georg e. fantner, roberto j. barbero, david s. gray, angela m. … · 2013-07-03 · georg e....

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nnano.2010.29

nature nanotechnology | www.nature.com/naturenanotechnology 1

Page S1 of 11

Kinetics of Antimicrobial Peptide Activity Measured on Individual

Bacterial Cells Using High Speed AFM

Georg E. Fantner, Roberto J. Barbero, David S. Gray, Angela M. Belcher

Supplemental Material:

AFM instrument:

The AFM instrument used for this study is a modified MultiMode with a Nanoscope V

controller (Veeco Metrology, Santa Barbara, CA). The main modification consisted of

replacing the regular optics head with an optics head designed for use with the small

cantilevers, as described in Figure S 1. This version is based on a previous design by

Hansma, et al. and a detailed description of the optics design can be found in US patent

6,871,527. In brief: in this design, the incoming laser beam passes though only one part

of the lens, off center from the optical axis (see Figure S 1). The laser spot is then focused

on the cantilever and reflected onto another part of the lens. A deflection of the cantilever

and therefore a change in the angle of the reflection, results in a parallel shift of the

exiting laser beam after the objective lens. The laser beam is reflected onto the photo

detector using a mirror, where the cantilever deflection is measured using the difference

in current between the top and the bottom elements of the photo detector. The laser diode,

objective lens and the mirror are rigidly coupled, with one degree of freedom along the

optical axis for coarse focus. The laser alignment onto the cantilever is done using a ζx

and ζy flexure. The excitation of the cantilever for tapping mode was done by a stack

piezo positioned directly underneath the cantilever, which resulted in an effective drive

mechanism and only a limited amount of parasitic resonance peaks. Experiments were

conducted using an open fluid cell, and fluid exchange was performed with a dual syringe

pump (see Figure S 1C). A high speed scanner was not required at these scan speeds and

surface features, so the experiments were performed on a MultiMode E type scanner

(without rounding).

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SUPPLEMENTARY INFORMATION doi: 10.1038/nnano.2010.29

Page S2 of 11

FigureS1Smallcantileveropticshead.A)Basicopticsdesign.B)CADdrawingofaprototypeheadbased

ontheopticsdesigninA.C)IntegrationofprototypeheadintoaMultiModeVsetupwithflowthrough

pump.D)Closeupofaprototypehead.

Time series of figure 2:

We chose to use the phase data for the evaluation of the changes in the bacterial surface

due to the increased contrast. Phase data can also include information about a change in

materials properties. At this point we cannot rule out that the contrast is increased by such

a change. However, a change in topography does have a big effect on the phase signal,

such that obtaining materials properties information from phase images of samples with




Page S3 of 11

significant topography is hardly possible. Also the fact that the changes in the phase data

are somewhat scan direction dependent (for example comparing trace and re-trace, data

not shown) makes us believe that the majority of the features in the phase signal are due

to a change in topography.

Figure S 2: Amplitude error data of time series described in figure 2A. Controller gains were set

aggressively to ensure proper tracking of the ROI (region of interest) on top of the bacteria, which

resultedinoscillationsinareaswherenobacteriawerepresent.Imagesare3umx3um.




Page S4 of 11

Highresolutionimagesaftertimesequenceoffigure4:

FigureS3showshigherresolutionimagesofthebacteriarecordedafterthetimeseriesoffigure4.

Thecolumnsrepresentheight,amplitudeandphasedataandtherowsshowdifferentmagnifications.

Inthehighestresolutionimages,smallfeaturescanbeseenwhichhavesimilardimensionstothose

reportedfortheporesformedbyCM15(2‐4nm).However,itisnotclearifthesefeaturesareindeed

pores formed by CM15, due to the large background variations and the limited depth an AFM tip

couldpenetrateintosuchsmallpores.

FigureS3:Highresolutionimagesofthebacteriainfigure4afterthetimeseries.Thecolumns

represent height, amplitude and phase data respectively. The rows represent different

magnifications.Thesmallfeatureshavedimensionsintherangeoftheporesizespredicted,

butitcannotbeunequivocallyconcludedthatthesefeaturesareinfactporesformedbythe

CM15.




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Negative Controls:

In order to ensure that the morphology changes seen in figure 2, 3 and 4 are in fact due to

the antimicrobial action of CM15 and not just the addition of a peptide, we used a peptide

called 2K1 with the sequence (GK)6 AS (GK)6 which has no antimicrobial action at the

concentration used. Figure S 4 shows phase images before addition of 2K1 (A and C)

and 45 minutes after addition of 2K1 (B and D). There is no apparent difference between

the surface morphology in the two bacteria as can be seen from Figure S 4 E.




Page S6 of 11

FigureS4:Negativecontrolwithapeptide,whichhasnoknownantimicrobialaction(2K1).FigureAand

Cweretakenbeforeadditionof2K1, figureBandDweretaken45minutesafteradditionof2K1.The

crosssections inpanelEshownosignificantdifference in thesurfacevariationsbeforeandafter2K1

addition.

We also tested the effect of a common antibiotic ampicillin on the surface morphology of

the bacteria. Figure S 5 shows the height, amplitude and phase data (columns) at different

times and resolutions (rows) after addition of ampicillin. No apparent change happens in

the first tens of minutes. After approximately 2 hours, subtle changes were observed on

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the surface of the bacteria, but these are not as extensive as the changes observed after

CM15 treatment.




Page S8 of 11

FigureS5:Negativecontrolwiththeantibioticampicillin.Thecolumnsrepresentheight,amplitude,and

phase data respectively. The rows represent different time points after addition of ampicillin (no

ampicillin, 2minutes, 108minutes and 112minutes after addition of ampicillin). After 112minutes,

minorchangeswerevisibleonthecellsurface,buttheyaremuchlessthanthatcausedbyCM15.




Page S9 of 11

Fulltimesequenceoffigure4:

FigureS6:phasedataoffulltimeseriesoffigure4.Rotated90degreescounter‐clockwisefromFigure4A.




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FigureS7:Amplitudedataoffulltimesequenceoffigure4.Rotated90degreescounter‐clockwisefrom

Figure4A.




Page S11 of 11

Analysis of height and amplitude changes of time series experiment of figure 4:

FigureS8:RMSvariationcalculatedfromheightandamplitudedata.Thedatashowsthesametrendas

thevaluescalculatedfromthephaseimages(Figure4C),butthemorepronouncedfeaturesinthephase

imagesresultinbettersignaltonoiseratio.A)RMSvariationcalculatedfromheightdataasafunctionof

imagesafteradditionofCM15normalizedtothemaximumvalue.B)Initialheightimagedirectlyafter

CM15additionindicatingtheareaswhichwereusedforcalculatingtheroughness.C)NormalizedRMS

variationascalculatedfromtheamplitudeimages.D)Initialamplitudeimagedirectlyafteradditionof

CM15.Theabsolutevaluesof the finalRMSroughnessof theheightdataare2030nmand thatof the

RMS value of the amplitude data is 50200nm. The absolute values however can vary between the

individualcellswhichcouldbearesultofthedifferentorientationsofthecellswithrespecttothefast

scanaxis.


georg e. fantner, roberto j. barbero, david s. gray, angela m. … · 2013-07-03 · georg e....

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