NANO LETTERS
AFM Nanodissection Reveals Internal Structural Details of Single Collagen Fibrils
2004 Vol. 4, No. 1 129-132
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 subnanometer 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 AFM’s 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 visualization 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 unfolded,4 antibody-antigen bonds physically disrupted,5 protein conformations physically modulated,6 and individual DNA molecules7 intentionally sliced. The AFM has allowed * Corresponding author. Tel: +1-416-978-6254; fax: +1-416-978-4526. E-mail:
[email protected]. 10.1021/nl034685n CCC: $27.50 Published on Web 11/20/2003
© 2004 American Chemical Society
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 manipulations, the exterior layers of a specific segment on the fibril were initially cut and then peeled away. This dissection technique exposed the fibril’s 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 structure’s 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 group’s 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 unaddressed. 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 fibril’s 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.
Figure 3. A deflection mode image of the same fibril in Figures 1 and 2 after compression. Area (1) marks the well-defined opened segment of the fibril; area (2) marks the location where compression took place. The scale bar represents 1 µm.
Figure 1. 3D height image plot of a FLS-type collagen fibril with a diameter of ∼210 nm prior to cutting. The characteristic ∼270 nm periodic cross-striated pattern is evident. Scale bar represents 1 µm in the x-y plane.
Figure 4. A 3D height image plot of the dissected area is presented. A hierarchical macromolecular architecture is observed, consisting of parallel-aligned fibrillar subcomponents that respectively maintain the characteristic FLS cross-striation pattern; the banding of each fibrillar subcomponent lies in register with that of its neighbors’. The scale bar represents 500 nm.
Figure 2. The depicted incision, marked by the dotted line (1), is ∼140 nm deep into the fibril. Making a set of successive indentations into the fibril produced the cut. Changing the force by which the indentations are made controls their depth. This is illustrated in the boxed area marked (2). The inset shows a 2× magnification of this area; lower indentation forces resulted in shallow penetrations (of 1-2 nm deep only). The scale bar represents 1 µm in the x-y plane. This is a 3D height image plot of the same fibril depicted in Figure 1. The inset is a deflection mode image.
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 130
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 pulled toward 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 fibril’s 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 Nano Lett., Vol. 4, No. 1, 2004
Figure 5. (A) Depicts the tip moving downward toward a fibril. (B) Depicts the lines of tensile stress that would be expected to be induced in the fibril during compression by the cantilever tip.
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 advantageous 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 nanodissection 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 fibril’s 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 Nano Lett., Vol. 4, No. 1, 2004
Figure 6. Deflection mode images of FLS type collagen fibril before (A) and after (B) scraping with the AFM probe. The box in (A) represents the area of scraping. Extensive damage is done to the fibril construct. The arrow in (B) points to an area of the fibril that appears to have been pulled away from its original arrangement during the scraping process. The scale bar represents 1 µm.
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. Unfortunately, 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, such frictional 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 visualization.8 In that work, observations are typically focused on the fibril’s 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 FLS131
type fibril’s 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 sizescale, 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 progressively 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 structure’s 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 are developed 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 µL 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 Instruments, Santa Barbara, CA). Images were captured in contactmode operation under ambient conditions and using square pyramidal silicon nitride cantilevers with nominal spring constants of 0.12 N/m (Digital Instruments). All nanodissection procedures, cutting and compressing, were performed under ambient conditions using ultrasharp silicon cantilevers (Silicon-MDT, Moscow, Russia) with nominal spring constants of 14 N/m, and applied forces, roughly on the order of 1.4-2.8 µN, were used. The nanodissection procedure was performed using the force-volume mode functions of the Nanoscope III software, version 4.43r8 (Digital Instru-
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ments). Fibril relocation, which allowed successive nanomanipulation 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. References (1) Binnig, G.; Quate, C. F.; Gerber, C. Atomic Force Microscope. Phys. ReV. Lett. 1986, 56, 930-933. (2) Czajkowsky, D. M.; Iwamoto, H.; Shao, Z. F. Atomic force microscopy in structural biology: From the subcellular to the submolecular. J. Electron Microsc. 2000, 49(3), 395-406. (3) Fotiadis, D.; Scheuring, S.; Mu¨ller, S. A.; Engel, A.; Mu¨ller, D. J. Imaging and manipulation of biological structures with the AFM. Micron 2002, 33, 385-397. (4) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 1997, 276, 1109-1112. Fisher, T. E.; Marszalek, P. E.; Fernandez, J. M. Stretching single molecules into novel conformations using the atomic force microscope. Nature Struct. Biol. 2000, 7(9), 719-724. (5) Mu¨ller, D. J.; Schoenenberger, C. A.; Bu¨ldt, G.; Engel, A. Immunoatomic force microscopy of purple membrane. Biophys. J. 1996, 70, 1796-1802. (6) Mu¨ller, D. J.; Bu¨ldt, G.; Engel, A. Force-induced conformational change of bacteriorhodopsin. J. Mol. Biol. 1995, 249, 239-243. (7) Hansma, H. G.; Vesenka, J.; Siegerist, C.; Kelderman, G.; Morrett, H.; Sinsheimer, R. L.; Elings, V.; Bustamante, C.; Hansma, P. K. Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science 1992, 256, 1180-1184. Henderson, E. Imaging and nanodissection of individual supercoiled plasmids by atomic force microscopy. Nucleic Acids Res. 1992, 20, 445-447. (8) Paige, M. F.; Rainey, J. K.; Goh, M. C. Fibrous Long Spacing Collagen Ultrastructure Elucidated by Atomic Force Microscopy. Biophys. J. 1998, 74, 3211-3216. Paige, M. F.; Rainey, J. K.; Goh, M. C. A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy. Micron 2001, 32, 341-353. (9) Thalhammer, S.; Stark, R. W.; Mu¨ller, S.; Wienberg, J.; Heckl, W. M. The atomic force microscope as a new microdissecting tool for the generation of genetic probes. J. Struct. Biol. 1997, 119, 232237. (10) Doyle, B. B.; Hukins, D. W. L.; Hulmes, D. J. S.; Miller, A.; Woodhead-Galloway, J. Collagen Polymorphism: Its Origins in the Amino Acid Sequence. J. Mol. Biol. 1975, 91, 79-99. Chapman, J. A.; Armitage, P. M. An analysis of Fibrous Long Spacing Forms of Collagen. ConnectiVe Tissue Res. 1972, 1, 31-37. (11) Rainey, J. K.; Wen, C. K.; Goh, M. C. Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy. Matrix Biol. 2002, 21, 647-660. (12) Markiewicz, P.; Goh, M. C. Identifying locations on a substrate for the repeated positioning of AFM samples. Ultramicroscopy 1997, 68, 215-221.
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