator can thereby avoid

artifacts (e.g., due to freezing and fixation). The SFM

allows imaging, measuring, and manipulating native coro-

nary artery tissue in its physiological context. Moreover,

there is great potential for directly observing changes in

individual vascular endothelial cells in response to mechan-

ical or pharmacological stimulation by time-lapse SFM. In

the context of studying atherosclerosis, plaques might not

only be localized but also differentiated in terms of their

vulnerability by measuring their local, scale-dependent

biomechanical properties, thereby depicting differences in

tissue elasticity and plasticity by an experimental approach

being similar to that reported previously [48,55].

Inspection of articular cartilage

Osteoarthritis is a painful and disabling joint disease that

affects millions of people by progressively degrading the

articular cartilage. Osteoarthritis poses a dilemma: it usually

begins by attacking different joints long before middle age,

but it cannot be diagnosed until it becomes symptomatic

decades later, at which point the structure and biomechan-

ical properties of the affected cartilage are usually irrevers-

ibly altered. Currently available devices for measuring the

biomechanical properties of cartilage typically work at the

millimeter scale or above [48,56-60] and hence are not able

to assess the cellular and molecular features of cartilage (i.e.,

the scale at which biomechanical processes occur and

pathological lesions typically start). In contrast, the SFM

can readily image cartilage morphology and measure its

biomechanical properties at the micrometer scale and below.

Thus, the SFM opens the exciting possibility to detect

pathological features of articular cartilage long before they

become symptomatic and cause a functional impairment of

the affected joint. Detection of OA at a presymptomatic

stage might be key to the development of effective therapies

to slow or stop its progression.

Recently, we used ex vivo IT SFM on articular cartilage

biopsies to measure the mechanical stiffness, which slowly

Fig 5. Imaging and measuring native articular cartilage. Surface topography of (A) normal articular cartilage, (B) osteoarthritic articular cartilage, and (C) the

corresponding elasticity measurements performed at the nanometer scale with a sharp pyramidal tip. A, The 67-nm axial repeat distance of individual collagen

fibrils was clearly resolved by SFM imaging. B, In contrast to the normal cartilage that exhibited a random orientation of the collagen fibril network, in the

diseased cartilage the collagen fibrils coalesced on top of each other and exhibited a preferred orientation. This orientation might follow the directed movement

within the joint more easily once the GAGs become digested in the course of the disease progression. C, Comparison of the two slopes of the corresponding

force displacement curves indicated stiffening of the osteoarthritic articular relative to the normal cartilage. (A and B, Scale bars = 1 lm).

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–6258

but definitely changes during progression of osteoarthritis.

Interestingly, when measured at the nanometer scale—but

not at the micrometer or millimeter scale—such stiffness

changes can be depicted long before clinical symptoms of

osteoarthritis (e.g., pain) arise. As illustrated in Figure 5, A,

normal articular cartilage exhibits a stochastic 3D orienta-

tion of the collagen fibrils, revealing a characteristic 67-nm

axial repeat. In contrast, osteoarthritic cartilage exhibits a

preferred orientation and a tendency of bundling of its

collagen fibrils, as indicated by the arrows in Figure 5, B.

Because of the mechanical stress, it is conceivable that upon

degradation of the interspersed glycosaminoglycan network,

the collagen fibrils are no longer spaced apart and coalesce

on top of each other, thereby slowly but definitely aligning

themselves in a direction representing the predominant joint

movement. As documented in Figure 5, C, when assessed

by IT SFM at the nanometer scale the osteoarthritic cartilage

appears mechanically significantly stiffer than the normal

cartilage. It should be noted, however, that there are also

stages and/or conditions of osteoarthritis where the cartilage

appears mechanically softer than normal cartilage, indicat-

ing that there exist multiple osteoarthritic sequelae, all

eventually leading to complete degeneration and/or disap-

pearance of the cartilage normally covering the moving

parts of a joint. Nevertheless, this finding opens the prospect

of early diagnosis, hopefully at a stage when disease

progression can still be halted.

Manipulating: using multifunctional SFM probes

as bfingersQ for minimally invasive therapeutic

interventions

Minimally invasive therapeutic interventions are increas-

ingly being performed using surgical tools with embedded

miniature sensors providing real-time information to im-

prove surgical results, hence benefiting both the patient and

the surgeon. Based on such tools, an envisioned scenario in

a future surgical theater might be the following: the

coronary arteries are first inspected by catheter-based SFM

for identifying and differentiating potential plaques into

bstableQ versus bvulnerableQ plaques based on their biome-

chanical properties. Next, a more detailed characterization

of the state of the vulnerable plaques might be achieved by

multifunctional SFM probes that will be able to measure

heat production or pH in the near field of individual plaques,

or can detect specific plaque surface markers using

fluorescent reporter molecules by a scanning near-field

optical microscopy (SNOM)-type probe being incorporated

into the SFM tip [61,62]. Obviously, the next step will be to

develop such multifunctional SFM probes into multipurpose

SFM-type actuators that will permit us to move from

diagnostics to treatment and, consequently, toward repairing

the diseased or damaged tissue locally.

Hence, novel, nanotechnology-based surgical devices are

now needed to permit the surgeon to perform interventions

that are currently not possible, for example, diagnosing and/

or treating a disease directly at the site where it occurs. In

this context, a minimally invasive, nanoactuator-type

bnanoscalpelQ would offer unprecedented precision for

performing delicate surgery at the level of individual cells

without damaging the surrounding tissue. Once a diseased

tissue can directly be targeted by a multifunctional nano-

device, a drug or a small amount of radioactive material

could be locally administered to the diseased site, destroy-

ing, for example, cancer cells one by one. The selectivity of

nanosurgery could even be further enhanced by tagging

specific cellular components with light-sensitive markers for

specific localization or by activating photolabile chemical

reactions by light-emitting local probes [63-65].

Ultimately, such surgical nanotools will adopt and

integrate a variety of functionalities that are currently being

explored in life science applications [66]. For example,

Fig 6. The arthroscopic SFM. A, Measurement by the arthroscopic SFM (arrows) in a phantom knee. B, Prototype of an arthroscopic SFM. C, The

arthroscopic SFM is a new tool that can be inserted into a standard arthroscopic tube for evaluating the health status (e.g., the elasticity) of cartilage and also for

quality control in follow-up examinations after cartilage replacements.

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–62 59

multifunctional SFM devices are being developed and

implemented that include SNOM and conductive scanning

probe microscopy (CSPM). Whereas SNOM permits local

detection of fluorescently tagged cell membrane targets at the

single-event level [67,68], CSPM probes allow detection of

currents through individual membrane channels [69]. Fur-

thermore, SFM probes have been built that can detect small,

local temperature changes [70], which are often an indicator

of a local inflammation. Next, these devices will be

customized for minimally invasive endoscopic or catheter-

based interventions, ultimately, with single-cell precision.

A more precise diagnostics and early therapeutic

intervention will provide an accurate detection of the

clinical relevant parameters even before a disease becomes

symptomatic. In addition, tissue engineering may solve the

problem of the limited amount of autologous tissue

available for transplantation. Tissue engineers are producing

bspare partsQ for replacing defective tissue in the human

body, such as bone, cartilage, or tendon. As yet, however,

no synthesized tissue can mimic the long-term mechanical

stability, durability, and biocompatibility of authentic tissue.

One major obstacle in trying to bwatch and copyQ nature

concerns the primary reliance of our perception on the

macroscopic world that is accessible to our senses. Hence,

almost everything ever created by humans is based on our

macroscopic understanding of the phenomena and actions to

which we are constantly exposed. For example, all human-

made technology follows the rule of assembling macroscale

components or by carving bulk pieces of matter into smaller

chunks. As yet, we by and large lack a rational understand-

ing of the self-assembly processes that nature uses to

generate hierarchically organized, multifunctional biological

matter from a finite set of elementary building blocks. In

fact, the elementary building blocks of biological matter

(e.g., the proteins) are nanometer-scale structures that are

endowed with a remarkable functional versatility. They are

used for creating supramolecular assemblies such as, for

example, cytoskeletal filaments, or higher order functional

units like ribosomes or the mitotic apparatus. It is these

distinct supramolecular assemblies acting in concert that, in

turn, render a tissue or organ highly versatile in terms of its

structure, plasticity, and functional adaptability.

Articular cartilage is a hierarchically organized biomate-

rial exhibiting distinct nanoscale, mesoscale, and tissue-scale

structural features. Unless being subjected to pathological

alterations, it is built to warrant lifelong performance within

the human body. Yet, cartilage is a finely tunable tissue: its

biomechanical properties, for example, may be affected by

(1) physical or chemical effectors, (2) overexpression or

knock down of some of its molecular constituents [71-73], or

(3) controlled enzymatic digestion [48,74-76]. However, for

capturing the thus induced structural and biomechanical

changes, we may need a better understanding body the old-

timer, tools that are capable to image and measure cartilage at

the sub-micrometer scale. IT SFM, for example, can measure

changes in the stiffness of articular cartilage when operated

with a sub-micrometer tip that goes undetected with

micrometer to millimeter indenters [48]. Moreover, sub-

micrometer IT SFMmay be very effective for controlling and

optimizing the quality of engineered cartilage [48].

As a next step, we are developing an arthroscopic SFM

(Figure 6) for direct, quantitative in situ inspection of articular

cartilage morphology and biomechanical properties at the

sub-micrometer scale [60,77]. In addition to serving as a

M. Stolz et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 53–6260

potential diagnostic nanotool, the arthroscopic SFMmay also

be used for quality control after cartilage transplantations.

Ultimately, this novel nanodevicemight permit the surgeon to

rapidly, minimally invasively, and reliably assess the health

status of articular cartilage prophylactically and, if necessary,

to prescribe therapeutic interventions that slow or even halt

disease progression at a presymptomatic stage.

Going beyond cartilage, similar nanotools may be

designed and customized to manipulate transplanted tissue

or stimulate de novo tissue formation directly in situ more

generally, and ultimately, to design and produce tissue-

engineered body parts that exhibit long-term mechanical

stability, functionality, and biocompatibility [78].

Outlook

As documented in the case of osteoarthritis, the prospects

for detecting progression of a disease in the human body well

before it becomes symptomatic is considerable, thus opening

completely new vistas for treatment of such a disease.

Because most diseases have their origin at the cellular or

even molecular level, capturing them at this stage requires

diagnostic tools that operate at the cellular or molecular scale.

Hence, the call for nanotools, such as, for example, SFM or

more generally, scanning probe microscopes (SPMs) that

permit the imaging, measuring, and manipulation of biolog-

ical matter at the nanometer to micrometer scale. Obviously, a

prerequisite for an SPM to operate is to bring it to the

presumed defect site, for example, into the knee joint by an

arthroscopic approach. More generally, SPMs will have to be

designed and customized for endoscopic or even catheter-

based direct in situ operation. This requirement, in turn,

confronts the individual appearing for a prophylactic

examination of this kind with the need for an invasive

procedure—a circumstance that may prompt him/her to

decide against it, particularly in the absence of any

symptoms. For such prophylactic interventions to eventually

being tolerated by patient, not only must they be ambulant

and minimally invasive, but they will require a change of

paradigm vis-a-vis the patient, namely to undergo an invasive

procedure without feeling sick—indeed a challenge for

nanomedicine and managed health care!

Acknowledgments

This work was supported by an NCCR program grant on

bNanoscale ScienceQ awarded by the Swiss National ScienceFoundation, the bEU Network of Excellence 3D-EMQproject LSHG -CT-2004-502828, the M.E. Mqller Founda-tion of Switzerland, and the Canton Basel-Stadt.

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