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ARTICLE IN PRESSG ModelMIC-1760; No. of Pages 9

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Contents lists available at SciVerse ScienceDirect

Micron

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canning ion conductance microscopy for imaging biological samples in liquid: comparative study with atomic force microscopy and scanning electronicroscopy

atsuo Ushikia,∗, Masato Nakajimaa, MyungHoon Choib, Sang-Joon Chob, Futoshi Iwatac

Division of Microscopic Anatomy, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8510, JapanPark Systems Corp., KANC 4F, Iui-Dong, 906-10, Suwon 443-270, Republic of KoreaDepartment of Mechanical Engineering, Faculty of Engineering, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan

r t i c l e i n f o

rticle history:eceived 24 December 2011eceived in revised form 25 January 2012ccepted 25 January 2012

eywords:canning ion conductance microscopytomic force microscopyollagen fibrilshromosomeseLa cells

a b s t r a c t

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM)for imaging different types of biological samples. For this purpose, we first applied SICM to image collagenfibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such sampleswith steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hoppingSICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolutionunder liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15 �m in height byARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellularprojections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demon-strated its applicability to study the morphological dynamics associated with biological processes on thetime scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surfaceof the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICMimages were comparable with those obtained by scanning electron microscopy. Although the dynamic
mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only thelatter can obtain contact-free images of samples with steep slopes, rendering it an important tool forobserving live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together,we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique withunique capabilities for imaging the three-dimensional topography of a range of biological samples under
queo
physiologically relevant a
. Introduction

Scanning probe microscopy (SPM) refers to a family oficroscopy techniques that scan a probing tip over a sample sur-

ace to provide information about the local characteristics of solidamples. Among various SPM techniques, atomic force microscopyAFM) has been widely utilized for biological studies since its inven-ion (Binnig et al., 1986), because it can obtain three-dimensional

Please cite this article in press as: Ushiki, T., et al., Scanning ion condcomparative study with atomic force microscopy and scanning electro

nformation on sample topography at resolutions ranging fromhe micrometer to nanometer scales (Casuso et al., 2011; Allisont al., 2010; Ushiki and Kawabata, 2008; Hörber and Miles, 2007;

Abbreviations: AC mode, alternating current mode; AFM, atomic forceicroscopy; ARS/hopping mode, approach-retract scanning, or hopping mode; SEM,

canning electron microscopy; SICM, scanning ion conductance microscopy; SPM,canning probe microscopy.∗ Corresponding author.

E-mail address: [email protected] (T. Ushiki).

968-4328/$ – see front matter © 2012 Published by Elsevier Ltd.oi:10.1016/j.micron.2012.01.012

us conditions.© 2012 Published by Elsevier Ltd.

Ushiki et al., 1996). In particular, since the AFM works in vari-ous environments (vacuum, air and liquid), many biologists havebeen interested in imaging soft, biological samples in physiologi-cally relevant aqueous conditions (Braet et al., 2001; Le Grimellecet al., 1998; Schoenenberger and Hoh, 1994; Butt et al., 1990). Wehave also utilized AFM to characterize various biological samplessuch as cultured cells and chromosomes (Ushiki et al., 2008, 2000,1999; Hoshi et al., 2006, 2004). Furthermore, recent advances inAFM have enabled high-speed imaging of soft samples (Picco et al.,2008, 2007; Ando et al., 2001), allowing the direct visualization ofbiomolecules and their movement in liquid conditions (Uchihashiand Ando, 2011; Kodera et al., 2010). However, it has remaineddifficult to image the nanoscale topography of larger meso- andmicroscale biological samples by AFM, because there may be arti-facts due to the force between the tip and sample, as well as other

uctance microscopy for imaging biological samples in liquid: An microscopy. Micron (2012), doi:10.1016/j.micron.2012.01.012

factors such as tip geometry and lateral forces (Zhang et al., 2011;Braet et al., 2001).

To overcome these technical difficulties, some previous inves-tigators have been interested in scanning ion conductance

dx.doi.org/10.1016/j.micron.2012.01.012


http://www.sciencedirect.com/science/journal/09684328

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Fig. 1. Schematic drawing of the operating princ

icroscopy (SICM). SICM is another type of SPM technique thatas first introduced by Hansma et al. (1989), and is based on a glassicropipette that serves as a sensitive probe. The signal is modu-

ated by an ion current that flows between an electrode locatedithin the pipette and a bath electrode for feedback control of


he pipette-sample distance (Fig. 1). The distance is maintainedt the radius of the pipette during scanning, which allows non-ontact (or contact-free) imaging of the sample topography underiquid conditions. Biological application of SICM was first reported

ig. 2. Comparison of SICM imaging in AC and ARS/hopping modes. Collagen fibrils werhen in ARS/hopping mode (above right). Arrows indicate corresponding structures of thendicated in the ARS/hopping mode image.

f scanning ion conductance microscopy (SICM).

by Korchev et al. (1997), who observed live cultured cells underliquid conditions. Since then, SICM has been used to image thesurfaces of different cultured cells such as neurons and cardiomy-ocytes (Gorelik et al., 2006, 2004; Happel et al., 2003; Pastré et al.,2001; Korchev et al., 2000). Recent advances in SICM have also


provided a hopping mode (also called a backstep mode or approach-retract scanning mode), in which the pipette is moved vertically andrepeatedly approaches and retracts from the sample surface (Novaket al., 2009; Happel and Dietzel, 2009). This mode was especially

e attached on the glass slide and observed with SICM in AC mode (above left) and AC and ARS/hopping mode images. Line profile analysis is based on the broken line



T. Ushiki et al. / Micron xxx (2012) xxx–xxx 3

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ig. 3. Comparison of SICM and AFM imaging capabilities under liquid conditions.

ICM (upper right image). The section profiles 1 and 2 are based on the lines labele

seful for non-contact imaging of the cell surface of cultured ratippocampal neurons with a resolution better than 20 nm (Novakt al., 2009; Klenerman et al., 2011). These studies provided us withhe idea that SICM can be widely used not only for imaging culturedells but also for various other biological samples at resolutionsomparable to that of AFM under liquid conditions.

In the present study, we introduce our results on SICM imagingf different biological samples and demonstrate the technique’sxcellent imaging capabilities by comparative analysis of SICMmages with those obtained by AFM and/or scanning electron

icroscopy (SEM).

. Instrumentation

.1. Scanning ion-conductance microscopy (SICM)

SICM images were obtained with a commercial SPM (XE-Bio sys-em, Park Systems Corp, Suwon, Korea). This system has an SPMystem on the stage of an inverted optical microscope (Eclipse Ti,ikon Corp., Tokyo, Japan). The SPM system has an xy flat scan-er (100 �m × 100 �m) and a separate SICM or AFM head with

25 �m z scanner. The SICM probe consists of a glass pipette


lled with electrolyte with an Ag/AgCl electrode plugged intot. The glass pipette was fabricated from borosilicate capillar-es (inner diameter 0.6 mm, outer diameter 1.0 mm, Narishige,okyo, Japan) using a CO2-laser-based micropipette puller (P-2000,

en fibrils on a glass slide were first imaged by AFM (upper left image), and then byfile 1” and “profile 2” in the upper two images.

Sutter Instruments, Novato, CA, USA); the inner and outer diame-ters of the pipette were about 100 nm and 200 nm, respectively.There are several operating modes for SICM. In the direct current(DC) mode, DC ion current is recorded during scanning, while, inthe alternating current (AC) mode, the amplitude of the AC ion cur-rent modulated by oscillating the pipette probe is detected by usinga lock-in amplifier (Pastré et al., 2001). In the hopping or backstepmode, ion current is recorded while the pipette is moved verticallyand repeatedly approaches and retracts from the sample surface(Novak et al., 2009; Happel and Dietzel, 2009). Thus, this mode isalso referred to as approach-retract scanning (ARS) mode. In thepresent study, images were primarily obtained in the ARS/hoppingmode, although the AC mode was also used in some cases. In theARS/hopping mode, the probe approached towards an insulatingsurface until the resistance exceeded a predefined threshold. Aresistance increase of 2% with respect to the basal resistance wasused in this experiment to stop the approach.

2.2. Atomic force microscopy (AFM)

In some cases, AFM images were obtained with the same SPMplatform after the head was exchanged for AFM imaging. This


microscope was operated in dynamic mode in liquid conditions.Reduction of the oscillation amplitude was used as the feedbackparameter by a slope detection technique. The cantilevers usedwere DNP-S (triangle cantilevers with a nominal spring constant of


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Fig. 4. Dense collagen fibril networks imaged by the ARS/hopping SICM mode. Line

ARTICLEMIC-1760; No. of Pages 9

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.32 N/m and resonance frequency of about 56 kHz, Veeco, Metrol-gy, Santa Barbara, CA, USA) or PNP-TR (triangle cantilever with aominal spring constant of 0.32 N/m and resonance frequency ofbout 67 kHz, Nanotools, München, Germany).

.3. Scanning electron microscopy (SEM)

After SICM imaging, some of the specimens were prepared forEM imaging. Briefly, the samples were made electron-conductivey staining with tannic acid and osmium based on the method ofurakami (1973). They were then dehydrated with an ascending

thanol series, transferred to isoamyl acetate, and dried in a criticaloint dryer using CO2 (HCP-2, Hitachi High-technologies, Tokyo,

apan). This was followed by mounting on aluminum stubs, coatingith platinum–palladium in an ion coater, and observation in a field

mission SEM (S-4300SE/N, Hitachi High-technologies,).

. Results and discussion

.1. Collagen fibrils

As AFM imaging of collagen fibrils has been reported by sev-ral investigators (Yamamoto et al., 2002, 2000, 1997; Baselt et al.,993), we attempted to examine the applicability of SICM for imag-

ng collagen fibrils. For this purpose, collagen fibrils were obtainedrom the tail tendon of adult Wistar rats (Japan SLC, Inc., Hama-

atsu, Japan) and stored in physiological saline with 1–10% tymol2-isopropyl-5-methylphenol) at 4 ◦C for a minimum of 1 day. Amall piece of the tendon was then stretched on the glass surfacend air dried overnight, followed by immersion of the sample inhysiological saline or phosphate buffer saline (PBS) before AFMnd/or SICM imaging.

At first, we compared the image quality obtained by SICM imag-ng in AC mode versus ARS/hopping mode. Fig. 2 is an example of theomparison images of the two modes, in which AC mode imagingreceded the ARS/hopping mode imaging for this sample. In the ACode, no collagen fibril was imaged in this portion of the sample,hereas there was a collagen fibril, about 530 nm in height, in theRS/hopping mode. These two images indicate that the collagenbrils likely moved away during scanning in AC mode as a result ofbril deformation by the probing pipette, but were well-visualized

n the ARS/hopping mode. On the other hand, spatial resolution isreater in the AC mode than in the ARS/hopping mode in our exper-ment, because structures in the range of 50–60 nm were clearlyisualized only in AC mode.

Next, we compared the dynamic mode of AFM with theRS/hopping mode of SICM. Fig. 3 is an example of AFM and SICM

mages on the same collagen fibrils. In these images, the heightf the collagen fibrils was 1.4 times greater in the SICM imagehan in the AFM image, suggesting that SICM is better suited foron-contact imaging than AFM; the height of the collagen fibrilsre underestimated by AFM (here by about 30%, but this differ-nce may depend on the AFM imaging force) compared with thosebtained by SICM. On the other hand, the width of collagen fib-ils was 0.7 times smaller in the SICM image than that of the AFMmage. Because collagen fibrils are cylindrical, the width is likelynfluenced by the shape of the probing chip. Our finding suggestshat the shape of the collagen fibrils is well-delineated in the SICMmage even though the inner radius of the pipette (about 100 nm)s much larger than the radius of the AFM probing tip. In the AFMmage, distortions are inherent due to the effect of a convolution


etween the pyramidal tip and sample (Meyer et al., 2004; Baseltt al., 1993).

To examine the applicability of the ARS/hopping mode for imag-ng biological samples with greater height gaps, dense collagen

profile analysis is based on the red line indicated in the SICM image. The width ofthe collagen fibril, as indicated by the two white arrowheads, is 328 nm. The heightgap between the top of the collagen fibril and the substrate is 1.549 �m.

fibril networks were examined by SICM. Fig. 4 is an example ofan SICM image of collagen fibril networks. The width of the indi-vidual fibrils varied from 50 to 470 nm. The section profile alsoshowed that the height difference between the glass surface andfibril (328 nm thickness), as indicated in Fig. 4, was 1.549 �m,suggesting that some fibrils were suspended over the glasssubstrate.

Because collagen fibrils with a width of 50 nm can be observedwith SICM imaging, the horizontal resolution is estimated to bein the range of 50 nm. However, grooves produced by D periodic-ity (60–67 nm) in the collagen fibrils were faint in SICM images,probably because the depth of the groove (2 nm or less) in liquidenvironment is beyond the vertical resolution of the pipette usedfor this SICM experiment.

3.2. Chromosomes

The higher order structure of chromosomes has been widelystudied by transmission and scanning electron microscopy, andrecently by AFM (Daban, 2011; Ushiki and Hoshi, 2008; Ushikiet al., 2002; de Grooth and Putman, 1992; Summer, 1991; Earnshawand Laemmli, 1983). The advantage of AFM is its ability toimage at high resolution in a liquid environment, which is alsoexpected by SICM. Thus, we applied SICM to imaging of chro-mosomes. Chromosomes of human lymphocytes were preparedaccording to the standard air-drying method for light microscopy


(Ushiki and Hoshi, 2008). Briefly, human lymphocytes after cul-tivation were arrested in metaphase by adding colcemid to theculture medium at a final concentration of 0.05 �g/ml for 1 h,exposed to 75 mM KCl for 30 min at room temperature and fixed


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ith Carnoy’s solution (methanol: acetic acid = 3:1, v/v). Chromo-ome spreads were then formed by dropping the cell suspensionnto glass slides, briefly dried in air in order to fix them ontohe glass slides. They were immersed in PBS and observed byICM and AFM.

Fig. 5 shows the SICM image of human chromosomes in the liq-id environment. The image quality appears qualitatively similaro that obtained previously by the dynamic mode of AFM (Ushikind Hoshi, 2008; Hoshi et al., 2006, 2004). In order to comparehe quality of AFM and SICM images more precisely, chromo-omes were imaged first by the dynamic mode of AFM, then by theRS/hopping mode of SICM and again by AFM. In Fig. 6, the heightrofiles obtained by sequential AFM, SICM and AFM measurementsere 473, 746 and 375 nm, respectively. These findings indicate

hat the height of the chromosome determined by SICM (c.a.50 nm) is much greater than that of AFM (roughly 400–500 nmepending on the AFM imaging force). At higher magnification,lobular or fibrous structures about 50 nm were observed by SICMmicrograph not presented), suggesting that SICM is applicable fortudying higher order structures of very soft chromosomes in liquidnvironments.


.3. Cultured cells

SICM imaging of cultured cells was first reported by Korchevt al. (1997). Since then, multiple studies have reported SICM

ig. 6. Comparison of AFM and SICM images on the same human chromosomes. Chromond finally by AFM again. Line profile analysis is based on the red line indicated in the cor

Fig. 5. Human chromosomes imaged by the ARS/hopping SICM mode.


imaging of cultured cells (Gorelik et al., 2004; Rheinlaender et al.,2011). These previous findings indicate that SICM is very useful forimaging the surface topography of living and fixed cultured cells atnanoscale resolution under liquid. To examine the image quality of

somes immersed in an aqueous solution were first imaged by AFM, then by SICM,responding image.




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chea. This image shows that both the ciliated and non-ciliated cellscan be imaged by SICM, even though the height difference is at least8 �m in this sample. The short cellular projections are clearly dis-cernible on the surface of non-ciliated cells. The thickness of cilia

ig. 7. HeLa cells imaged by the ARS/hopping SICM mode. The sample was fixed

ndicated in the upper right image.

ICM for cultured cells, fixed and live HeLa cells were imaged byRS/hopping SICM in this study.

At first, HeLa cells were grown on cover slips in culture dishes50 mm in diameter) for 24–48 h in a CO2 incubator at 38 ◦C, fixedith 1% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4

or overnight at 4 ◦C, and observed by SICM. The height of HeLaells was estimated to be about 15 �m in the SICM image of Fig. 7.icroprojections on the cell surface were visualized on the cell sur-

ace, suggesting that the ARS/hopping mode of SICM can obtainigh-resolution topographic image of cultured cells with mini-al deformation in liquid conditions. By contrast, the steep slope

f the cell morphology renders it difficult to observe such softtructures by AFM. Fig. 8 is a higher magnification image of theeLa cell surface, in which microprojections with various irregular

hapes are observed on the cell surface; the height and thick-ess of these projections are roughly 0.5–1 �m and 100–150 nm,espectively.

We further tried to obtain SICM images of live cells. Fig. 9 showsequential time-lapsed SICM images of live HeLa cells. The datacquisition time per image was about 10 min in Fig. 9, but weound that the time can be reduced to about 6 min when 128 by28 pixel images were appropriately collected with our instrument.he time resolution is thus comparable with AFM imaging of livingells (Ushiki et al., 1996, 2000), even though high-speed AFM imag-ng is also introduced for specimens such as chromosomes (Piccot al., 2008). In addition, the ARS/hopping mode has the advantagef minimizing deformation of cellular structures by the probingip compared with AFM. Thus, the ARS/hopping mode of SICM isxpected to be widely useful for live cell imaging, especially inelation to the movement of cellular processes on a time scale ofinutes under liquid conditions.

.4. Bulky tissues and tissue blocks


Because the ARS/hopping mode of SICM can obtain images ofamples with surface irregularities, we examined the applicabilityf SICM on a variety of samples, including the imaging of bulky

1% glutaraldehyde before SICM imaging. Line profile analysis is based on the line

tissue blocks with irregular surfaces. For this purpose, tracheawas obtained from Wistar rats, which were transcardially fixedwith 2% glutaraldehyde in PB. A small piece of the trachea wasthen mounted on the glass slide with an adhesive (Aron Alphacyanoacrylate instant adhesives, Toagosei Co. Ltd., Tokyo, Japan)and observed by the ARS/hopping mode of SICM.

Fig. 10 shows an SICM image of the luminal surface of the tra-


Fig. 8. Projections on the surface of HeLa cell. Lamellar projections with a rangeof topographies are clearly observed on the surface of the HeLa cell. Image is amagnified view of Fig. 7.



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ig. 9. Repeated observation of live HeLa cells by the ARS/hopping SICM mode. Therocesses indicated by arrows and arrowheads.

s about 100–150 nm in the SICM image. To compare the SICM and


EM images, the samples were observed by SICM followed by obser-ation of the same portion by SEM after sample preparation. Fig. 11resents SICM and SEM images of the same portion of the trachea.hese images indicate that SICM images are comparable with SEM

Fig. 10. Luminal surface of rat trachea imaged by the ARS/hopping SICM mode. L

time of each image (128 by 128 pixels) was 10 min. Note the movement of cellular

images, although the thickness of cilia is somewhat smaller in SICM


images (c.a. 100 nm) than in SEM images (c.a. 200 nm). The differ-ence in thickness of cilia between the SICM and AFM images is notclear but it may be partially explained by the drift of cilia duringscanning. Further studies are needed to clarify this point.

ine profile analysis is based on the line indicated in the upper right image.




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ig. 11. Comparison of SICM and SEM images of the luminal surface of rat trachea. Afehydrated, subjected to critical point drying, coated with metal, and observed by S

. Conclusion

The present study shows our results to test the applicability ofICM for imaging different types of biological samples. SICM imag-ng of live and fixed cultured cells has been repeatedly reported,ince Korchev and his colleagues first applied SICM for this purposeRheinlaender et al., 2011; Novak et al., 2009; Gorelik et al., 2004;orchev et al., 2000, 1997). However, there has been no report on

he application of SICM to image other types of biological samples.n this paper, we show for the first time that SICM can be used tomage collagen fibrils and chromosomes as well as cultured cells.

e also introduce the applicability of SICM for studying the surfaceopography of thick tissue samples. Although Novak et al. (2009)eported SICM imaging of auditory hair cells in the cultured organf Corti explants, this is the first report that shows the applicabilityf SICM for imaging tissue blocks with irregular surfaces.

Comparison of SICM with AFM images also revealed that SICMan be utilized for obtaining contact-free images of the surfaceopography of biological samples. The ARS/hopping mode of SICM


s especially powerful for imaging samples with steep slopesNovak et al., 2009). We showed that the lateral resolution of theRS/hopping SICM mode is about 50 nm, which is comparable to

he finding by previous investigators (Novak et al., 2009; Happel

M imaging by the ARS/hopping mode, the samples were made electron-conductive,

and Dietzel, 2009). However, the actual resolution of SICM imagingis still a matter of debate (Rheinlaender and Schaffer, 2009). Theo-retical studies of SICM will be required to consider the resolution inrelation to the pipette radius and other parameters, although thereare a few papers already in this field (Edwards et al., 2009).

While it may be true that image resolution is better in thedynamic mode of AFM than in the ARS/hopping mode of SICMin some samples, only the latter can obtain contact-free images.As such, SICM is an excellent tool for observing live cells andunfixed soft samples. Taken together, SICM imaging, especially byARS/hopping mode, is expected to be useful for obtaining imageson the three-dimensional topography of biological samples.

Acknowledgements

This study was supported in part by Grant-in-Aid for ScientificResearch (B) (no. 21390051 for TU) by Japan Society for the Promo-tion of Science (JSPS). The part of development of instrument wasalso sponsored by the Industrial Source Technology Development


Programs (ISTDP10033633) in Ministry of Knowledge Economy inKorea. HeLa cells were kindly provided by Dr. Hirota, Department ofExperimental Pathology, Cancer Institute of the Japanese Founda-tion for Cancer Research, Tokyo, Japan The authors are also grateful


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