Research ArticleApplication of Environmental Scanning ElectronMicroscope-Nanomanipulation System onSpheroplast Yeast Cells Surface Observation

Maryam Alsadat Rad1 Mohd Ridzuan Ahmad1 Masahiro Nakajima2 Seiji Kojima3

Michio Homma3 and Toshio Fukuda2

1Department of Control and Mechatronic Engineering Faculty of Electrical Engineering Universiti Teknologi Malaysia81310 Skudai Johor Malaysia2Department of Micro-Nano Systems Engineering Nagoya University Nagoya Japan3Division of Biological Science Graduate School of Science Nagoya University Nagoya Japan

Correspondence should be addressed to Mohd Ridzuan Ahmad ridzuanfkeutmmy

Received 17 February 2017 Accepted 29 March 2017 Published 27 April 2017

Academic Editor Renato Buzio

Copyright copy 2017 Maryam Alsadat Rad et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope(ESEM) The spheroplasting conversion was successfully confirmed qualitatively by the evaluation of the morphological changebetween the normal W303 cells and the spheroplast W303 cells and quantitatively by determining the spheroplast conversionpercentage based on the OD

800absorbance data From the optical microscope observations as expected the normal cells had an

oval shape whereas spheroplast cells resemble a spherical shape This was also confirmed under four different mediums that isyeast peptone-dextrose (YPD) sterile water sorbitol-EDTA-sodium citrate buffer (SCE) and sorbitol-Tris-Hcl-CaCl

2(CaS) It was

also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile watermediums The OD

800absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after

about 15 minutes The observations of the normal and the spheroplast W303 cells were then performed under an environmentalscanning electron microscope (ESEM)The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-likesurface after the loss of its integrity when removing the cell wall

1 Introduction

Over the years traditional scanning electron microscope(SEM)made a lot of contributions in the imaging ofmaterialswith a detailed description of their structure and surfaces [1ndash4] However the need for high vacuum operating environ-ment and complex sample preparation steps made the appli-cation on biological samples and specimen unfavorable [5ndash7] Environmental Scanning Electron Microscope (ESEM)has overcome many of the drawbacks found in SEM andintroduced a new advantage in biological research [8ndash11]Theability of this tool to operate in wet and gaseous atmospheresmade the nonconductive samples conductive thus overcom-ing the need for coating samples prior to characterization and

preserving samples original features for further testing andmanipulation [12ndash14] In addition the capability to controlwater vapor pressure inside the microscope while operatingkeeps the samples hydrated which increases the chances ofsurvival [15 16]

One of the earliest experiments on biological specimenswas done by Collins et al [17]They explained the applicationand advantages of using ESEM on microorganisms ESEMhas found its potentials in tissue engineering and biomaterialsstudies because it supports the observation of cell and theirtopography in hydrated atmospheres [18] Furthermore thereduced sample preparation steps are useful for investigatingmammalian cells and biomaterial interactions [5 19ndash23] Kirket al have imagedmammalian cells using ESEMshowing very

HindawiScanningVolume 2017 Article ID 8393578 7 pageshttpsdoiorg10115520178393578

2 Scanning

Unit 2 Unit 3(Cooling Stage)

Unit 1

1 cm

Z

X

Y

(X-Y-Z-)

(X-Y-Z)

Figure 1 Nanorobotic manipulator of ESEM

fine detail of delicate features such as filopodia andmembraneruffles [24]They also showed that the cells survived the initialstages of sample preparation but experienced some damageduring the dehydration stage They have suggested cells withstronger cell wall could be imaged in their living states

One of the main criteria of ESEM is the real life obser-vation and monitoring of biological cells This could be anadvantage by integrating tools such as nanomanipulatorsor patch clamp to analyze cells and their characteristicswhile performing experiments From our previous work ananomanipulator was successfully incorporated inside ESEM(Figure 1) for various single cell manipulation and character-ization [25 26]

One potential application of ESEM is to study the ionchannel current measurement of cells by combining it witha patch-clamp system The conventional planar patch-clampsystem requires a bath solution to perform the measurementHowever this could result in having come errors in themeasurement because of the differences between the bathsolution chemical properties and the cytoplasm inside the cell[27] To overcome that instead of using the bath solution theelectrodes could be injected into the cell and the cytoplasmcould be used as the medium for the current to flow duringthe current recording Spheroplasting is one of the early stepsrequired in the ion channel measurements experiments [28]Thus in order to perform the patch-clamp experiment insideESEM it is important to confirm the ability of ESEM toobserve spheroplast cells successfully before further manip-ulation could be carried out

Nanomanipulation is an effective strategy for the charac-terization of basic properties of individual nanoscale objectsand to construct nanoscale devices quickly and effectivelyWehave constructed a hybrid nanorobotic manipulation systemintegrated with a transmission electron microscope- (TEM-)nanoroboticmanipulator (TEMmanipulator) and a scanningelectronmicroscope- (SEM-) nanoroboticmanipulator (SEMmanipulator) [29]This system allows effective sample prepa-ration inside SEM with wide working area and many degreesof freedom (DOFs) of manipulation It has high resolution

measurement and evaluation of samples inside a TEM capa-bility The sample chambers of these electron microscopesare set under the high vacuum (HV) condition to reducethe disturbance of electron beam for observation To observethe water-containing samples for example biocells dryingtreatment processes are additionally needed Hence directobservations of water-containing samples are normally quitedifficult in these electron microscopes

In the present study we used the nanorobotic manipu-lators inside an ESEM [30] It has been constructed with 3units and 7 degrees of freedom (DOFs) in total (Figure 1)The ESEM enables direct observation of water-containingsamples with nanometer high resolution by a specially builtsecondly electron detector The evaporation of water iscontrolled by both the sample temperature (0ndash40∘C) andsample chamber pressure (10minus2600 Pa) The temperature ofthe sample is controlled by the cooling stage unit (Unit3) The detailed specifications of the manipulator and theESEM can be obtained from our previous paper [31] Thefollowing experiments have been conducted through thissystem The observation and comparison of W303 cells andspheroplast W303 were successfully performed using ESEMSpheroplastW303 cells were obtained by enzymatic digestionwhich were confirmed qualitatively by the comparison ofcell morphology between the cells and spheroplast cells andquantitatively by the OD

800absorbance data The successful

observation of spheroplast cells opens the possibility for anew way in the single ion channel current measurement

2 Materials and Methods

21 Cell Cultures Wild type yeast cells (W303 strains) wereused for the observations and measurements under opticalmicroscope and ESEM systemTheW303 cells were culturedon aYPDplate (1 yeast extract 2peptone 2 glucose and2 agar) in a 37∘C incubator for 48 hours A single colonywas then picked from the cultured plate and then dipped intoa tube containing 10mL of YPD media The tube was thenincubated overnight in 30∘C at 200 rpm The OD600 valuesof the samples were measured by a spectrophotometer andsamples that had OD600 values between 02 and 03 wereused for spheroplasting

22 Preparation of W303 Spheroplasts Spheroplasts wereprepared using Pichia spheroplast kit In brief logarithmicgrowingW303 wild type yeast cells (OD

600value between 02

and 03 in 1mL of culture) were harvested by centrifugationat 6000 rpm for 5 minutes at 30∘C and then washed with1mL of sterile water Cells were pelleted by centrifugation at6000 rpm for 5 minutes at 30∘C

The cell pellets were washed by resuspending in 1mL ofSED buffer containing 1M sorbitol 25mM EDTA pH 80and 1M dithiothreitol and then centrifuged at 6000 rpm for5 minutes at 30∘C The cells then were washed with 1mL of1M sorbitol and centrifuged at 6000 rpm for 5 minutes at30∘CThen theywere resuspended by swirling in 1mL of SCEmedium A 3 120583L of cell wall hydrolyzing enzyme Zymolyasewas added to the cells The cells were then incubated at 30∘C

Scanning 3

(c)(b)(a)

(d)(e)(f)

Figure 2 Schematic diagram illustration of the two phases in spheroplast formation (a)ndash(d) Cell turns into prospheroplastThe appearanceof spheroplast can be seen when the prospheroplast changes into a spherical shape ((e) (f))

for about 1 hour The spheroplasts were washed with 1mL of1M sorbitol and collected by centrifugation at 6000 rpm for 5minutes at 30∘CThey were then resuspended in 1mL of CaS

A schematic diagram illustrating the spheroplast conver-sion is presented in Figure 2 In the first phase theW303 cellswere transformed into prospheroplasts The prospheroplastswere extruded at one end where partial digestion of the cellwall occurred (Figures 2(a)ndash2(d)) retaining its shape despitethe apparent loss of a supporting wall In the second phasethe prospheroplasts rapidly transformed into spheroplastswhere a spherical shape became dominant (Figures 2(e) and2(f))

23 W303 Spheroplast Observation by an Optical MicroscopeThe spheroplast cells were observed in four different medi-ums YPD sterile water SCE and CaS using Olympus IX-71optical microscope at room temperature (20ndash25∘C) in 100timesoil immersion objective lens The initial 1mL spheroplastcells inside CaS medium were aliquoted into four microcen-trifuge tubes The first aliquot was the spheroplasts insideCaS medium The other three aliquots were centrifuged at6000 rpm for 5 minutes in 30∘C and the CaS supernatant wasdiscarded completely The spheroplast pellets inside each ofthe three tubes were then resuspended with 250 120583L of YPDmedium 250 120583L of sterile water and 250120583L of SCE mediumrespectively

24 W303 Spheroplasts Observations by ESEM The ESEMsystem can perform direct observation of water-containingsamples with nanometer high resolution by specially builtsecondary electron detectorThe evaporation of water is con-trolled by adjusting the samplersquos temperature (sim0ndashsim40∘C) andsamplersquos chamber pressure (10ndash2600 Pa) The temperature ofthe sample is controlled by the cooling stage unit that isUnit3 as shown in Figure 1 ESEM system has a capability tocontrol the chamberrsquos pressure from high vacuum (sim10minus4 Pa)to high humidity (10ndash2600 Pa) The detailed specifications ofthe manipulator and the ESEM can be obtained from ourprevious paper [26] The nanomanipulator has a tungstenprobe which has been used to transfer the single cell usingthe adhesion force This force is produced between the microprobe and cell In fact the nanomanipulator system cancontrol the position of a single cell

3 Results and Discussion

31 Cells Morphology under Optical Microscope Figures 3and 4 show a comparison between the morphology of W303cells before and after spheroplasting inside four differentmediums YPD sterile water SCE and CaS From theobservations themorphology of theW303 cells inside all fourmediums had an oval shape

YPD medium provided the best living medium for thecells followed by sterile water SCE and CaS mediums(Figures 3(a)ndash3(d)) The reduction in number of the W303cells in SCE and CaS media compared to the YPD (1 yeastextract 2 peptone 2 glucose and 2 agar) is due tothe absence of yeast extract The yeast extract will typicallycontain all the amino acids necessary for growth Figure 4shows the morphology of spheroplast cells inside four thefour mediums It is clear that the spheroplast cells displayeda spherical shape inside all mediums However the conditionand visibility of cells inside YPD and sterile water mediumswere poor as compared to the SCE and CaSmedium (Figures4(a)ndash4(d))The spherical shape for the spheroplast yeast cellswas also reported by [32]

32 Spheroplast Conversion Percentage Based on the OD800Absorbance Data The percentage of the spheroplast cellsconversion can be determined from the following equation

Spheroplast = 100 minus [( (OD8001003816100381610038161003816time=119905)(OD

800

1003816100381610038161003816time=0)) times 100] (1)

Figure 5 shows the values of OD800

absorbance data for50 minutes and its corresponding spheroplast conversionpercentage From the graph it is shown that the cells rapidlychanged into spheroplast cells 85 of the cells convertedinto spheroplasts in 2 minutes after the addition of thedigestion enzyme The whole W303 cells completely adaptedthe spheroplast cells conditions in 15 minutes From thisdata it is concluded that the spheroplasting experiment wassuccessfully achieved

33 Surface Characteristic of W303 Cells and SpheroplastW303 Cells under ESEM Figure 6 shows the surface topol-ogy of the cells under ESEM The observations were per-formed at 30 kV and 100120583A The environmental parameters

4 Scanning

(a) (b)

(c) (d)

Figure 3Themorphology of theW303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaS Bar scale is 5 120583m

(a) (b)

(c) (d)

Figure 4 The morphology of the spheroplast W303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaSArrow marks are added to indicate the position of the spheroplast cells inside the first two mediums that is YPD and sterile water Bar scaleis 5 120583m

settings were 600 Pa and 0∘CThewhole cells were kept insidethe YPD medium and diluted with distilled water From theobservation the surfaces of theW303 cells were very smoothas can be seen (top view (Figure 6(a)) side view (Figure 6(b))and closed-up side view (Figure 6(c)))This is also confirmedin previous literature [25 26]

Figure 7 represents the surface topology of the sphero-plast cells under an ESEM For the first time the observations

of spheroplast cells were performed without the need ofcomplex sample preparation and sample coating This willensure the viability of the spheroplast cells that will enablefurther characterization and analysis to be carried out Asexpected the surface topology of the spheroplast cells wasnot smooth as cells having a cell wall The significance ofthe cell wall as a cell shaper was highly acknowledged fromthese observations In addition to the nonsmooth surface

Scanning 5

0

001002

003

004005

006

007008

009

0 10 20 30 40 50Time (minutes)

OD

800

abso

rban

ce0102030405060708090100

Sphe

ropl

astin

g pe

rcen

tage

()

Figure 5 The OD800

absorbance data and its corresponding spheroplast conversion percentage for 50 minutes of treatment using digestionenzyme Zymolyase on W303 cells

4m

(a)

5m

(b)

500nm

(c)

Figure 6The surface topology of theW303 cells under an ESEM from different views (a) top view (b) side view and (c) closed-up side view

4m

(a)

2m

(b)

500nm

(c)

Figure 7The surface topology of the spheroplastW303 cells under an ESEM fromdifferent views (a) top view (b) side view and (c) closed-upside view

topology the spheroplast cells experienced some reductionin size as can be seen in top view image of spheroplast cell(Figure 7(a)) From the side view images (Figures 7(b) and7(c)) it is noticed that the surface had several bleb structures

4 Conclusion

The advantages of the integrated ESEM-nanomanipulationsystem rely on its capability to perform in situ local direct

observation and manipulation of biological sample andthe ability to control the environmental conditions Theobservations of the spheroplast without prior coating foran electron microcopy observation have been highlightedin this work To the best of our knowledge this workis the first to attempt to observe spheroplast cells underelectron microscope without the need of sample coatingThespheroplasting was verified qualitatively and quantitativelyby observing the cellrsquos morphological change under optical

6 Scanning

microscope observations and the spheroplast conversion per-centage based on the OD

800absorbance dataThe spheroplast

cells showed a spherical shape as compared to the oval shapefor the normal cells The electron microscope observationrevealed the bleb-like surface of the spheroplasts as comparedto the very smooth surface of the normal cells This workcould be extended to perform single ion channel currentmeasurements on the spheroplast W303 cells inside theESEM-nanomanipulation system

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The researchwas supported by theMinistry of Higher Educa-tion ofMalaysia (Grant nos 4L640 and 4F351) and UniversitiTeknologi Malaysia (Grant nos 03E11 03G47 02G46 03H82and 03H80) the authors thank them for funding this projectand for their endless support In addition they would liketo thank Professor Toshifumi Inada at Nagoya University forproviding the W

303yeast cell stains

References

[1] M Yu M J Dyer G D Skidmore et al ldquoThree-dimensionalmanipulation of carbon nanotubes under a scanning electronmicroscoperdquo Nanotechnology vol 10 no 3 pp 244ndash252 1999

[2] M A Sutton N Li D C Joy A P Reynolds and X LildquoScanning electron microscopy for quantitative small and largedeformation measurements Part I SEM imaging at magnifica-tions from 200 to 10000rdquo Experimental Mechanics vol 47 no6 pp 775ndash787 2007

[3] J I Goldstein D E Newbury P Echlin et al Scanning ElectronMicroscopy and X-Ray Microanalysis Springer US BostonMass USA 1992

[4] K C A Smith and C W Oatley ldquoThe scanning electronmicroscope and its fields of applicationrdquo British Journal ofApplied Physics vol 6 no 11 article 304 pp 391ndash399 1955

[5] L Muscariello F Rosso G Marino et al ldquoA critical overviewof ESEM applications in the biological fieldrdquo Journal of CellularPhysiology vol 205 no 3 pp 328ndash334 2005

[6] K Hrubanova O Samek A Haronikova et al ldquoMorphologicaland production changes in stressed red yeasts monitored usingsem and raman spectroscopyrdquo Microscopy and Microanalysisvol 22 no S3 pp 1146ndash1147 2016

[7] Y Zhang H Wu X Yu F Chen and J Wu ldquoMicroscopicobservations of the lotus leaf for explaining the outstandingmechanical propertiesrdquo Journal of Bionic Engineering vol 9 no1 pp 84ndash90 2012

[8] L Bergmans P Moisiadis B Van Meerbeek M Quirynen andP Lambrechts ldquoMicroscopic observation of bacteria Reviewhighlighting the use of environmental SEMrdquo InternationalEndodontic Journal vol 38 no 11 pp 775ndash788 2005

[9] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[10] M R Ahmad M Nakajima T Fukuda S Kojima andM Homma ldquoSingle cells electrical characterizations using

nanoprobe via ESEM-nanomanipulator systemrdquo in Proceedingsof the 9th IEEE Conference on Nanotechnology (IEEE NANOrsquo09) pp 589ndash592 Genoa Italy July 2009

[11] Y Shen M Nakajima M R Ahmad T Fukuda S Kojimaand M Homma ldquoSingle cell injection using nano pipette viananorobotic manipulation system inside E-SEMrdquo in Proceed-ings of the 9th IEEEConference onNanotechnology (IEEENANOrsquo09) pp 518ndash521 Genoa Italy July 2009

[12] J M Manero F J Gil E Padros and J A Planell ldquoApplicationsof environmental scanning electron microscopy (ESEM) inbiomaterials fieldrdquo Microscopy Research and Technique vol 61no 5 pp 469ndash480 2003

[13] A M Donald ldquoThe use of environmental scanning electronmicroscopy for imaging wet and insulating materialsrdquo NatureMaterials vol 2 no 8 pp 511ndash516 2003

[14] G D Danilatos ldquoIntroduction to the ESEM instrumentrdquoMicroscopy Research and Technique vol 25 no 5-6 pp 354ndash361 1993

[15] C Gilpin andD C Sigee ldquoX-raymicroanalysis of wet biologicalspecimens in the environmental scanning electron microscope1 Reduction of specimen distance under different atmosphericconditionsrdquo Journal of Microscopy vol 179 no 1 pp 22ndash281995

[16] B Little P Wagner R Ray R Pope and R Scheetz ldquoBiofilmsan ESEM evaluation of artifacts introduced during SEM prepa-rationrdquo Journal of Industrial Microbiology vol 8 no 4 pp 213ndash222 1991

[17] S P Collins R K Pope R W Scheetz R I Ray P A Wagnerand B J Little ldquoAdvantages of environmental scanning electronmicroscopy in studies of microorganismsrdquoMicroscopy Researchand Technique vol 25 no 5-6 pp 398ndash405 1993

[18] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoA study of the spheroplast observations for W303 sin-gle cells under environmental-SEMrdquo in Proceedings of the Inter-national Symposium on Micro-NanoMechatronics and HumanScience MHS 2008 with Symposium on ldquoCOE for Educationand Research of Micro-Nano Mechatronicsrdquo Symposium onldquoSystem Cell Engineering by Multi-scale Manipulationrdquo pp 89ndash92 Nagoya Japan November 2008

[19] M Baguneid D Murray H J Salacinski et al ldquoShear-stresspreconditioning and tissue-engineering-based paradigms forgenerating arterial substitutesrdquo Biotechnology and Applied Bio-chemistry vol 39 no 2 pp 151ndash157 2004

[20] A Motta C Migliaresi F Faccioni P Torricelli M Fini andR Giardino ldquoFibroin hydrogels for biomedical applicationsPreparation characterization and in vitro cell culture studiesrdquoJournal of Biomaterials Science Polymer Edition vol 15 no 7pp 851ndash864 2004

[21] Y Shen M Nakajima M R Ahmad S Kojima M Hommaand T Fukuda ldquoEffect of ambient humidity on the strengthof the adhesion force of single yeast cell inside environmental-SEMrdquo Ultramicroscopy vol 111 no 8 pp 1176ndash1183 2011

[22] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoBuckling nanoneedle for characterizing single cellsmechanics inside environmental SEMrdquo IEEE Transactions onNanotechnology vol 10 no 2 pp 226ndash236 2011

[23] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoMechanical properties characterization of individualyeast cells using environment-SEM nanomanipulation systemrdquoin Proceedings of the IEEERSJ International Conference onIntelligent Robots and Systems (IROS rsquo07) pp 596ndash601 RaleighNC USA November 2007

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

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ThermodynamicsJournal of

Scanning 3

(c)(b)(a)

(d)(e)(f)

Figure 2 Schematic diagram illustration of the two phases in spheroplast formation (a)ndash(d) Cell turns into prospheroplastThe appearanceof spheroplast can be seen when the prospheroplast changes into a spherical shape ((e) (f))

for about 1 hour The spheroplasts were washed with 1mL of1M sorbitol and collected by centrifugation at 6000 rpm for 5minutes at 30∘CThey were then resuspended in 1mL of CaS

A schematic diagram illustrating the spheroplast conver-sion is presented in Figure 2 In the first phase theW303 cellswere transformed into prospheroplasts The prospheroplastswere extruded at one end where partial digestion of the cellwall occurred (Figures 2(a)ndash2(d)) retaining its shape despitethe apparent loss of a supporting wall In the second phasethe prospheroplasts rapidly transformed into spheroplastswhere a spherical shape became dominant (Figures 2(e) and2(f))

23 W303 Spheroplast Observation by an Optical MicroscopeThe spheroplast cells were observed in four different medi-ums YPD sterile water SCE and CaS using Olympus IX-71optical microscope at room temperature (20ndash25∘C) in 100timesoil immersion objective lens The initial 1mL spheroplastcells inside CaS medium were aliquoted into four microcen-trifuge tubes The first aliquot was the spheroplasts insideCaS medium The other three aliquots were centrifuged at6000 rpm for 5 minutes in 30∘C and the CaS supernatant wasdiscarded completely The spheroplast pellets inside each ofthe three tubes were then resuspended with 250 120583L of YPDmedium 250 120583L of sterile water and 250120583L of SCE mediumrespectively

24 W303 Spheroplasts Observations by ESEM The ESEMsystem can perform direct observation of water-containingsamples with nanometer high resolution by specially builtsecondary electron detectorThe evaporation of water is con-trolled by adjusting the samplersquos temperature (sim0ndashsim40∘C) andsamplersquos chamber pressure (10ndash2600 Pa) The temperature ofthe sample is controlled by the cooling stage unit that isUnit3 as shown in Figure 1 ESEM system has a capability tocontrol the chamberrsquos pressure from high vacuum (sim10minus4 Pa)to high humidity (10ndash2600 Pa) The detailed specifications ofthe manipulator and the ESEM can be obtained from ourprevious paper [26] The nanomanipulator has a tungstenprobe which has been used to transfer the single cell usingthe adhesion force This force is produced between the microprobe and cell In fact the nanomanipulator system cancontrol the position of a single cell

3 Results and Discussion

31 Cells Morphology under Optical Microscope Figures 3and 4 show a comparison between the morphology of W303cells before and after spheroplasting inside four differentmediums YPD sterile water SCE and CaS From theobservations themorphology of theW303 cells inside all fourmediums had an oval shape

YPD medium provided the best living medium for thecells followed by sterile water SCE and CaS mediums(Figures 3(a)ndash3(d)) The reduction in number of the W303cells in SCE and CaS media compared to the YPD (1 yeastextract 2 peptone 2 glucose and 2 agar) is due tothe absence of yeast extract The yeast extract will typicallycontain all the amino acids necessary for growth Figure 4shows the morphology of spheroplast cells inside four thefour mediums It is clear that the spheroplast cells displayeda spherical shape inside all mediums However the conditionand visibility of cells inside YPD and sterile water mediumswere poor as compared to the SCE and CaSmedium (Figures4(a)ndash4(d))The spherical shape for the spheroplast yeast cellswas also reported by [32]

32 Spheroplast Conversion Percentage Based on the OD800Absorbance Data The percentage of the spheroplast cellsconversion can be determined from the following equation

Spheroplast = 100 minus [( (OD8001003816100381610038161003816time=119905)(OD

800

1003816100381610038161003816time=0)) times 100] (1)

Figure 5 shows the values of OD800

absorbance data for50 minutes and its corresponding spheroplast conversionpercentage From the graph it is shown that the cells rapidlychanged into spheroplast cells 85 of the cells convertedinto spheroplasts in 2 minutes after the addition of thedigestion enzyme The whole W303 cells completely adaptedthe spheroplast cells conditions in 15 minutes From thisdata it is concluded that the spheroplasting experiment wassuccessfully achieved

33 Surface Characteristic of W303 Cells and SpheroplastW303 Cells under ESEM Figure 6 shows the surface topol-ogy of the cells under ESEM The observations were per-formed at 30 kV and 100120583A The environmental parameters

4 Scanning

(a) (b)

(c) (d)

Figure 3Themorphology of theW303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaS Bar scale is 5 120583m

(a) (b)

(c) (d)

Figure 4 The morphology of the spheroplast W303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaSArrow marks are added to indicate the position of the spheroplast cells inside the first two mediums that is YPD and sterile water Bar scaleis 5 120583m

settings were 600 Pa and 0∘CThewhole cells were kept insidethe YPD medium and diluted with distilled water From theobservation the surfaces of theW303 cells were very smoothas can be seen (top view (Figure 6(a)) side view (Figure 6(b))and closed-up side view (Figure 6(c)))This is also confirmedin previous literature [25 26]

Figure 7 represents the surface topology of the sphero-plast cells under an ESEM For the first time the observations

of spheroplast cells were performed without the need ofcomplex sample preparation and sample coating This willensure the viability of the spheroplast cells that will enablefurther characterization and analysis to be carried out Asexpected the surface topology of the spheroplast cells wasnot smooth as cells having a cell wall The significance ofthe cell wall as a cell shaper was highly acknowledged fromthese observations In addition to the nonsmooth surface

Scanning 5

0

001002

003

004005

006

007008

009

0 10 20 30 40 50Time (minutes)

OD

800

abso

rban

ce0102030405060708090100

Sphe

ropl

astin

g pe

rcen

tage

()

Figure 5 The OD800

absorbance data and its corresponding spheroplast conversion percentage for 50 minutes of treatment using digestionenzyme Zymolyase on W303 cells

4m

(a)

5m

(b)

500nm

(c)

Figure 6The surface topology of theW303 cells under an ESEM from different views (a) top view (b) side view and (c) closed-up side view

4m

(a)

2m

(b)

500nm

(c)

Figure 7The surface topology of the spheroplastW303 cells under an ESEM fromdifferent views (a) top view (b) side view and (c) closed-upside view

topology the spheroplast cells experienced some reductionin size as can be seen in top view image of spheroplast cell(Figure 7(a)) From the side view images (Figures 7(b) and7(c)) it is noticed that the surface had several bleb structures

4 Conclusion

The advantages of the integrated ESEM-nanomanipulationsystem rely on its capability to perform in situ local direct

observation and manipulation of biological sample andthe ability to control the environmental conditions Theobservations of the spheroplast without prior coating foran electron microcopy observation have been highlightedin this work To the best of our knowledge this workis the first to attempt to observe spheroplast cells underelectron microscope without the need of sample coatingThespheroplasting was verified qualitatively and quantitativelyby observing the cellrsquos morphological change under optical

6 Scanning

microscope observations and the spheroplast conversion per-centage based on the OD

800absorbance dataThe spheroplast

cells showed a spherical shape as compared to the oval shapefor the normal cells The electron microscope observationrevealed the bleb-like surface of the spheroplasts as comparedto the very smooth surface of the normal cells This workcould be extended to perform single ion channel currentmeasurements on the spheroplast W303 cells inside theESEM-nanomanipulation system

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The researchwas supported by theMinistry of Higher Educa-tion ofMalaysia (Grant nos 4L640 and 4F351) and UniversitiTeknologi Malaysia (Grant nos 03E11 03G47 02G46 03H82and 03H80) the authors thank them for funding this projectand for their endless support In addition they would liketo thank Professor Toshifumi Inada at Nagoya University forproviding the W

303yeast cell stains

References

[1] M Yu M J Dyer G D Skidmore et al ldquoThree-dimensionalmanipulation of carbon nanotubes under a scanning electronmicroscoperdquo Nanotechnology vol 10 no 3 pp 244ndash252 1999

[2] M A Sutton N Li D C Joy A P Reynolds and X LildquoScanning electron microscopy for quantitative small and largedeformation measurements Part I SEM imaging at magnifica-tions from 200 to 10000rdquo Experimental Mechanics vol 47 no6 pp 775ndash787 2007

[3] J I Goldstein D E Newbury P Echlin et al Scanning ElectronMicroscopy and X-Ray Microanalysis Springer US BostonMass USA 1992

[4] K C A Smith and C W Oatley ldquoThe scanning electronmicroscope and its fields of applicationrdquo British Journal ofApplied Physics vol 6 no 11 article 304 pp 391ndash399 1955

[5] L Muscariello F Rosso G Marino et al ldquoA critical overviewof ESEM applications in the biological fieldrdquo Journal of CellularPhysiology vol 205 no 3 pp 328ndash334 2005

[6] K Hrubanova O Samek A Haronikova et al ldquoMorphologicaland production changes in stressed red yeasts monitored usingsem and raman spectroscopyrdquo Microscopy and Microanalysisvol 22 no S3 pp 1146ndash1147 2016

[7] Y Zhang H Wu X Yu F Chen and J Wu ldquoMicroscopicobservations of the lotus leaf for explaining the outstandingmechanical propertiesrdquo Journal of Bionic Engineering vol 9 no1 pp 84ndash90 2012

[8] L Bergmans P Moisiadis B Van Meerbeek M Quirynen andP Lambrechts ldquoMicroscopic observation of bacteria Reviewhighlighting the use of environmental SEMrdquo InternationalEndodontic Journal vol 38 no 11 pp 775ndash788 2005

[9] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[10] M R Ahmad M Nakajima T Fukuda S Kojima andM Homma ldquoSingle cells electrical characterizations using

nanoprobe via ESEM-nanomanipulator systemrdquo in Proceedingsof the 9th IEEE Conference on Nanotechnology (IEEE NANOrsquo09) pp 589ndash592 Genoa Italy July 2009

[11] Y Shen M Nakajima M R Ahmad T Fukuda S Kojimaand M Homma ldquoSingle cell injection using nano pipette viananorobotic manipulation system inside E-SEMrdquo in Proceed-ings of the 9th IEEEConference onNanotechnology (IEEENANOrsquo09) pp 518ndash521 Genoa Italy July 2009

[12] J M Manero F J Gil E Padros and J A Planell ldquoApplicationsof environmental scanning electron microscopy (ESEM) inbiomaterials fieldrdquo Microscopy Research and Technique vol 61no 5 pp 469ndash480 2003

[13] A M Donald ldquoThe use of environmental scanning electronmicroscopy for imaging wet and insulating materialsrdquo NatureMaterials vol 2 no 8 pp 511ndash516 2003

[14] G D Danilatos ldquoIntroduction to the ESEM instrumentrdquoMicroscopy Research and Technique vol 25 no 5-6 pp 354ndash361 1993

[15] C Gilpin andD C Sigee ldquoX-raymicroanalysis of wet biologicalspecimens in the environmental scanning electron microscope1 Reduction of specimen distance under different atmosphericconditionsrdquo Journal of Microscopy vol 179 no 1 pp 22ndash281995

[16] B Little P Wagner R Ray R Pope and R Scheetz ldquoBiofilmsan ESEM evaluation of artifacts introduced during SEM prepa-rationrdquo Journal of Industrial Microbiology vol 8 no 4 pp 213ndash222 1991

[17] S P Collins R K Pope R W Scheetz R I Ray P A Wagnerand B J Little ldquoAdvantages of environmental scanning electronmicroscopy in studies of microorganismsrdquoMicroscopy Researchand Technique vol 25 no 5-6 pp 398ndash405 1993

[18] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoA study of the spheroplast observations for W303 sin-gle cells under environmental-SEMrdquo in Proceedings of the Inter-national Symposium on Micro-NanoMechatronics and HumanScience MHS 2008 with Symposium on ldquoCOE for Educationand Research of Micro-Nano Mechatronicsrdquo Symposium onldquoSystem Cell Engineering by Multi-scale Manipulationrdquo pp 89ndash92 Nagoya Japan November 2008

[19] M Baguneid D Murray H J Salacinski et al ldquoShear-stresspreconditioning and tissue-engineering-based paradigms forgenerating arterial substitutesrdquo Biotechnology and Applied Bio-chemistry vol 39 no 2 pp 151ndash157 2004

[20] A Motta C Migliaresi F Faccioni P Torricelli M Fini andR Giardino ldquoFibroin hydrogels for biomedical applicationsPreparation characterization and in vitro cell culture studiesrdquoJournal of Biomaterials Science Polymer Edition vol 15 no 7pp 851ndash864 2004

[21] Y Shen M Nakajima M R Ahmad S Kojima M Hommaand T Fukuda ldquoEffect of ambient humidity on the strengthof the adhesion force of single yeast cell inside environmental-SEMrdquo Ultramicroscopy vol 111 no 8 pp 1176ndash1183 2011

[22] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoBuckling nanoneedle for characterizing single cellsmechanics inside environmental SEMrdquo IEEE Transactions onNanotechnology vol 10 no 2 pp 226ndash236 2011

[23] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoMechanical properties characterization of individualyeast cells using environment-SEM nanomanipulation systemrdquoin Proceedings of the IEEERSJ International Conference onIntelligent Robots and Systems (IROS rsquo07) pp 596ndash601 RaleighNC USA November 2007

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of

4 Scanning

(a) (b)

(c) (d)

Figure 3Themorphology of theW303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaS Bar scale is 5 120583m

(a) (b)

(c) (d)

Figure 4 The morphology of the spheroplast W303 cells inside four different mediums (a) YPD (b) sterile water (c) SCE and (d) CaSArrow marks are added to indicate the position of the spheroplast cells inside the first two mediums that is YPD and sterile water Bar scaleis 5 120583m

settings were 600 Pa and 0∘CThewhole cells were kept insidethe YPD medium and diluted with distilled water From theobservation the surfaces of theW303 cells were very smoothas can be seen (top view (Figure 6(a)) side view (Figure 6(b))and closed-up side view (Figure 6(c)))This is also confirmedin previous literature [25 26]

Figure 7 represents the surface topology of the sphero-plast cells under an ESEM For the first time the observations

of spheroplast cells were performed without the need ofcomplex sample preparation and sample coating This willensure the viability of the spheroplast cells that will enablefurther characterization and analysis to be carried out Asexpected the surface topology of the spheroplast cells wasnot smooth as cells having a cell wall The significance ofthe cell wall as a cell shaper was highly acknowledged fromthese observations In addition to the nonsmooth surface

Scanning 5

0

001002

003

004005

006

007008

009

0 10 20 30 40 50Time (minutes)

OD

800

abso

rban

ce0102030405060708090100

Sphe

ropl

astin

g pe

rcen

tage

()

Figure 5 The OD800

absorbance data and its corresponding spheroplast conversion percentage for 50 minutes of treatment using digestionenzyme Zymolyase on W303 cells

4m

(a)

5m

(b)

500nm

(c)

Figure 6The surface topology of theW303 cells under an ESEM from different views (a) top view (b) side view and (c) closed-up side view

4m

(a)

2m

(b)

500nm

(c)

Figure 7The surface topology of the spheroplastW303 cells under an ESEM fromdifferent views (a) top view (b) side view and (c) closed-upside view

topology the spheroplast cells experienced some reductionin size as can be seen in top view image of spheroplast cell(Figure 7(a)) From the side view images (Figures 7(b) and7(c)) it is noticed that the surface had several bleb structures

4 Conclusion

The advantages of the integrated ESEM-nanomanipulationsystem rely on its capability to perform in situ local direct

observation and manipulation of biological sample andthe ability to control the environmental conditions Theobservations of the spheroplast without prior coating foran electron microcopy observation have been highlightedin this work To the best of our knowledge this workis the first to attempt to observe spheroplast cells underelectron microscope without the need of sample coatingThespheroplasting was verified qualitatively and quantitativelyby observing the cellrsquos morphological change under optical

6 Scanning

microscope observations and the spheroplast conversion per-centage based on the OD

800absorbance dataThe spheroplast

cells showed a spherical shape as compared to the oval shapefor the normal cells The electron microscope observationrevealed the bleb-like surface of the spheroplasts as comparedto the very smooth surface of the normal cells This workcould be extended to perform single ion channel currentmeasurements on the spheroplast W303 cells inside theESEM-nanomanipulation system

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The researchwas supported by theMinistry of Higher Educa-tion ofMalaysia (Grant nos 4L640 and 4F351) and UniversitiTeknologi Malaysia (Grant nos 03E11 03G47 02G46 03H82and 03H80) the authors thank them for funding this projectand for their endless support In addition they would liketo thank Professor Toshifumi Inada at Nagoya University forproviding the W

303yeast cell stains

References

[1] M Yu M J Dyer G D Skidmore et al ldquoThree-dimensionalmanipulation of carbon nanotubes under a scanning electronmicroscoperdquo Nanotechnology vol 10 no 3 pp 244ndash252 1999

[2] M A Sutton N Li D C Joy A P Reynolds and X LildquoScanning electron microscopy for quantitative small and largedeformation measurements Part I SEM imaging at magnifica-tions from 200 to 10000rdquo Experimental Mechanics vol 47 no6 pp 775ndash787 2007

[3] J I Goldstein D E Newbury P Echlin et al Scanning ElectronMicroscopy and X-Ray Microanalysis Springer US BostonMass USA 1992

[4] K C A Smith and C W Oatley ldquoThe scanning electronmicroscope and its fields of applicationrdquo British Journal ofApplied Physics vol 6 no 11 article 304 pp 391ndash399 1955

[5] L Muscariello F Rosso G Marino et al ldquoA critical overviewof ESEM applications in the biological fieldrdquo Journal of CellularPhysiology vol 205 no 3 pp 328ndash334 2005

[6] K Hrubanova O Samek A Haronikova et al ldquoMorphologicaland production changes in stressed red yeasts monitored usingsem and raman spectroscopyrdquo Microscopy and Microanalysisvol 22 no S3 pp 1146ndash1147 2016

[7] Y Zhang H Wu X Yu F Chen and J Wu ldquoMicroscopicobservations of the lotus leaf for explaining the outstandingmechanical propertiesrdquo Journal of Bionic Engineering vol 9 no1 pp 84ndash90 2012

[8] L Bergmans P Moisiadis B Van Meerbeek M Quirynen andP Lambrechts ldquoMicroscopic observation of bacteria Reviewhighlighting the use of environmental SEMrdquo InternationalEndodontic Journal vol 38 no 11 pp 775ndash788 2005

[9] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[10] M R Ahmad M Nakajima T Fukuda S Kojima andM Homma ldquoSingle cells electrical characterizations using

nanoprobe via ESEM-nanomanipulator systemrdquo in Proceedingsof the 9th IEEE Conference on Nanotechnology (IEEE NANOrsquo09) pp 589ndash592 Genoa Italy July 2009

[11] Y Shen M Nakajima M R Ahmad T Fukuda S Kojimaand M Homma ldquoSingle cell injection using nano pipette viananorobotic manipulation system inside E-SEMrdquo in Proceed-ings of the 9th IEEEConference onNanotechnology (IEEENANOrsquo09) pp 518ndash521 Genoa Italy July 2009

[12] J M Manero F J Gil E Padros and J A Planell ldquoApplicationsof environmental scanning electron microscopy (ESEM) inbiomaterials fieldrdquo Microscopy Research and Technique vol 61no 5 pp 469ndash480 2003

[13] A M Donald ldquoThe use of environmental scanning electronmicroscopy for imaging wet and insulating materialsrdquo NatureMaterials vol 2 no 8 pp 511ndash516 2003

[14] G D Danilatos ldquoIntroduction to the ESEM instrumentrdquoMicroscopy Research and Technique vol 25 no 5-6 pp 354ndash361 1993

[15] C Gilpin andD C Sigee ldquoX-raymicroanalysis of wet biologicalspecimens in the environmental scanning electron microscope1 Reduction of specimen distance under different atmosphericconditionsrdquo Journal of Microscopy vol 179 no 1 pp 22ndash281995

[16] B Little P Wagner R Ray R Pope and R Scheetz ldquoBiofilmsan ESEM evaluation of artifacts introduced during SEM prepa-rationrdquo Journal of Industrial Microbiology vol 8 no 4 pp 213ndash222 1991

[17] S P Collins R K Pope R W Scheetz R I Ray P A Wagnerand B J Little ldquoAdvantages of environmental scanning electronmicroscopy in studies of microorganismsrdquoMicroscopy Researchand Technique vol 25 no 5-6 pp 398ndash405 1993

[18] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoA study of the spheroplast observations for W303 sin-gle cells under environmental-SEMrdquo in Proceedings of the Inter-national Symposium on Micro-NanoMechatronics and HumanScience MHS 2008 with Symposium on ldquoCOE for Educationand Research of Micro-Nano Mechatronicsrdquo Symposium onldquoSystem Cell Engineering by Multi-scale Manipulationrdquo pp 89ndash92 Nagoya Japan November 2008

[19] M Baguneid D Murray H J Salacinski et al ldquoShear-stresspreconditioning and tissue-engineering-based paradigms forgenerating arterial substitutesrdquo Biotechnology and Applied Bio-chemistry vol 39 no 2 pp 151ndash157 2004

[20] A Motta C Migliaresi F Faccioni P Torricelli M Fini andR Giardino ldquoFibroin hydrogels for biomedical applicationsPreparation characterization and in vitro cell culture studiesrdquoJournal of Biomaterials Science Polymer Edition vol 15 no 7pp 851ndash864 2004

[21] Y Shen M Nakajima M R Ahmad S Kojima M Hommaand T Fukuda ldquoEffect of ambient humidity on the strengthof the adhesion force of single yeast cell inside environmental-SEMrdquo Ultramicroscopy vol 111 no 8 pp 1176ndash1183 2011

[22] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoBuckling nanoneedle for characterizing single cellsmechanics inside environmental SEMrdquo IEEE Transactions onNanotechnology vol 10 no 2 pp 226ndash236 2011

[23] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoMechanical properties characterization of individualyeast cells using environment-SEM nanomanipulation systemrdquoin Proceedings of the IEEERSJ International Conference onIntelligent Robots and Systems (IROS rsquo07) pp 596ndash601 RaleighNC USA November 2007

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of

Scanning 5

0

001002

003

004005

006

007008

009

0 10 20 30 40 50Time (minutes)

OD

800

abso

rban

ce0102030405060708090100

Sphe

ropl

astin

g pe

rcen

tage

()

Figure 5 The OD800

absorbance data and its corresponding spheroplast conversion percentage for 50 minutes of treatment using digestionenzyme Zymolyase on W303 cells

4m

(a)

5m

(b)

500nm

(c)

Figure 6The surface topology of theW303 cells under an ESEM from different views (a) top view (b) side view and (c) closed-up side view

4m

(a)

2m

(b)

500nm

(c)

Figure 7The surface topology of the spheroplastW303 cells under an ESEM fromdifferent views (a) top view (b) side view and (c) closed-upside view

topology the spheroplast cells experienced some reductionin size as can be seen in top view image of spheroplast cell(Figure 7(a)) From the side view images (Figures 7(b) and7(c)) it is noticed that the surface had several bleb structures

4 Conclusion

The advantages of the integrated ESEM-nanomanipulationsystem rely on its capability to perform in situ local direct

observation and manipulation of biological sample andthe ability to control the environmental conditions Theobservations of the spheroplast without prior coating foran electron microcopy observation have been highlightedin this work To the best of our knowledge this workis the first to attempt to observe spheroplast cells underelectron microscope without the need of sample coatingThespheroplasting was verified qualitatively and quantitativelyby observing the cellrsquos morphological change under optical

6 Scanning

microscope observations and the spheroplast conversion per-centage based on the OD

800absorbance dataThe spheroplast

cells showed a spherical shape as compared to the oval shapefor the normal cells The electron microscope observationrevealed the bleb-like surface of the spheroplasts as comparedto the very smooth surface of the normal cells This workcould be extended to perform single ion channel currentmeasurements on the spheroplast W303 cells inside theESEM-nanomanipulation system

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The researchwas supported by theMinistry of Higher Educa-tion ofMalaysia (Grant nos 4L640 and 4F351) and UniversitiTeknologi Malaysia (Grant nos 03E11 03G47 02G46 03H82and 03H80) the authors thank them for funding this projectand for their endless support In addition they would liketo thank Professor Toshifumi Inada at Nagoya University forproviding the W

303yeast cell stains

References

[1] M Yu M J Dyer G D Skidmore et al ldquoThree-dimensionalmanipulation of carbon nanotubes under a scanning electronmicroscoperdquo Nanotechnology vol 10 no 3 pp 244ndash252 1999

[2] M A Sutton N Li D C Joy A P Reynolds and X LildquoScanning electron microscopy for quantitative small and largedeformation measurements Part I SEM imaging at magnifica-tions from 200 to 10000rdquo Experimental Mechanics vol 47 no6 pp 775ndash787 2007

[3] J I Goldstein D E Newbury P Echlin et al Scanning ElectronMicroscopy and X-Ray Microanalysis Springer US BostonMass USA 1992

[4] K C A Smith and C W Oatley ldquoThe scanning electronmicroscope and its fields of applicationrdquo British Journal ofApplied Physics vol 6 no 11 article 304 pp 391ndash399 1955

[5] L Muscariello F Rosso G Marino et al ldquoA critical overviewof ESEM applications in the biological fieldrdquo Journal of CellularPhysiology vol 205 no 3 pp 328ndash334 2005

[6] K Hrubanova O Samek A Haronikova et al ldquoMorphologicaland production changes in stressed red yeasts monitored usingsem and raman spectroscopyrdquo Microscopy and Microanalysisvol 22 no S3 pp 1146ndash1147 2016

[7] Y Zhang H Wu X Yu F Chen and J Wu ldquoMicroscopicobservations of the lotus leaf for explaining the outstandingmechanical propertiesrdquo Journal of Bionic Engineering vol 9 no1 pp 84ndash90 2012

[8] L Bergmans P Moisiadis B Van Meerbeek M Quirynen andP Lambrechts ldquoMicroscopic observation of bacteria Reviewhighlighting the use of environmental SEMrdquo InternationalEndodontic Journal vol 38 no 11 pp 775ndash788 2005

[9] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[10] M R Ahmad M Nakajima T Fukuda S Kojima andM Homma ldquoSingle cells electrical characterizations using

nanoprobe via ESEM-nanomanipulator systemrdquo in Proceedingsof the 9th IEEE Conference on Nanotechnology (IEEE NANOrsquo09) pp 589ndash592 Genoa Italy July 2009

[11] Y Shen M Nakajima M R Ahmad T Fukuda S Kojimaand M Homma ldquoSingle cell injection using nano pipette viananorobotic manipulation system inside E-SEMrdquo in Proceed-ings of the 9th IEEEConference onNanotechnology (IEEENANOrsquo09) pp 518ndash521 Genoa Italy July 2009

[12] J M Manero F J Gil E Padros and J A Planell ldquoApplicationsof environmental scanning electron microscopy (ESEM) inbiomaterials fieldrdquo Microscopy Research and Technique vol 61no 5 pp 469ndash480 2003

[13] A M Donald ldquoThe use of environmental scanning electronmicroscopy for imaging wet and insulating materialsrdquo NatureMaterials vol 2 no 8 pp 511ndash516 2003

[14] G D Danilatos ldquoIntroduction to the ESEM instrumentrdquoMicroscopy Research and Technique vol 25 no 5-6 pp 354ndash361 1993

[15] C Gilpin andD C Sigee ldquoX-raymicroanalysis of wet biologicalspecimens in the environmental scanning electron microscope1 Reduction of specimen distance under different atmosphericconditionsrdquo Journal of Microscopy vol 179 no 1 pp 22ndash281995

[16] B Little P Wagner R Ray R Pope and R Scheetz ldquoBiofilmsan ESEM evaluation of artifacts introduced during SEM prepa-rationrdquo Journal of Industrial Microbiology vol 8 no 4 pp 213ndash222 1991

[17] S P Collins R K Pope R W Scheetz R I Ray P A Wagnerand B J Little ldquoAdvantages of environmental scanning electronmicroscopy in studies of microorganismsrdquoMicroscopy Researchand Technique vol 25 no 5-6 pp 398ndash405 1993

[18] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoA study of the spheroplast observations for W303 sin-gle cells under environmental-SEMrdquo in Proceedings of the Inter-national Symposium on Micro-NanoMechatronics and HumanScience MHS 2008 with Symposium on ldquoCOE for Educationand Research of Micro-Nano Mechatronicsrdquo Symposium onldquoSystem Cell Engineering by Multi-scale Manipulationrdquo pp 89ndash92 Nagoya Japan November 2008

[19] M Baguneid D Murray H J Salacinski et al ldquoShear-stresspreconditioning and tissue-engineering-based paradigms forgenerating arterial substitutesrdquo Biotechnology and Applied Bio-chemistry vol 39 no 2 pp 151ndash157 2004

[20] A Motta C Migliaresi F Faccioni P Torricelli M Fini andR Giardino ldquoFibroin hydrogels for biomedical applicationsPreparation characterization and in vitro cell culture studiesrdquoJournal of Biomaterials Science Polymer Edition vol 15 no 7pp 851ndash864 2004

[21] Y Shen M Nakajima M R Ahmad S Kojima M Hommaand T Fukuda ldquoEffect of ambient humidity on the strengthof the adhesion force of single yeast cell inside environmental-SEMrdquo Ultramicroscopy vol 111 no 8 pp 1176ndash1183 2011

[22] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoBuckling nanoneedle for characterizing single cellsmechanics inside environmental SEMrdquo IEEE Transactions onNanotechnology vol 10 no 2 pp 226ndash236 2011

[23] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoMechanical properties characterization of individualyeast cells using environment-SEM nanomanipulation systemrdquoin Proceedings of the IEEERSJ International Conference onIntelligent Robots and Systems (IROS rsquo07) pp 596ndash601 RaleighNC USA November 2007

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of

6 Scanning

microscope observations and the spheroplast conversion per-centage based on the OD

800absorbance dataThe spheroplast

cells showed a spherical shape as compared to the oval shapefor the normal cells The electron microscope observationrevealed the bleb-like surface of the spheroplasts as comparedto the very smooth surface of the normal cells This workcould be extended to perform single ion channel currentmeasurements on the spheroplast W303 cells inside theESEM-nanomanipulation system

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The researchwas supported by theMinistry of Higher Educa-tion ofMalaysia (Grant nos 4L640 and 4F351) and UniversitiTeknologi Malaysia (Grant nos 03E11 03G47 02G46 03H82and 03H80) the authors thank them for funding this projectand for their endless support In addition they would liketo thank Professor Toshifumi Inada at Nagoya University forproviding the W

303yeast cell stains

References

[1] M Yu M J Dyer G D Skidmore et al ldquoThree-dimensionalmanipulation of carbon nanotubes under a scanning electronmicroscoperdquo Nanotechnology vol 10 no 3 pp 244ndash252 1999

[2] M A Sutton N Li D C Joy A P Reynolds and X LildquoScanning electron microscopy for quantitative small and largedeformation measurements Part I SEM imaging at magnifica-tions from 200 to 10000rdquo Experimental Mechanics vol 47 no6 pp 775ndash787 2007

[3] J I Goldstein D E Newbury P Echlin et al Scanning ElectronMicroscopy and X-Ray Microanalysis Springer US BostonMass USA 1992

[4] K C A Smith and C W Oatley ldquoThe scanning electronmicroscope and its fields of applicationrdquo British Journal ofApplied Physics vol 6 no 11 article 304 pp 391ndash399 1955

[5] L Muscariello F Rosso G Marino et al ldquoA critical overviewof ESEM applications in the biological fieldrdquo Journal of CellularPhysiology vol 205 no 3 pp 328ndash334 2005

[6] K Hrubanova O Samek A Haronikova et al ldquoMorphologicaland production changes in stressed red yeasts monitored usingsem and raman spectroscopyrdquo Microscopy and Microanalysisvol 22 no S3 pp 1146ndash1147 2016

[7] Y Zhang H Wu X Yu F Chen and J Wu ldquoMicroscopicobservations of the lotus leaf for explaining the outstandingmechanical propertiesrdquo Journal of Bionic Engineering vol 9 no1 pp 84ndash90 2012

[8] L Bergmans P Moisiadis B Van Meerbeek M Quirynen andP Lambrechts ldquoMicroscopic observation of bacteria Reviewhighlighting the use of environmental SEMrdquo InternationalEndodontic Journal vol 38 no 11 pp 775ndash788 2005

[9] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[10] M R Ahmad M Nakajima T Fukuda S Kojima andM Homma ldquoSingle cells electrical characterizations using

nanoprobe via ESEM-nanomanipulator systemrdquo in Proceedingsof the 9th IEEE Conference on Nanotechnology (IEEE NANOrsquo09) pp 589ndash592 Genoa Italy July 2009

[11] Y Shen M Nakajima M R Ahmad T Fukuda S Kojimaand M Homma ldquoSingle cell injection using nano pipette viananorobotic manipulation system inside E-SEMrdquo in Proceed-ings of the 9th IEEEConference onNanotechnology (IEEENANOrsquo09) pp 518ndash521 Genoa Italy July 2009

[12] J M Manero F J Gil E Padros and J A Planell ldquoApplicationsof environmental scanning electron microscopy (ESEM) inbiomaterials fieldrdquo Microscopy Research and Technique vol 61no 5 pp 469ndash480 2003

[13] A M Donald ldquoThe use of environmental scanning electronmicroscopy for imaging wet and insulating materialsrdquo NatureMaterials vol 2 no 8 pp 511ndash516 2003

[14] G D Danilatos ldquoIntroduction to the ESEM instrumentrdquoMicroscopy Research and Technique vol 25 no 5-6 pp 354ndash361 1993

[15] C Gilpin andD C Sigee ldquoX-raymicroanalysis of wet biologicalspecimens in the environmental scanning electron microscope1 Reduction of specimen distance under different atmosphericconditionsrdquo Journal of Microscopy vol 179 no 1 pp 22ndash281995

[16] B Little P Wagner R Ray R Pope and R Scheetz ldquoBiofilmsan ESEM evaluation of artifacts introduced during SEM prepa-rationrdquo Journal of Industrial Microbiology vol 8 no 4 pp 213ndash222 1991

[17] S P Collins R K Pope R W Scheetz R I Ray P A Wagnerand B J Little ldquoAdvantages of environmental scanning electronmicroscopy in studies of microorganismsrdquoMicroscopy Researchand Technique vol 25 no 5-6 pp 398ndash405 1993

[18] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoA study of the spheroplast observations for W303 sin-gle cells under environmental-SEMrdquo in Proceedings of the Inter-national Symposium on Micro-NanoMechatronics and HumanScience MHS 2008 with Symposium on ldquoCOE for Educationand Research of Micro-Nano Mechatronicsrdquo Symposium onldquoSystem Cell Engineering by Multi-scale Manipulationrdquo pp 89ndash92 Nagoya Japan November 2008

[19] M Baguneid D Murray H J Salacinski et al ldquoShear-stresspreconditioning and tissue-engineering-based paradigms forgenerating arterial substitutesrdquo Biotechnology and Applied Bio-chemistry vol 39 no 2 pp 151ndash157 2004

[20] A Motta C Migliaresi F Faccioni P Torricelli M Fini andR Giardino ldquoFibroin hydrogels for biomedical applicationsPreparation characterization and in vitro cell culture studiesrdquoJournal of Biomaterials Science Polymer Edition vol 15 no 7pp 851ndash864 2004

[21] Y Shen M Nakajima M R Ahmad S Kojima M Hommaand T Fukuda ldquoEffect of ambient humidity on the strengthof the adhesion force of single yeast cell inside environmental-SEMrdquo Ultramicroscopy vol 111 no 8 pp 1176ndash1183 2011

[22] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoBuckling nanoneedle for characterizing single cellsmechanics inside environmental SEMrdquo IEEE Transactions onNanotechnology vol 10 no 2 pp 226ndash236 2011

[23] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoMechanical properties characterization of individualyeast cells using environment-SEM nanomanipulation systemrdquoin Proceedings of the IEEERSJ International Conference onIntelligent Robots and Systems (IROS rsquo07) pp 596ndash601 RaleighNC USA November 2007

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of

Scanning 7

[24] S E Kirk J N Skepper and A M Donald ldquoApplicationof environmental scanning electron microscopy to determinebiological surface structurerdquo Journal of Microscopy vol 233 no2 pp 205ndash224 2009

[25] M R Ahmad M Nakajima S Kojima M Homma and TFukuda ldquoThe effects of cell sizes environmental conditions andgrowth phases on the strength of individual W303 yeast cellsinside ESEMrdquo IEEE Transactions on Nanobioscience vol 7 no3 pp 185ndash193 2008

[26] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn situ single cell mechanics characterization ofyeast cells using nanoneedles inside environmental SEMrdquo IEEETransactions on Nanotechnology vol 7 no 5 pp 607ndash616 2008

[27] D Ogden ldquoMicroelectrode techniques the Plymouth Work-shop handbookrdquo Company of Biologists Limited 1994

[28] M Sokabe F Sachs and Z Jing ldquoQuantitative videomicroscopy of patch clamped membranes stress straincapacitance and stretch channel activationrdquo BiophysicalJournal vol 59 no 3 pp 722ndash728 1991

[29] M Nakajima F Arai and T Fukuda ldquoIn situ measurement ofyoungrsquos modulus of carbon nanotubes inside a TEM througha hybrid nanorobotic manipulation systemrdquo IEEE Transactionson Nanotechnology vol 5 no 3 pp 243ndash248 2006

[30] M Nakajima F Arai and T Fukuda ldquoNanofixation withlow melting metal based on nanorobotic manipulationrdquo inProceedings of the 6th IEEE Conference on Nanotechnology(IEEE-NANO rsquo06) pp 925ndash928 Ohio USA June 2006

[31] M R Ahmad M Nakajima S Kojima M Homma andT Fukuda ldquoIn-situ single cell mechanical characterization ofW303 yeast cells inside environmental-SEMrdquo in Proceedingsof the 7th IEEE International Conference on Nanotechnologymdash(IEEE-NANO rsquo07) pp 1022ndash1027 Hong Kong China August2007

[32] S Darling J Theilade and A Birch-Andersen ldquoKinetic andmorphological observations on Saccharomyces cerevisiae dur-ing spheroplast formationrdquo Journal of Bacteriology vol 98 no2 pp 797ndash810 1969

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

FluidsJournal of

Atomic and Molecular Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Condensed Matter Physics

OpticsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstronomyAdvances in

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Superconductivity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Statistical MechanicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GravityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AstrophysicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Physics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Solid State PhysicsJournal of

Computational Methods in Physics

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PhotonicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Biophysics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ThermodynamicsJournal of