L'Ancien d'Algérie, May 2011

8/3/2019 L'Ancien d'Algrie, May 2011

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AFM Nanodissection Reveals InternalStructural Details of Single CollagenFibrils

Chuck K. Wen and M. Cynthia Goh*

Department of Chemistry, UniVersity of Toronto, Toronto, Ontario, Canada M5S 3H6

Received August 20, 2003; Revised Manuscript Received November 7, 2003

ABSTRACT

We utilize the atomic force microscope (AFM) to perform a careful nanodissection procedure on a single collagen fibril. By precisely incising

the fibril and then peeling away exterior layers, its internal structure was successfully exposed for direct visualization. This methodology has

the advantage of avoiding unnecessary rough contact with the specific area being examined as well as high lateral frictional forces, both of

which can hinder attempts to dissect delicate biological specimens with the AFM.

Applying the atomic force microscope (AFM) toward studies

in the realm of the biological sciences has been an exciting

and fast-growing trend over the past decade. Boasting sub-

nanometer resolutions, the ability to operate in solution, and

only minimal sample preparation prerequisites compared to

other microscopy techniques, it is easy to understand why it

has become the popular visualization tool that it presently

is. Since its discovery only seventeen years ago,1 its efficacy

for the imaging of various biological systems is already well

established and documented.2

The contribution of AFM though, is not restricted to the

typical domain of other microscopy methods: That is, the

AFM is more than simply an aid for the eye. Recall, an AFM

picture is generated by monitoring the reaction of a flexible

cantilever tip as it is precisely raster scanned over a sample

surface by a computer-controlled piezo-crystal. Effectively,

the microscope is feeling the sample (rather than seeing it)

during the imaging process; topographical maps are readily

constructed from this. Therefore, the AFMs instrumentation

can essentially use an extremely sharp stylus to exactly feel

or touch a sample with sub-nanometer precision; this ability

affords unique opportunities outside the function of visual-

ization alone.This ability of the AFM to precisely touch the nanoworld

has opened a vast storehouse of exciting new possibilities

for the biological scientist. Among other striking examples,3

single proteins have been individually stretched and un-

folded,4 antibody-antigen bonds physically disrupted,5 pro-

tein conformations physically modulated,6 and individual

DNA molecules7 intentionally sliced. The AFM has allowed

researchers to manipulate and literally play with the miniature

world of molecular biology in a manner previously only

dreamed of. In this paper, we present work that sought to

take hold of this opportunity.

Herein, an account describing how we carefully dissected

a single biological fibril is presented. Using careful manipu-

lations, the exterior layers of a specific segment on the fibril

were initially cut and then peeled away. This dissection

technique exposed the fibrils previously hidden internal

anatomy, and thus made it available for direct visualization

for the first time. The observations afforded by this allowed

new data concerning the structures internal organization to

be gained.

The biological fibril chosen to be dissected is known as a

fibrous long spacing (FLS) type collagen fibril. This choice

was made due to our groups ongoing interest in the topic

of collagen structure. Previously, we have utilized the AFM

to produce topographical maps of FLS-type fibrils in order

to elucidate their ultrastructure.8 Though this former work

was fruitful for discerning the surface features of these fibrils,

questions about its interior architecture remained unad-

dressed. How this unique dissection procedure was performed

is now presented.As alluded to earlier, an incision into the exterior layers

of a collagen fibril was initially made. This was produced

by making a linear set of closely distanced indentations along

the fibrils surface. Each indentation was independently made

by pressing down the cantilever tip into the fibril surface at

a specified coordinate with the same predefined depth.

Together, this can effectively produce a single cut that

partially severs the fibril through this stitching-like action.

Figure 1 depicts the fibril prior to the cut, while Figure 2

depicts it after the cut.* Corresponding author. Tel: +1-416-978-6254; fax: +1-416-978-4526.

E-mail: [email protected].

NANO

LETTERS

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10.1021/nl034685n CCC: $27.50 2004 American Chemical SocietyPublished on Web 11/20/2003


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This stitching-like indentation method is useful for the

production of cuts into particular specimens in a readily

controllable manner. Though the depicted incision is 140

nm deep, the depth of the slice can be readily varied by

simply changing the predefined depth to which the tip is

pressed into the fibril at each indentation point. For example,

when a relatively shallower tip-penetration depth is chosen

(which corresponds to a lower indentation force), a shallower

cut is made into the fibril. This is exemplified by the 1-2

nm deep perforated cut demarcated by the white line, and

the corresponding inset close-up, in Figure 2.

Following the production of this cut though, we then

desired to peel away the scored exterior layers of the fibril

in order to expose its interior domains. This was performed

by applying a compressing force on a nearby area of the

fibril. The force was applied by repeatedly squeezing down

on the fibril with the tip over a segment that was axially

removed from the original line of incise. By doing so, we

can see that the cut outer layers of the fibril became pulledtoward the segment that was compressed, and away from

the original line of cutting. A well-defined area that allowed

the interior structure of the fibril to be directly imaged in

the context of the larger form was produced (see box 1 in

Figure 3 and Figure 4). Thus we can see that, with proper

technique, AFM nanodissection can lead to internal structural

information of individual fibrils, not limited to the surface

level only.

In applying this compressing force, we envision that the

cut layers were peeled away in the following manner: The

force would be expected to produce a tensile stress axially

directed toward the point of squeezing and unevenly felt in

the cross-section of the fibril (Figure 5). This stress would

be most strongly experienced in the fibrils radial outermost

layers. As these exterior layers are then deformed during

the compression, one would expect these previously scored

outer layers to be pulled toward the point of squeezing and

away from the original line of incise during this process.

Though the compressed segment of the fibril is sacrificed

in this process (see area 2 in Figure 3A), this method has

the significant advantage of doing no damage to the area of

interest (see area 1 in Figure 3A). The now-exposed interior

domain is distinct and readily visualized. It is not excessively

Figure 1. 3D height image plot of a FLS-type collagen fibril witha diameter of210 nm prior to cutting. The characteristic 270nm periodic cross-striated pattern is evident. Scale bar represents1 m in the x-y plane.

Figure 2. The depicted incision, marked by the dotted line (1), is140 nm deep into the fibril. Making a set of successiveindentations into the fibril produced the cut. Changing the forceby which the indentations are made controls their depth. This isillustrated in the boxed area marked (2). The inset shows a 2magnification of this area; lower indentation forces resulted inshallow penetrations (of 1-2 nm deep only). The scale barrepresents 1 m in the x-y plane. This is a 3D height image plotof the same fibril depicted in Figure 1. The inset is a deflectionmode image.

Figure 3. A deflection mode image of the same fibril in Figures

1 and 2 after compression. Area (1) marks the well-defined openedsegment of the fibril; area (2) marks the location where compressiontook place. The scale bar represents 1 m.

Figure 4. A 3D height image plot of the dissected area is presented.A hierarchical macromolecular architecture is observed, consistingof parallel-aligned fibrillar subcomponents that respectively maintainthe characteristic FLS cross-striation pattern; the banding of eachfibrillar subcomponent lies in register with that of its neighbors.The scale bar represents 500 nm.

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complicated by the damage done to the nearby surrounding

areas, nor is it obscured by any damage that may have been

directly incurred during the nanomanipulation process. The

fact that no tip-sample contact is ever directly made in the

dissected area of specific interest may be especially advanta-

geous for delicate biological specimens that may otherwise

be readily deformed due to such immediate contact by the

typically much-harder cantilever tip. For example, the

methodology we have described may be useful for the

dissection of specimens such as individual biological cells:

Selected local regions of a cell may be carefully and gently

opened up to a specific depth, to reveal its underlying and

internal anatomy at that targeted space.The method we have herein outlined differs from the

techniques previously employed. Typically, AFM nanodis-

section has been performed by repetitively running the

cantilever tip against the particular specimen of interest, with

high loading forces in a contact-mode-like fashion. Such an

action effectively produces a scraping effect that can do

damage to the sample, and then sweep away those damaged

or frayed portions. This has been exemplified in work such

as that done by Thalhammer et al. where the AFM tip was

used to scrape off a particular section on an isolated human

chromosome.9

This scraping technique, however, did not prove fruitful

in our nanodissection attempts of FLS-type fibrils. In those

attempts, overly excessive damage to the fibril was often

observed. Significant distortion of the fibril was often seen,

even in areas that were axially removed from the local

segment of scraping. This is illustrated in Figure 6. Such

effects confounded our attempts to produce a well-defined

dissected area that could serve as a window into the fibrils

interior structure.

This more typical nanodissection technique may have

failed us in our work due to the high frictional forces that

are incurred during the process. These forces are likely the

reason for the heavily distorted and pulled appearance of

the fibril after scraping (as demarcated by the arrow in Figure

5B); they deform the scraped structures too much. Unfor-

tunately, though, these frictional forces are unavoidable; they

are a byproduct of the high loading forces that are necessary

if the cantilever tip is to get into the tough exterior of these

dried biological fibrils via a scraping motion. As an analogy,

we envision this difficulty to be likened to that experienced

by a person who desires to make a precise and defined cut

utilizing a dull blade: considerable lateral forces are exerted

on the particular object being sliced due to such a motion,

which can undesirably warp and twist it away from its

original form.

In the technique we have described in this letter, suchfrictional forces are minimized. Recall, all nanomanipulation

procedures utilized here move the tip in an up-and-down

fashion, as opposed to a side-to-side one. In making the

original incision, a stitching-like motion is used to make the

cut, and in the pulling away of the scored outer layers of

the fibril a compressing motion is utilized. As has been

demonstrated, such movements are readily controllable, and

they avoid the production of undesirable frictional forces that

may lead to excessive and uncontrolled damage to the

specimen.

In the context of our own studies on collagen fibril

structure, this technique of AFM nanodissection has afforded

unique and useful empirical results. For noncrystallizing

fibrous proteins such as this, there are relatively few

experimental techniques that are suitable for the study of

such supramolecular structure. Previous studies of FLS-type

fibril structure have been confined to the techniques of

electron microscopy10 (EM) or traditional AFM visualiza-

tion.8 In that work, observations are typically focused on the

fibrils surface or ultrastructure only. However, by utilizing

the unique opportunities the AFM has afforded us as an

investigative nanodissection tool, we have managed to

produce three-dimensional topographical maps into the FLS-

Figure 5. (A) Depicts the tip moving downward toward a fibril.(B) Depicts the lines of tensile stress that would be expected to beinduced in the fibril during compression by the cantilever tip.

Figure 6. Deflection mode images of FLS type collagen fibrilbefore (A) and after (B) scraping with the AFM probe. The box in(A) represents the area of scraping. Extensive damage is done tothe fibril construct. The arrow in (B) points to an area of the fibrilthat appears to have been pulled away from its original arrangementduring the scraping process. The scale bar represents 1 m.

Nano Lett., Vol. 4, No. 1, 2004 131


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type fibrils core when a suitable methodology is utilized.

These new results well supplement and very much add to

the past observations obtained using those other techniques.

Though this investigative nanodissection procedure was

demonstrated on an FLS-type fibril specifically, we envision

this procedure to be general in its potential applicability.

There does not appear to be anything particularly specific

or unique for the case of this FLS-type fibril that makes it

any more suitable as a subject for nanodissection than another

potential specimen. Even with respect to the working size-scale, the procedure we have presented would be expected

to be readily scalable. However, as is typical for other AFM

functions, we acknowledge that we would expect a generally

increasing level of difficulty when working with progres-

sively smaller size scales.

In this letter, a general nanodissection technique has been

described on an isolated FLS-type collagen fibril. This

procedure has demonstrated its unique investigative utility

for the elucidation of the structures internal anatomy and

organization. By utilizing the unique nanomanipulation

capabilities of the AFM, novel insights that other techniques

would be hard pressed to obtain have been made on this

system. As our techniques and abilities with the AFM aredeveloped and refined, we are excited about the new potential

such work may hold for our ability to reach out and directly

interact with these very small systems that are operative at

diminutive size scales.

In this work, dried FLS-type collagen fibril samples were

prepared as previously described.11 Briefly, an acidified 1

mg/mL Type I collagen (99% purity, Sigma, St. Louis,

MO) monomer solution was dialyzed against a water bath

of milli-Q deionized water at 21 C overnight; after which,

a 10 L aliquot sample was removed, diluted 10-fold with

milli-Q deionized water, and then deposited on a freshly

cleaved piece of mica surface where it was allowed to dry

under ambient conditions. All AFM work was performed

on a Nanoscope III multi-mode instrument (Digital Instru-

ments, Santa Barbara, CA). Images were captured in contact-

mode operation under ambient conditions and using square

pyramidal silicon nitride cantilevers with nominal spring

constants of 0.12 N/m (Digital Instruments). All nanodis-

section procedures, cutting and compressing, were performed

under ambient conditions using ultrasharp silicon cantilevers

(Silicon-MDT, Moscow, Russia) with nominal spring con-

stants of 14 N/m, and applied forces, roughly on the order

of 1.4-2.8 N, were used. The nanodissection procedure

was performed using the force-volume mode functions of

the Nanoscope III software, version 4.43r8 (Digital Instru-

ments). Fibril relocation, which allowed successive nanoma-

nipulation and imaging to be performed on the same single

fibril, was enabled by using an indexed TEM grid underneath

the mica substrate.12

Acknowledgment. We acknowledge the support of

NSERC and the Materials and Manufacturing Ontario

Emerging Materials Knowledge fund. We thank Dr. Rich

McAloney for helpful AFM advice. C.K.W. thanks NSERC

for a PGSA fellowship.

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L'Ancien d'Algérie, May 2011