dmr-0116398 non-technical abstract the university of...

A Networked Scanning Probe Microscope for Research Training.

University of New England SPM MRI,

DMR-0116398

Non-Technical AbstractThe University of New England (UNE) requests NSF funds for the acquisition of a

“networked” scanning probe microscope (SPM) for undergraduate research training in the fieldsof biophysics, biochemistry and microbiology. UNE is a non-Ph.D. granting institution thatseeks to provide its students undergraduate research experiences as a “capstone” part of theeducational experience. The core SPM facility will help to provide a multi-disciplinary researchfacility that will help to bridge the artificial divide between life and physical sciences. We willactively recruit diversity in student researchers from courses in advanced biology and chemistrylaboratories. Access by advanced life and physical science courses with large enrollments willbe through networked remote operations. Direct undergraduate use will be undertaken under theguidance of UNE faculty in their respective research areas.

Technical AbstractThe University of New England requests NSF funds for the acquisition of a “networked”

scanning probe microscope (SPM-Digital Instruments NanoIII) for undergraduate researchtraining in the analysis of quadruplex DNA, biofilms and metallocarbohedrenes. Optimal use ofthe NT-based instrument will be accomplished by “Virtual Network Computing”(http://www.uk.research.att.com/vnc/) freeware that will enable undergraduates in organic ,physical chemistry and microbiology to access to the SPM via remote computers. Ourenvironmental microbiology Co-PI will examine the growth processes of biofilms responsiblefor fouling surfaces exposed to running water. Our physical-inorganic chemist co-PI is interestedin the structure and electronic properties of metallocarbohedrenes. The PI’s interest focuses onthe growth kinetics and structure of a novel four-stranded “G-wire” DNA and research training.



A Networked Scanning Probe Microscope for Research Training.Cover Sheet

Follows NSF Grant Proposal Guide NSF 01-2

Program announcement/solicitation number: NSF 01-7 Major Research Instrumentation programsolicitation.NSF Unit Consideration: Directorate for Biological Sciences OR Directorate for Mathematicaland Physics Sciences.: Division of Materials Research



Project Summary

Project GoalsThe University of New England (UNE) requests $157,835 in NSF funds for the acquisition

of a “networked” scanning probe microscope (SPM) for undergraduate research training in thefields of biophysics, biochemistry and microbiology. UNE is a non-Ph.D. granting institutionthat seeks to provide its students undergraduate research experiences as a “capstone” part of theeducational experience. The core SPM facility will help to provide a multi-disciplinary researchfacility that will help to bridge the artificial divide between life and physical sciences. We willactively recruit diversity in student researchers from courses in advanced biology and chemistrylaboratories. The proposed instrumentation is part of a concerted effort to recruit women intophysical science majors.

To optimize instrumentation usage our effort is two pronged. First we will be providingnetworked SPM access to larger class activities (between 10 and 20 students) and secondly weare supporting intensive individual research projects. Students participating in advanced coursescan efficiently be trained on the SPM through the use of remote operations using from anadjoining computer work station facility. The undergraduate majors in these courses range frombasic sciences to health related fields. These courses will culminate in class research projectsthat emphasize research training tailored to each student’s need. Students participating inindividual biology, chemistry and physics research projects will run the SPM instrumentationdirectly from the small lab space devoted to the equipment

Our research focus will reflect, in part, UNE’s heavy investment in the life sciences. A Co-PI in the area of environmental microbiology will examine the growth processes of biofilmsresponsible for fouling surfaces exposed to running water. The time scale under which thisgrowth takes place (µm/min) is well suited to SPM examination. Our physical-inorganic chemistco-PI is interested in the structure and electronic properties of metallocarbohedrenes. Both co-PIsteach instrumentation as an integral part of their advanced laboratory courses. In these largelaboratory classes the Co-PIs plan to create a culminating class research project in which groupsof students are assigned tasks needed to complete the entire project. The PI’s interest focuses onthe growth kinetics and structure of a novel four stranded “G-wire” DNA and research training.The proposed SPM provides a simple way to monitor real-time growth of the G-wires, andmodeling of the self-assembly process. The G-wires also present themselves as an interestingcandidate for a biological “nanowire.” The advanced near-field detecting capabilities of theproposed instrumentation are essential for the above proposed research. Providing ourundergraduates relevant research training in SPM technology is essential for making our studentscompetitive in Maine’s flourishing biotechnology and semiconductor industries.



a) Results from Prior NSF Support

DUE 9750942: Networked Scanning Probe MicroscopeBrief Summary:

James Vesenka (JV) was awarded an NSF Instructional Laboratory Improvement award todevelop a networked scanning probe core facility at California State University, Fresno. Thefacility was designed to serve large remote audiences, on and off campus, through broad band orinternet connections, and to serve as a research training tool for advanced undergraduates atCSUF. JV secured two microscopes, a Park Scientific BioProbe for large sample imaging (e.g.cells), and a Molecular Imaging Picoscope™ for molecular and atomic resolution.

Contributions to disciplineAfter 9750942 was first funded, a broad band modulator was purchased (as part of the

matching effort by CSU-Fresno) enabling remote presentations of SPM images in real-time ontothe campus broad band network. The major drawback of this approach was in that the “pipeline”was only one way, i.e. no feedback from remote audiences was possible. Remote operationswere limited to a commercial software package “PC Anywhere,” with limited platform abilitiesin 1997. In addition, the commercial licensing fee of “PC Anywhere” made usage by theintended audiences of faculty and students from other CSUs, community colleges, and local highschools prohibitive. A free-ware package called virtual network computing (VNC1) by AT&Tlab associates was discovered on the internet in 1998. VNC allowed any trained user of theSPMs to access the networked microscope from a remote site with a password. Though asignificant improvement over one-way transmission, the finite transfer rate (about 10 kb/sec) fellfar short of the feedback during on-site operations. There were faster alternatives.Asynchronous transfer mode (ATM) was capable of running a thousand times faster, but at aprohibitive cost ($3000/month). Cable technology appeared to be the most cost-effectivesolution, but cable standards were not in place by the time of grant completion. In sum, wefollowed the available technology and tried to develop a strategy to secure the widest possibleand appropriate usage. One of the larger difficulties was securing adequate operational support.

Major findingsIn addition to training all students and faculty in microscope operations and maintaining the

SPMs, JV taught two to three classes per semester, and was active in physics education researchas well as molecular biology research. He was NOT provided any release time to support theremote operation activities. JV submitted three grants to secure post-doctoral and/or technicalsupport over the life of the ILI grant. The first was submitted jointly to a California StateUniversity technology development division with CSU-Hayward, the second to the NSF'sEngineering Experiences program and the last to the NSF MBRS program. All grants wereunsuccessful. Funding for staff support through CSUF was not possible because of staffingfreezes.

Although JV achieved the overall objective of developing a remotely operatedinstrumentation core facility within the first two years of the grant, the ultimate objective ofcreating a self-sustaining imaging facility was not realized during his tenure. In the spring of2000 JV resigned from CSUF to help his family in New England, leaving the equipment in thecapable hands of his colleagues. Continuation of the project’s objectives are being undertakenby one of the grant’s senior personnel, Dr. Alejandro Calderon-Urrea (CSUF Biology). He has



received MBRS funding and will be able to sustain a trained SPM operator to run the SPMfacility for the next four years.

What research and teaching skills and experience has the project helped provide to thosewho worked on the project?

Over 20 students received research training on the SPM during the life of the grant. Theimprovement in student research skills associated with the networked SPM was dramatic. Eachparticipant involved in research was required to present a paper at either a local or nationalresearch symposium. In all but two cases, the research influenced or directly aided the student'sgraduate school aspirations. In addition, three faculty members were trained in the operations ofthe microscopes. These include a senior personnel at CSUF; a developmental biologist, Dr.Calderon; an entomologist and microscopist, Dr. Fred Schreiber; and a biochemist, Dr. DaveChester from Fresno Pacific University. The impact on Dr. Calderon’s research was profound,since the inverted optical microscope, seeded in part by the ILI grant, helped him secure a four-year $400,000 NSF-MBRS award to undertake research in cell pathogenesis. He is currently oneof two active research faculty in Biology at CSUF. Dr. Schreiber has since become the biologydepartment chair and has been a driving force in continued use of the instrumentation. Dr.Chester obtained some outstanding initial data that he is in the process of analyzing for a paper.The collaboration with Dr. Chester was exactly the kind of outreach to area universities the granthad originally proposed.

What outreach activities have you undertaken to increase public understanding of, andparticipation in, science and technology?

Outreach came through three activities: public seminars, teacher education, and a web site.Public seminars were held in the Department of Physics at CSUF each fall and spring of theacademic year, discussing progress in development of remote microscopy. An average of fivegeneral public participants appeared at each of these seminars over the four years of the grant.Teacher education, funded through a NSF Urban Systems Initiative grant to Co-PI DaveAndrews, was used as a forum to advertise the availability, and experiment with the utility ofremote microscopy. The integrated science program involved 160 teachers in the summer of1998 and 1999 mostly from the Fresno Unified School District. Two of Paul Lake’s studentsfrom the Clovis Unified School District, undertook part time summer research in the lab. Lastlythe web site received over 10,000 hits in the four years of operation athttp://maxwell.phys.csufresno.edu:8001/~csufspm/csufspm.htmlThe web site has since been closed due to JV’s departure from CSUF.



b) Research ActivitiesPI: James Vesenka (JV), University of New England biophysics, surface and material science:Introduction:

Rapid advances in electronics miniaturization demands the development of a new generationof micro-electronic circuitry capable of single electron delivery.2,3,4 The carbon nanotubeappears to be the strongest “nano-wire” candidate. However, nanotubes dramatically changetheir electronic character when only a single carbon atom is out of place, or if the tubes are evenslightly deformed. Though there may be a bright future of nanotubes as semiconductor devices,biopolymers still have and edge in molecular wiring.5

The robust character of duplex DNA has long been examined for its potential to address themolecular wire question. Very low conductivity through short DNA sequences (~15 base pairs)has been established by photo-induced and flash-quench techniques,6 while electron hopping hasbeen established as the dominant form of electron migration in longer segments of DNA7. Underhydrated conditions, LCSTM reveals that duplex DNA is a poor electron carrier compared to thehydrated surface of mica8. Furthermore duplex DNA appears to collapse on the surface of micaand other silicates (e.g. glassware). Such behavior would affect electron transfer along thetwisted backbone-shape9, raising continuity concerns. Biomolecular templates have beenemployed as masks and scaffolding to create traditional miniature metalized conductors.However, the large grain-size of the conductive metals make the resulting structures highlyirregular, and commercially unappetizing10. An ideal “nano-wire” would combine the flexibilityof a biopolymer, the uniformity of integrated circuit technology and conductivity of metals.Description:

For the past several years I have been interested in characterizing the molecular andelectronic structure of a novel four-stranded self-assembled DNA. These forms of nucleic acidcomplexes have been discovered in the end regions (telomeres) of chromosomes. The hairpinstructures, comprised of guanine quartets, are thought to help signal a DNA protein activity(telomerase) necessary for DNA replication11. Marsh and Henderson established that self-assembled Guanine-rich tetraplex DNA, termed “G-wires,” could be grown to micrometerlengths in large quantities through the overlap of the repeated G4T2G4 oligonucleotide (oligo)sequence found in the G-quartets12. The ionic conditions under which G-wires are growndetermined the type of caged metal cations (e.g. Mg, K, or Na), integrated into the structure13. Incomparison to double-stranded DNA, the integrated metal cations surrounded by the four-stranded phosphate backbone plus extensive hydrogen bonding of G-wires might facilitate lateralconductivity over their uniform 2.4 nm diameter and micrometer-lengths. The uninterruptednanometer-scale morphology of G-wire DNA, as characterized in my former lab at CaliforniaState University Fresno (CSUF), make them an exciting candidate as a molecular wire.

Atomic force microscopy (AFM) and low current scanning tunneling microscopy (LCSTM)measurements made at CSUF indicated that G-wires can be reproducibly imaged at tunnelingcurrents above a picoampere at high humidity14. To our knowledge these observations were thefirst images of duplex and quadruplex DNA reported at such high levels of tunneling current.The contrast mechanism for non-conductive biomolecules by LCSTM is now well understood.Tunneling stems from the hydration layer on top of a hygroscopic substrate such as mica inhumid air, depending only upon the applied “bias” voltage15. Under low voltages, high-resolution imaging is maintained by conduction through the hydration layer. At high voltagesballistic tunneling takes place through the air gap into the hydration layer, at the expense ofresolution. What made our initial results interesting is the factor of 10 to 100 increase in



tunneling current is attained in the presence of G-wire DNA compared to double stranded DNA.The implication was that the G-wires are assisting conductivity over the substrate. One ofthe questions of interest was whether the conductivity might be parallel to the G-wire chain(Figure 1).

My colleagues, from the Institute of Physical High Technology in Jena Germany, havedeveloped microfabricated substrates for biopolymer conductivity measurements16. Thebiopolymers need to be several micrometers in length in order to span the gold contacts, or to beattached to the contacts through electron beam deposition17. After establishing an SPMlaboratory at the University of New England,18 my lab set about growing G-wires from acommercially available, purified oligo sequence. Two undergraduates undertook preliminarystudies this past summer to establish the time needed for the growth of long G-wires forsimplified macroscopic electronic characterization. Several problems were immediately evident.The zero time “pure” G4T2G4 oligo sequence was contaminated with self-assembled G-wiresaveraging 30 nm in length (Figure 2). Approximately 40 oligos are bound up in this length ofmolecule. The pure oligo should have a length of about 3.4-nm, below the 5-nm lateralresolution limit of the best contact AFM imaging. In principle pre-melting (heating the G-wiresbriefly to near boiling temperatures) can reduce partially assembled G-wires into their oligobuilding blocks. This is non-trivial matter, since heating the oligo can also lead to irreversibledissociation. The entropically driven process of self-assembly should not be affected by pre-existing G-wires. However, as we grew the G-wires we were unable to obtain the micrometerlengths published by Marsh, Vesenka, and Henderson12. Figure 3 is a plot from preliminary datarelating the “reduced” mean length of the G-wires, measured by the AFM, as a function ofgrowth time. The experimental fit to the data leads to a power law with the general equation:

<LengthG-wire> (nm) = (11-nm/√day)*t0.5 (√day)According to this model a 10-µm length would take 2300 years to grow! Since I was involved inthe original studies with Marsh et al.9, where we grew micrometer length G-wires in a fewmonths time at Iowa State University, there is clearly an error somewhere in our samplepreparation at UNE. After we independently confirmed that purity of the oligomer was not inquestion, obvious areas of scrutiny were the growth cocktail media and SPM sample preparation.Additionally, we undertook a literature review of molecular self-assembly growthcharacterization to help define baselines but found no discussion applicable to our system.

We propose a two-year undergraduate research project to establish the conditions forconductivity measurements.

1. What are the conditions for optimal G-wire growth?2. How do we characterize G-wire growth rates?3. How do we characterize G-wire electronic structure?4. What is/are the mechanism(s) of G-wire conductivity?

Solving the first question will help us establish the procedures, concentrations, and time scalesneeded to grow practical size (micrometer length) G-wires. In particular, an answer to the firstquestion will provide us with better samples to address the issues of conductivity. All fourquestions involve extensive use of the proposed major research instrumentation.Objectives:1. High-resolution, native-state G-wire characterization

The proposed equipment will have the essential AC imaging mode of intermittent contact(a.k.a. TappingMode™) in both gasses and liquids. This will enable us to take high-resolutionimages of the G-wires both in dry air and liquids. These measurements are essential to establish



accurate estimates of mean G-wire lengths as part of our time study. Intermittent contact,especially when undertaken in buffered media, helps to mitigate the artificial broadening of thelateral features of biological samples due to the finite tip geometry19. One of the enormousadvantages of imaging in buffered media is the ability to use G-wire cocktails at their fullconcentrations. G-wire dilutions of about a factor of 1000 are needed, in PURE WATER, forimaging in air due to the artifact-induced contamination from residual dried buffer ions. Suchdilutions are likely to be deleterious to the G-wires because of their entropically driven growthprocesses. Dilution drives disassembly of the molecules into their constituent building blocks,the guanine oligonucleotide tetramer. However, imaging in the G-wire growth medium andconcentrations assures mean G-wire lengths reflective of current equilibrium conditions.Imaging G-wires in their growth medium is NOT speculative. I have confirmed this capabilityduring a preliminary study on the same equipment we are proposing. Details are provided insection b on “Description of Research Instrumentation Needs”.2. Fundamental question: Growth rate characterization

Establishment of a growth rate constant for a self-assembled structure is complicated. Wewill compare our length-time studies above with numerical computations. The nature of theproblem can be seen in Figure 4. The total equilibrium constant is the product of individualconstants for different ladder lengths “i”.

K(t) = P Ki(t)To simplify the problem we assume the rate-limiting building block to be the tetramer ladder.This assumption is based on evidence from assays of telomeric DNA in which the binding of theG4T2G4 oligo into hairpin or looped structures with the telomeric Guanine-quartet is kineticallymore favorable than remaining in the oligo state. This system of n-linear equations requiresnumerical solutions. With the proposed instrumentation, we will have the means to take accurategrowth measurements. Co-PI, Computational Chemist, Dr. Clinton Nash, will assist in thenumerical simulations. An important issue at stake is how to deal with the time dependency ofeach individual equilibrium constant of G-wires with different chain lengths. At a minimum, wewill insert the known time dependence with empirical fits to the data, such as shown in Figure 4.3. Estimating G-wire conductivity

Attachment of G-wires to electrodes will involve direct deposition between gold contacts.The microfabricated conduction grids are labeled for optical identification. When a candidate G-wire, identified by long range AFM scan, lie across the electrodes, we can easily locate the gridposition for macroscopic current measurements. Even if long G-wires are not fortuitouslyconnected between contacts, we can employ electron beam deposition (the contamination residuefrom repeated line tracing with an electron microscope) with the UNE scanning electronmicroscope to complete a biopolymer circuit7. Control experiments include the comparison ofthe current through G-wires, duplex DNA, and electron beam deposited (EBD) scanning electronmicroscope carbon contamination traces using a transistor-type electrode set-up (Figure 5) withthe LCSTM. Conductivity will likely be a function of ambient relative humidity and residualbuffer ions. Humidity/ion control is extremely important not only to limit SPM-induced sampledamage but also to avoid electrophoresis (migration due to charge separation) of the G-wires.We seek to characterize conductivity both perpendicular and parallel to the G-wire DNA chain.Since different forms of molecular bonding, the location and number of “caged” (i.e. limitedmovement) ions may be responsible for transverse and lateral conductivity, these experimentscould provide fundamental information surrounding G-wire structure and its relation to electrontransfer through four stranded DNA.



4. Future question: Conduction mechanism.The caged metal cations (Figure 4) integrated into a hydrated G-wire molecule may have somemobility. In addition, the metal cations are sufficiently close to each other (estimated between0.3-0.7 nm) to support electron tunneling. Lastly, the p-bonding of adjacent Guanine-quartetsmay overlap enough to enhance electron mobility. All of these mechanisms can contribute to thelateral conductivity we are trying to characterize. The experiments mentioned above cannotdistinguish between these different conduction modes. Future efforts will be aimed at 1)“locking-out” different conduction modes by the construction of G-wires around less mobilemetal cations, or 2) enhancing p-bonding along the length of the G-wire chain through theaddition of complementary adenine bases binding to the vacant thymine sites.Dissemination:

The students are expected to give poster presentations at UNE and local and/or nationalmeetings of my professional organizations, Council of Undergraduate Research and theBiophysical Society meetings. These results will then be converted into web site presentationsand appended under the “student research” link at my UNE faculty web site(faculty.une.edu/cas/jvesenka/DrVIndex.HTM). Suitably publishable results will be submittedeither to the Journal of Vacuum Science and Technology or the Biophysical Journal.Time Line:

Summer 2001: Continue baseline length-time studies on existing older contact imaging SPMto help establish optimal G-wire growth conditions. Undergraduate research stipends areindependently provided by UNE.

Academic Year 2001-2: Train students in powerful techniques on proposed MRI SPMoperation and recruit potential summer research participants from microbiology (BIO226) and/orphysical chemistry (CHE327).

Summer 2002: Fluid-cell AC imaging of G-wires for rigorous characterization of growthrates. Undertake preliminary conductivity assays.

AY 2002-3: Continue conductivity assays with select honors and or independent researchstudents. Repeat training and recruiting of undergraduates from advanced courses.

Summer 2003: Undertake full-scale conductivity characterization. Estimated 2-4undergraduate research assistants, again all supported by UNE stipends.

Figure 1: a) Cross-section schematic of electron tunneling path between tip and sample, likely tobe through a thin aqueous layer containing residual buffer salts and G-wires. b) Top viewschematic of the possible tunneling paths through the G-wires (black lines) and hydration layer(white lines) from tip (circle). Top-view example of G-wire DNA and residual salt imaged at82% relative humidity, -7V bias voltage and 3.0 pA tunneling current. Vertical height is 0 - 5nm from dark to light color. Scale marker is 100 nm long.

pA

V

Dielectric substrate

DNA w/ moisture layer

a b



Figure 2: The “pure” solution of ten nucleotide oligomer was imagedby the atomic force microscope at t = 0 (i.e. at the instant they wereplunged into the growth cocktail). Imaged at about 10 nN of constantforce and 10 % relative humidity. The G-wires had an average lengthof 28-nm. The vertical color scale indicates the G-wire rise anaverage of 2.0 nm above the mica substrate, whereas the lateralresolution is only good at best 5 nm because of the finite tipgeometry. In this image the G-wires are broadened an average of 20nm, seriously affecting our length measurements. The scale markeris 100 nm long.

Figure 3: PreliminaryG-wire growth study.Raw data (roundcircles) with error bars(standard deviation ofmean length) plottedversus days. N=100 foreach data point .Diamonds correspond to“reduced length” ,achieved by subtractingthe t = 0 mean lengthfrom the raw data. Anexponential fit to thereduced length datareveals:

Reduced Length (nm) = (11 nm/√day)t0.5

Though this is a typical function for the simple molecular equilibrium process, note the errorsbars are so large that a variety of other function (e.g. linear) could also be used to fit the data.

K1 = [L1]/[GGGGTTGGGG]2

K2 = [L2]/[L1]2

K3 = [L3]/[L1][L2],… Kn = [Ln]/[L1][Ln-1], K = K1K2K3… = PKi

Figure 4: The above diagram shows the steps involved in defining an overall equilibriumconstant for the growth of the G-wires. A system of “n” simultaneous first order linear equations

GGGGTTGGGG

GGGGTTGGGGGGGGTTGGGG + GGGGTTGGGG L1

L1 + L2L3

G-wire Growth Study

0

20

40

60

80

100

120

0 5 10 15 20Days

Mea

n L

engt

h (n

m)

GGGGTTGGGGGGGGTTGGGG

GGGGTTGGGG

L1 + L1

GGGGTTGGGG

L2

= caged metal cation of Mg2+ or K+



is generated, requiring numerical analysis. One of the assumptions is that the addition of L1 to aG-wire chain is the most dominant process. A hidden complication is the equilibrium constantsbeing time dependent because the mean concentrations of the ladder components change in time,as seen in Figure 3.

Figure 5: Side view of conductivity measurements of G-wires (brown) made through twoelectrodes (gold) or three point contacts of the two electrodes plus SPM tip (green). Three-point-contact, i.e. biasing the G-wires and measuring any leakage current using the LCSTM tip, willenable us to characterize any non-ohmic behavior of the G-wires. Two pico-ammeters arerequired, one for macroscopic current measurement, the other is built into the low current STM.

Co-PI: Mark Johnson (MJ), UNE microbiology and ecology:Introduction:

A biofilm is the matrix of microorganisms and mucopolysaccharide that covers surfaces;especially in aquatic habitats.

Drawing by Mark Weincek20

Biofilms are important components of lotic, lentic, and oceanic ecosystems, providing aprimary grazing surface for moluscan and arthropod grazers which themselves subsequentlyprovide food for higher organisms. Much study has been done on the role that algae andcyanobacteria play in stream biofilms. However, the structuring of such communities dependsupon the earliest colonists of a newly cleared surface, which are often the bacteria. Research hasbeen difficult in determining how contact and colonization proceeds because the resolution of

pA

pA

V



light microscopy is inadequate to see this stage, and various electron microscopic approaches tothe subject require fixation and metal coating which disturbs the matrix and produce artifacts.

Of possibly more practical use is increasing our understanding of the process of bacterialcolonization of painted, plastic, teflon, metal, and other surfaces which form the submersedportions of boat hulls. Understanding how biofouling of boats, ships, and other man-madesurfaces occurs is an important area for research and of particular interest to the commercial boatmanufacturing industry in the State of Maine. Biofilms also form inside the human (and otheranimal) bodies; on teeth for example. Contact and colonization occurs well before the origin ofdental carries.

The scale of contact and primary colonization is in terms of nanometers up to 3 micrometers.Acoustically driven intermittent contact atomic force microscopy appears to be ideally suited tothis range of scale, and provides the possibility of imaging live organisms and relativelyundisturbed mucopolysaccharides.Class Projects:

At the University of New England I teach courses in microbial ecology and independentresearch among others. Since I require that students do actual research in these courses, theylend themselves to students using scanning probe microscopes (SPM) to find out how biofilmsare generated. As a starting point for dealing with the formation of these structures, I proposethat a number of different bacteria are isolated from the water column in local harbors, streams,and ponds, in addition to the open ocean. Cultures of these organisms would then be placed inproximity to an appropriate surface where colonization can be observed and recorded with theSPM in fluid environments. We can even simulate low flow rates in the SPM imaging chamberthrough input/output ports of the fluid cell. Low flow rates are necessary to avoid disturbing theimaging tip.

Not all bacteria are the same. Neither are surfaces. Selecting appropriate bacteria andsurfaces for testing will be critical in defining the value of the studies. Imaging rough surfaces isdifficult given the limitations of the vertical range (10 µm) of the equipment's scanner. Mostprotozoa and algae are not suitable for the same reason. However, there are some surfaces andmicroorganisms that are appropriate for this equipment and these kinds of questions. Mica,being a common mineral in the rocks found in streams in this area, also happens to be one of thestandard substrata for use in SPM because it provides such a smooth surface. Glass surfacescoated with fine grades of paint may also be possible, and would provide a model system formarine fouling communities. Many bacteria found in natural communities do not grow inculture, so in addition to specific isolates of bacteria from various habitats, whole samples ofmixed natural communities will also be used.

Many bacteria require other species in their close proximity to survive or thrive. Thesesymbiotic groups of bacteria are called consortia. One of the questions that we wish to address isthe differential efficacy of colonization by individual species of bacteria – versus consortia ofbacteria working together. Developmental rates of mucopolysaccharide will be determined bytime course studies using the SPM. Spatial arrangements of several bacterial morphotypes willprovide an indication of which system is quicker in producing a “colonizable” film, for secondstage biofilm development. Additional comparisons of mucopolysaccharide development undervarious nutrient and dissolved carbon enrichments will also be done on the same basis todetermine the effects of fertilizers and other pollutants on initial stages of biofilm formation.

In addition to adherence to a surface, mucopolysaccharide exudates may function as aholding medium for compounds that provide a competitive advantage to the initial colonists.



Pulsed experiments, where one species is added first followed by a second after the first has laiddown an initial mucopolysaccharide layer – contrasted with systems where both organisms gainaccess to the surface simultaneously – should provide some evidence for alternate hypotheses.The implications for these studies are not only insights into the development of anti-foulingagents, but also to inform stream, lake, and ocean ecologists of the relevance of successionalmodels to this type of micro-habitat development.

The variety of possible research projects using this equipment makes it ideal for a focus inmicrobial ecology classes. Several teams can use the same instrument to focus on differentaspects or questions. The various projects allow for students to follow their own interests whileat the same time, linking or coordinating the knowledge they gain with others focusing ondifferent aspects of the same system. Thus, in a way the student researchers get an introductionto the collaborative nature of modern research within the microcosm of student research projects.

Co-PI: Clinton Nash (CN), UNE inorganic and physical chemistry:Introduction:

Since the discovery several years ago of a class of extraordinarily stable carbon clusters nowidentified as the fullerenes,21 there has been a great deal of effort invested and success attained inthe characterization and understanding of the structure-property relationships inherent in thesecluster molecules.

Fullerenes have been defined as cluster species consisting of closed cages of carbon atomsthat conform to the so-called isolated pentagon rule. This rule is a statement that special stabilityis conferred upon such cage molecules that include exactly twelve facial pentagons, or C5 rings,none of which have any atoms in common. According to these criteria, the smallest possible,and by now the best known example, of such a species is C60 that is also the prototypicalfullerene in that it was the first to be recognized as a unique and distinct allotrope of pure carbon.The isolated pentagon rule is somewhat restrictive and is perhaps more appropriate thatfullerenes be classified more generally by only the presence of the twelve pentagonal facesrequired to close the cluster surface upon itself. The isolation of pentagons ensures that no singleatom resides in any two pentagonal faces and results in maximal aromatic stabilization as thefusion of five-membered rings results in a Hückel antiaromatic cycle of eight.

Although others are known, a particularly interesting exception to the isolated pentagon ruleis the remarkable stability of certain metal containing clusters of the general formula M8C12,reported first for M = Ti by Castleman, et al., and dubbed metallocarbohedrenes.22 Theseclusters were originally proposed to adopt a cage structure of Th symmetry analogous to theunstable fullerene C20 that has recently been identified in the gas phase.23 The proposed structureof this species exemplifies an extreme case of violation of the isolated pentagon rule as everyatom is included in the maximum possible number (three) of five-membered rings. Recentquantum chemical investigations have suggested that the actual structure of neutral Ti8C12

geometry is of Td symmetry which represents a Jahn-Teller distortion of the idealized Th

configuration.24

Target System:The increased stability of this metal cage structure over that of its all-carbon analog must be

due, at least in part, to some intrinsic property of the metal atoms. The most obvious candidate isthe presence of energetically accessible metallic d valence orbitals that must in some wayenhance the "aromaticity" of the molecule. Support of this notion is provided by the fact thatdodecahedrane, C20H20, the fully hydrogenated derivative of the twenty-vertex fullerene is a



known compound adopting the idealized icosahedral geometry of the dehydrogenatedcompound.25 As a saturated compound there can be no antiaromatic destabilization fordodecahedrane. It must be concluded that the ephemeral existence of C20 is a result ofunfavorable p-orbital interactions rather than any sort of carbon-carbon bond angle or ring-straineffects, because these should be roughly the same in both compounds. When metals aresubstituted for certain carbon atoms in these structures, the unfavorable effects are reduced oreliminated thereby leading to the stability seen in Castleman's and others' results. This is asituation reminiscent of the phenomenon of metalloaromaticity which leads, for example, to theexistence of metallocyclobutadienes, whereas no four-carbon conjugated ring is known to beother than transiently stable.26 Unfortunately, there is not yet available direct informationconcerning the structure of Ti8C12 or any other metallocarbohedrene. The limited and indirectinformation that is available comes from mass spectrometric ion mobility analysis of laser-ablated titanium carbide soots. However, this has demonstrated that indeed the molecule doesadopt a hollow cage structure superficially reminiscent of fullerenes.27

Class Research Project:We propose to use nanometer scale atomic force microscopy (AFM) and scanning tunneling

microscopy (STM) to directly assess the structural characteristics of metallocarbohedrenes (Met-cars), and Ti8C12 in particular. Met-cars are relatively easily produced in inert atmospheres byusing titanium-carbon electrodes in an 'arc-welding' apparatus of the type frequently used toproduce C60 and higher fullerenes. We have access to an Edwards evaporative coating apparatusthat has the control of temperature and gas needed to generate monolayer Ti/C soots evaporateddirectly only highly oriented pyrolitic graphite substrates. The Ti/C soots produced in such away will contain both the cage-like molecules Ti8C12 and Ti8C13 (the latter a met-car containingan endohedral carbon atom) as well as titanium carbide nanocrystals of rock-salt likestoichiometry, Ti12C13. A recent transmission electron microscopy investigation Ti/C soot by Yuand Huber was unable to distinguish met-cars from the nanocrystals.28 We propose to use bothAFM and STM to examine Ti/C soots and thereby directly characterize the structures of thetitanium metallocarbohedrene. As a necessary first step in this process, we will conduct similarstudies on quantites of air-stable dodecahedrane that can be relatively easily obtained in gramquantities. As mentioned earlier, C20H20 has a similar size and analogous structure to those ofthe proposed metallocarbohedrenes and should therefore provide an excellent proof-of-concept.Such a study would represent a substantial advance in fullerene science as it enhances ourunderstanding of the electronic and structural characteristics of non-carbon fullerene-likestructures thereby expanding their potential inclusion among the palate of elements to be used infullerene science and technology.

An undertaking such as this will serve as both a capstone in our development of a PhysicalChemistry laboratory curriculum for our major students and an engaging vehicle forundergraduate research. The project itself represents an important investigation into the natureof this particular class of cluster compounds and provides a number of achievable milestonesalong the way. The very nature of project involving repeated sample comparisons is well suitedfor a large class project. The University of New England is fairly unique among primarilyundergraduate colleges in that we currently have a capacity to perform work having at its centerthe tools of AFM. The enhancement of this capacity to include STM will allow us to orient thephysical chemistry laboratory experience around research training, as has been successfullyachieved elsewhere29. At the same time we seek to provide opportunities for our students towork with powerful, cutting edge tools with which to do promising science.



c) Description of Research Instrumentation Needs:Research Training (all senior faculty): In selecting an SPM manufacturer we sought an

instrument that is robust, reliable, easy-to-use and well supported. We feel the DigitalInstruments Nanoscope IIIa controller and MultiMode™ scanning probe microscope is exactlythis tool. The Nanoscope operates on a Windows NT platform, providing a hardy operatingsystem capable of withstanding lots of accidental abuse. NT is supported by a remote operationssoftware, Virtual Network Computing, essential for large-scale research training. Trainingstudents for independent operation is facilitated by a user-friendly software interface andsensibly engineered hardware. We are able to reduce the purchase cost of the microscope by$30,000 (non-matching funds) by trading in our old SPM controller (Nanoscope E) from thesame manufacturer. A detailed quote from Digital Instruments is included in the SupplementaryDocuments section.

Mark Johnson, biofilms: Imaging large surfaces for bacterial colonization requires 100 µmscan sizes and imaging in fluid environments with a non-invasive AC intermittent contact (a.k.a.TappingMode™-an acoustically driven vibrating tip feedback mechanism) to avoid dislodgingthe bacterial growth. Since adhesion of the bacteria to the surface is a central question in MJ’sresearch, being able to record phase differences (measurements of adhesion properties) hidden inthe intermittent contact signal and force-position measurements (Young’s moduluscharacterization) are essential. The proposed equipment includes a vertical engage “J” scannerfor long range imaging and fast approach to quickly monitor numerous positions over a biofilmsurface, and a phase box accessory to extract adhesion information. Our current configuration, aNanoscope E controller with lateral force microscope and mid range scanner cannot be used forundertaking any of the above proposed experiments.

Clinton Nash: metallocarbohedrenes: High resolution mapping of metal-substituted sootparticles is best suited for scanning tunneling and low current scanning tunneling microscopy.The MultiMode™ SPM is a modular microscope, requiring only a change in the detector toswitch operations from one near field imaging technique to the (e.g. AFM to STM). Theimportant piezoelectric scanners, lying underneath the detectors, can be used in any imagingmode. We currently have 1.0 µm and 10 µm scanners from our old lateral force microscope thatcan also be used on the proposed SPM, at a savings of several thousand dollars to the proposalbudget. These scan ranges will be used to establish specimen purity and high-resolutionimaging. The LCSTM TipView detector, requiring the Phase Extender to operate, is included inthe MRI proposal.

James Vesenka – Four stranded DNA characterization: Our existing SPM has the ability tomonitor “contact” topography, lateral force, deflection (error signal) and make tip-sample forcemeasurements. We have been measuring G-wire lengths as a function of growth time to helppredict the incubation times needed to construct long G-wires. These contact AFM images arevery hard on the biological samples, which must be thoroughly dried to avoid degradation duringimaging. No images of G-wires have been successfully captured in buffered media or otherliquids in contact imaging mode. Though fluid cell imaging can greatly reduce imaging forceson DNA, these forces (≈ 1.0 - 0.1 nN) are still high enough to sweep the G-wires off the surfacein buffered media. Imaging G-wires in alcohol, which causes duplex DNA to precipitate,denatures the four-stranded DNA. This is not to say that imaging in buffered media can not bedone. JV has successfully and routinely imaged G-wires in a fluid cell using DI’sTappingMode™ technology. These observations were made during visits to a colleague at IBMAlmaden while JV was an assistant professor of physics at California State University, Fresno.



Another imaging mode that was forfeited during JV’s cross-country move was low currentscanning tunneling microscopy (Figure 1). The hardware for this instrumentation was securedthrough a NSF ILI grant to CSUF (cf. Section “a”-Results from prior NSF support.) andremained at CSUF after his move. The proposed instrumentation includes LCSTM, fluid cellAC imaging technology, and Phase Box Extender essential for the proposed research. Thesehardware configurations overlap with the other two proposals such that there are noinstrumentation requests that are unique to a single researcher. Both silicon tapping tips (forhigh resolution imaging in air) and silicon nitride tips (for high resolution imaging in liquid) arealso included in the budget.Existing instrumentation

The University of New England currently supports a Digital Instruments (Santa Barbara, CA)“Nanoscope E” controller and a lateral force microscope (LFM). The controller will be used toleverage an upgrade path ($30,000 non-cost sharing credit) towards securing the above describedequipment. This controller and microscope reflect commercially available 1995 technology.The SPM software is DOS-based, taking over the computer’s high memory, and is therefore non-networkable. We have a dedicated off-line analysis computer for image processing, allowingmultiple users to be taking data and image processing simultaneously. This system is also DOS-based and non-networkable.

The PI (JV) and a Co-PI (MJ) share the same lab space, greatly enhancing cooperation andtraining of research students. Our lab is supplied with an array of basic molecular biology tools(tabletop centrifuge, protein and DNA separation, fume hood, etc.) The lab also has a Leitzmetallographic inverted microscope for the examination of geological specimens, and a Leicadifferential interference microscope for large biological samples. These are used by the Co-PIfor his geology and microbiology courses. The other Co-PI (CN) is a theoretician who issecuring parallel process computing resources needed to undertake rate constant calculations.The College of Osteopathic Medicine has a low-resolution scanning electron microscope(TopCon) for histology purposes, and Edwards evaporative coater that we can use to generatesoot particles, as well as several fluorescence microscopes used for muscle potential research.The department of Chemistry also has the following laboratory holdings.

Spectroscopy holdings: Perkin-Elmer lambda-20 Scanning UV-Vis, Beckman DU-2 withGilson upgrades Single-wavelength UV-Vis and Spectronic 20 Vis Spectrophotometers,Aminco-Bowman Series 2 and Jasco Scanning Spectrofluorometers, InstrumentationLaboratories 251 and Perkin Elmer 3110 Atomic Absorption/Emission (flame), BioRad FTS-7Fourier-transform Infrared, Varian T-60 Nuclear Magnetic Resonance

Electrochemistry holdings: BioAnalytical Systems CV-50W Multipurpose ElectrochemicalAnalyzer, Misc ISEs; computer-interfaced pH/ion mV meters Potentiometry, ElectrogravimetricAnalyzer

Chromatography holdings: Hewlett-Packard GCD GC-Mass Spectrometer and Hewlett-Packard 5890 GC with FID and TCD Capillary Gas Chromatography, Perkin-ElmerAutoSystem, GC Gow-Mac Model 350 dual column (2 units) Gow-Mac Model 550 Packed-column Gas Chromatography, : Waters pump & injector w/Varian variable wavelength UVdetector, Perkin-Elmer w/PE variable wavelength UV and refractive index detectors, Beckmanpump & Waters conductivity detector (ion chromatography) High Performance LiquidChromatography



d) Impact of Infrastructure ProjectsThe educational objectives for graduates of UNE are encapsulated by a “core” curriculum.

The final part of the core sequence involves a “capstone” experience. For science students thistypically consists of a research project that ties together the different threads of their educationaltraining. The chemistry/physics department was recently (since this past year) split off from theDepartment of Life Sciences at UNE. The department’s instrumentation holdings consist ofstandard older model undergraduate laboratory tools. We have little in the way high-resolutionanalytical equipment. Any sophisticated instrumentation analysis is typically sent off camps.For example, all high resolution NMR imaging is farmed out to large research institutions, theclosest being the University of New Hampshire over 90 minutes away.

Our program proposes to integrate the proposed scanning probe microscope into the capstoneexperiences in two ways. The traditional approach involves individual, faculty-guided integratedresearch projects. These projects would start in the fall of 2001, involving life and health sciencemajors in a microbiology course (BIO226). A class project, involving the real-time analysis ofbiofilm growth, will examine a range of practical problems, such as the fouling of boat hulls tothe construction of organic matrices needed to support life on aquatic surfaces. In the spring of2002 our biochemistry majors will examine metallocarbohedrenes as part of their physicalchemistry (CHE327) class project. They will use high resolution scanning tunneling microscopyunder inert environments to see the differences in atomic structure induced by metalsubstitutions. During the summer the above two projects are expected to continue with highlymotivated undergraduate research assistants recruited from these courses. In addition,undergraduates will have research opportunities in self-assembled DNA as well as examiningfour stranded DNA electronic properties. The proposed SPM would dramatically increase theopportunities for capstone experiences demanded by the core curriculum at UNE.

Scanning probe microscopy has evolved from its origins as a simple high-resolution three-dimensional topographic imaging tool to a powerful array of sister technologies sensitive to avariety of “near-field” detection schemes. SPM technology is routinely used in the research andcommercial enterprises ranging from the measurement of individual rupture forces of moleculesto microfabricated wafer characterization. When JV arrived at UNE he was able to bring hisolder Nanoscope E controller and lateral force microscope. Maine is home to a growing numberof silicon and biotechnology enterprises. However, in the state of Maine UNE is one of only afew universities that have any SPM tools. Securing a late-model SPM with non-invasiveimaging abilities and a broad range of near-field detection mechanisms will dramatically expandthe range of our students’ research opportunities AND the number of students impacted (est.twenty-fold increase). These skills will translate to competitive applications for graduate schooland improve our graduates’ marketability in Maine’s growing high technology industries. Weanticipate improving the representation of women in chemistry through the recruitment efforts inthese advanced courses. Taught by all three senior personnel the top students from our generalservice courses at UNE are dominated by women who tend to shy away from the biochemistrymajor. We are particularly eager to recruit women from the pool of health and life sciencemajors (60% women) into biochemistry.



e) Project and Management Plans:The principal investigator, JV, will be responsible for the SPM maintenance and the research

training of faculty and students in SPM operations. UNE’s finite resources and majorrequirements rule out the use of undergraduates or staff as SPM operators. On the other hand,the PI’s extensive experience with the SPM’s manufacturer and training faculty and students atfour universities (cf. vita) make him an obvious choice for staffing the proposed equipment. ThePI has the expertise in computer hardware and operating systems to trouble shoot most problemson the proposed SPM. Based on a standard 9-credit/semester faculty load JV will have a 1-credit/semester duty to operate and maintain the SPM during the academic year. He will also beresponsible for heightened operations, maintenance and training of the SPM during the summer,an estimated 2 credits/summer. This corresponds to one month of support out of the three-monthsummer research period providing JV two thirds of his summer time to pursue his ownundergraduate research projects. The cost of supporting the maintenance and operations of theSPM by the PI is less than a half time staff position. The level of support will continue after theconclusion of the grant (cf. letter of support from the Dean). No manufacturer’s maintenancecontracts are needed because of the outstanding support Digital Instruments provides.

Instrument time during the academic year will be prioritized first towards research trainingfor large classes and secondly toward individual undergraduate research projects. The latter havethe flexibility of using the equipment during off-hours. Furthermore, individualized advanceduser training will be undertaken during the summer and shared equally amongst the researchgroups. Large class training sessions will be facilitated by Virtual Network Computing (VNC)1software, enabling each class member to monitor and/or operate the networked SPM from theirown individual workstation located in an adjacent computer laboratory. This laboratory also hasa large wet lab bench space for sample preparation purposes. Individual student instruction willbe undertaken directly in the SPM lab. In deference to the minimal resources our researchcommunity has, no user fees will be charged. We will optimize outreach through recruitment ofstudents and faculty in advanced courses, dissemination during our yearly on-campus researchsymposia, and through weekend professional development workshops supported our dean. Weseek to grow the use of the SPM laboratory facilities through training and, ultimately,autonomous monitored operations to a level of about four hours daily during the academic yearand eight hours/day during the summer. A timeline for the first two years is discussed below.

Fall 2001: Installation of Digital Instruments Nanoscope IIIa controller and MultimodeScanning Probe Microscope. Training of one Co-PI (MJ) and his BIO226 students in the SPMoperations. Estimated involvement of one faculty and 18 undergraduate students.

Spring 2002: Training of one Co-PI (CN) and his CHE327 students in the SPM operations.Estimated involvement of one faculty and 12 undergraduate students.

Summer 2002: Core group of capstone activity students will participate in intensive summerresearch projects. Estimated involvement of 2-3 faculty and 6-9 undergraduate students. JVrecruits one more UNE faculty in SPM research as part of capstone and/or research educationalexperiences. Remote monitoring of trained faculty and students using VNC software.

Fall/Spring 2002-3: Outreach to the new faculty member and their students as part ofgrowing SPM usage and training in SPM operations. Completion of any unfinished capstoneresearch experiences. Co-PIs will consult with JV as necessary in SPM use but are expected tooperate equipment autonomously. Estimated involvement of 1 new faculty and 24-36undergraduate students.



Biographical Sketches: Mark Johnson

Biological Sciences (Mark Johnson – MJ): Mark undertakes research in locations rangingfrom the swamps in west Alabama to the pack ice of Antarctica. The unifying theme to all of hisresearch has been curiosity about the dynamics of microorganisms in their natural environments.The incredible diversity of the organisms in the sub-visible world, the "Micro-cosmos," is one ofthe least understood realms in ecology. The assays Mark performs on the microorganisms in hisresearch involve a basic understanding of photosynthesis and the motion of these systemsthrough fluids. The latter requires his students to have a basic understanding of fluid dynamics,the former requires understanding the nature of photon-matter interactions.

In Antarctica one of the systems Mark investigated was the bacteria, protozoa and algae whoform a complex community in the minute fissures in pack ice filled with highly saline brine. Inthe Alabama wetlands he tracked the populations of ciliates, flagellates, amoebas, algae, andbacteria and their productivity through various seasonal changes to get one of the most completepictures of microbial populations ever done. An unexpected finding there was that smallflagellate protists increased in numbers during the winter when just about every other measure oflife dimmed. In addition, Mark has worked on the development of a technique to determineactivity levels in bacteria. Putting these two factors together is something worth investigatinghere in Maine. Currently, Mark is setting up incubation chambers to do controlled lab studies ofthe effects of temperature on the behavior of ciliates from our local ponds and wetlands; as wellas to try rehydrating naturally freeze-dried cyanobacterial mat communities from Bratina Island,Antarctica. He has undertaken extensive research characterizing macroscopic affects of biofilmgrowth in riverine systems. He is pursuing a better understanding of the microscopic processesinvolved in the development of biofilms.



Biographical Sketches: Clinton Nash

Physical/Inorganic Chemistry (Clinton Nash - CN): His primary research interests involvethe theoretical chemistry of heavy elements, particularly those in which relativistic effects aremanifest such as the actinides and transactinides. CN’s Ph.D. work focused on elucidating theperiodic relationships among these transactinides and their familial congeners as well as thesource of disruptions in this periodic behavior. Although he still consider this “generalchemistry of the transactinides” to be interesting and will continue to explore it in future work,CN has begun to apply the same advanced computational methods to problems of moreimmediate environmental interest such as aqueous actinide chemistry. More generally, he isinterested in the application of computational methods to all branches of chemistry, includingbiochemistry, especially as they impact current technological and environmental issues.

CN is also pursuing an analysis of the relationship between fullerenes and the related metal-containing species known as metallocarbohedrenes or met-cars. He teaches the biochemistrymajors advanced courses in inorganic and physical chemistry. CN intends to take advantage ofthe small departmental structure of the University of New England to break down the artificialbarriers that can often be erected between the physical sciences. As it currently stands, there isno laboratory component to the physical chemistry course at UNE. One opportunity forintegration that presents itself is to use our local capacity to do scanning probe microscopy toexamine the structures of surfaces. Addressing questions surrounding the electronic structures ofmaterials are fundamental to both CN’s research and to the approach he takes to lectures. To beable to take students into the physics laboratory and demonstrate the nature of electron tunnelingand illuminate, in a very concrete way, the consequences of atomic and molecular structure is avery exciting prospect. An example of this will include the AFM and STM examination offullerenes, materials in which the molecular and electronic structures are intimately intertwined.Students would perform the experiments to determine an approximate molecular structure forthese materials and then justify their structures in terms of molecular orbital theory (e.g.Giancarlo et al.29.



Biographical Sketches: James Vesenka

Biophysics, Material Sciences (James Vesenka – JV): JV teaches general physics andundertakes research in molecular self-assembly and physics education. He has 11 years ofexpertise in the area of scanning probe microscopy (SPM), including the successful developmentof a networked SPM core facility at California State University, Fresno (see section “a” onResults from Prior NSF Support.) JV received post-doctoral training at the University of Oregonat the Institute for Molecular Biology where he developed tools for imaging duplex DNA withthe AFM in the laboratory of Carlos Bustamante (now at UC Berkeley). The power of SPMtechnology lay in its ability to image biological specimens without coatings or freezing, i.e. inthe DNA’s native state. JV had two papers published in Science which detailed the procedurefor imaging DNA in liquid30 and measuring DNA melting-angles during RNA polymerasetranscription31.

He continued his post-doctoral research as part of the Signal Transduction Training Group atIowa State University in the laboratory of Eric Henderson. At ISU JV developed verticalcalibration standards32 and image reconstruction software with Professor Richard Miller from theDepartment of Mathematics33. The two papers combined to provide reliable recreation ofsurface topography artificially broadened by the finite geometry of the SPM imaging tip. JV alsoassisted in high resolution imaging of nucleo-proteins, discovering the conditions under whichdetailed observations of histone-DNA packing in chromatin were possible34. In 1995 JV also co-authored an article providing images of a novel form of four-stranded DNA12, and started anassistant professor position of Physics at California State University Fresno (CSUF).

At CSUF JV concentrated his effort on using the SPM as an undergraduate research trainingtool, publishing several articles with his students that used the G-wires as a target system. Aspart of a CSU system-wide effort, JV secured NSF ILI funds for a network scanning probemicroscope anchored at CSUF. Access to the equipment was provided on campus to individualusers and to larger audiences through remote operations using virtual network computing. Inaddition to his own professional research interests, JV has maintained and operated the SPM’sever since his first exposure to scanning probe technology over 11 years ago. He has trainedalmost 50 graduate and undergraduate students in SPM operations and helped them developresearch skills that helped them to move on to professional school or to secure Ph.D. in sciencefields ranging from Molecular Biology and Materials Science.

JV will assume all duties for maintaining and operating the proposed SPM. Vita attached atend.



James Philip Vesenka

University of New EnglandDepartment of Chemistry and Physics11 Hills Beach RoadBiddeford, ME 04005Office: (207) 283-0170 Ext. 2560, Fax: (207) 282-6279Email: [email protected]: faculty.une.edu/cas/jvesenka/DrVIndex.HTM

Education & Research ExperiencePost-graduate Researcher, Iowa State University, 8/92-6/95 P.I.: Prof. Eric HendersonPost-graduate Researcher, University of Oregon, 10/90-6/92. P.I.: Prof. Carlos BustamantePost-graduate Researcher, University of California, 10/89-9/90. P.I.: Prof. Yin YehPh.D., Physics, University of California, Davis, September 1989.M.Sc., Physics, University of California, Davis, March 1986.B.A., Physics/Chemistry, Clark University, Worcester, Massachusetts, May 1982.

Teaching ExperienceAssistant Professor, Chem/Phys Department, University of New Enlgand (8/99 – present)Assistant Professor, Physics Department, California State University Fresno, (8/95-6/99)Adjunct Assistant Professor, Physics Department, Iowa State University, (8/94-12/94)SPM Instructor, Zoology and Genetics Department, Iowa State University, (9/92-6/95)SPM Instructor, Institute for Molecular Biology, University of Oregon, (9/89-8/92)

SPM Operations and MaintenanceDigital Instruments (Santa Barbara, CA) Nanoscope E w/ lateral force microscope. Responsible for the

maintenance of the SPM and trained four undergraduates in its operation at the University of New England (8/99 –present).

Park Scientific (Sunnyvale, CA) SPM controller w/ BioProbe biological Atomic Force Microscope. MolecularImaging (Tempe, AZ) Picoscope controller and SPM. Digital Instruments Nanoscope E w/ Lateral ForceMicroscope. Responsible for the maintenance of all SPM and training of 6 graduate students and 14 undergraduatesin the operation at California State University Fresno (8/95 – 6/99).

Digital Instruments Nanoscope III w/ Bioscope biological Atomic Force Microscope. Digital InstrumentsNanoscope II atomic force microscope and scanning tunneling microscope. Responsible for the maintenance of thetwo machines in addition to training four graduate students and two undergraduates in their operation at Iowa StateUniversity (9/92-6/95).

Digital Instruments Nanoscope II w/ atomic force microscope. Brief experience with Digital InstrumentsNanoscope I scanning tunneling microscope. Responsible for the maintenance of the SPMs and trained fivegraduate students and one post-doc in their operations at the University of Oregon (9/89-8/92).

Professional AffiliationsAmerican Association of Physics TeachersCouncil on Undergraduate ResearchSigma-Xi, undergraduate research societyProject Kaleidescope

Seven published student-initiated abstracts:C. Wilson & J. Vesenka, "Atomic Force Microscopy of Olivine" Scanning 18:3, 254 (1996).J. Stafford & J. Vesenka, "An SPM Internet Site" Scanning 18:3, 252-253 (1996).C. West, I. Kumar, & J. Vesenka, Scanning 19:3, journal cover (1997).D. Detweiler, S. Laslovich,. & J. Vesenka, “General microscopy” Scanning 19:3, 205 (1996).I. Kumar, C. West,. & J. Vesenka, “Orientation of G-wires”, Scanning 19:3, 234-235 (1997).J. Root,... & J. Vesenka, “LCSTM of G-wires”, Scanning 19:3, 243-244 (1997).C. Vellandi,... & J. Vesenka, “Inexpensive Tapping SPM”, Scanning 19:3, 246 (1997).



Selected List of Recent Professional Presentations: (> 50 lifetime presentations)J. Vesenka, “Remote Microscopy operation at CSU Fresno, Scanning. 21 53-54 (1999). Organized and chaired asession on remote operations of networked equipment, Scanning ’99, Chicago IL (4/99).J. Vesenka, “Potential Applications of Scanning Probe Microscopy in Gene Therapy”, Scanning ‘98, Baltimore, MD(4/98).J. Vesenka, I. Kumar, & C. West, “The orientation of G-wires on mica.” 44th American Vacuum Society meeting,San Jose, CA (11/97).J. Vesenka, T.C. Marsh, J. Root, W. Han, S.M. Lindsay, E. Henderson, “Electronic Properties of ‘G-wire’ DNAinvestigated by Low Current Scanning Tunneling Microscopy,” 44th American Vacuum Society meeting, San Jose,CA (11/97).

Last 10 Publications from 38 Lifetime: “*” = Student Participant38. I. Kumar*, T. Muir*, B. Garcia*, W.H. Han, J. Zhu, T. Marsh, J. Bolonick, E. Henderson, & J. Vesenka

“Orientation of G-wires on Mica”, Under revision to Biophys. J.

37. T. Muir, E. Morales, J. Root, I. Kumar, B. Garcia, C. Vellandi, D. Jenigian, T. Marsh, E. Henderson, & J.Vesenka “The morphology of duplex and quadruplex DNA on mica.” J. Vac. Sci. Technol. A. 16, 1172-1177(1998).

36. C. Wilson* & J. Vesenka, “Atomic Force Microscopy of Olivine”, In press, AFM/STM III.

35. J. Vesenka & E. Morales* “Scanning Probe Microscopy in Biology with Potential Applications in Forensics.”In press, AFM/STM III.

34. J. Vesenka, C. Vellandi, I. Kumar*, T. Marsh, & E. Henderson, “The diameter of duplex and quadruplex DNAmeasured by Scanning Probe Microscopy.” In press, Scanning Microscopy (1997).

33. Yang, G., Vesenka, J.P., and Bustamante, C. Effects of Tip-sample Forces and Humidity on the Imaging ofDNA with a Scanning Force Microscope. Scanning 18 (5), (1996).

32. W. Fritzsche, L. Martin, D. Dobbs, D. Jondle*, R. Miller, J. Vesenka, E. Henderson, “Reconstruction ofRibosomal Subunits and rDNA Chromatin Imaged by Scanning Force Microscopy”, J. Vac. Sci. Technol. B14, (1996).

31. J. Vesenka, T. Marsh, R. Miller, & E. Henderson, "High Resolution Atomic Force Microscopy Reconstructionof G-wire DNA." J. of Vac. Sci. Technol. B 14, 1413-1417 (1996).

30. J. Vesenka, “Facile Procedure for Screening Nucleoproteins for Imagibility”, H. Gaub Module Ed., Accepted toProcedures in Scanning Probe Microscopies (1996), J. Wiley & Sons, Ltd.

29. W. Fritzsche, J. Vesenka, & E. Henderson, "Scanning Force Microscopy of Chromatin", Scanning Microscopy,9, 729-739 (1995).

PatentsE. Henderson & J. Vesenka, “Decontamination Device and Method Thereof”, Submitted, U.S. Paten

Application Serial N. 08/766,871, United States Patent and Trademark Office (1998)R. Miller & J. Vesenka, “Reconstructing the Shape of an Atomic Microscope Probe”, United States Patent No.

5,591,903, United States Patent and Trademark Office (1997).



Revised Budget Impact Statement

Item NSF-RequestSummer staffing wages and benefits $14,860

The PI has 11 years of experience with Digital Instruments SPMs. He anticipates training 10students/year and three faculty/year in SPM operations during the academic year and summers.In addition the PI anticipates training an additional 30 students/year in advanced courses(microbiology and physical chemistry) via networked operations during the academic year. TheDean’s Office from the College of Arts and Science at UNE is dedicated to the long-term supportof this equipment (see attached letter of support).

125 µm Vertical engage scanner $9000The scanner is essential for biofilm analysis to help characterize the differences betweenbacterial “clustering” versus uniform surface covering. The long-range scanner will also benecessary for the characterization of long G-wires for conductivity measurements. Extender electronics (phase box): $8,000The extender electronics are essential for monitoring the adhesion characteristics of the bacterialcolonies for biofilm growth.

Low current STM detectors $6,350The extender electronics is also essential for extracting low current STM signal and electroniccharacterization of the G-wires and metallocarbohedrenes.

Nanoscope E to Nanoscope IIIa Control Station Upgrade: $45,000Upgrade path from UNE’s existing Nanoscope E SPM reduces the cost of the equipment by$30,000. The controller operates the scanners and feedback systems. This piece of equipment isneeded to be able to handle the variety of imaging modes essential to the proposed research (ACmethods such as intermittent contact and low current STM).

Nanoscope Multimode SPM with fluid cell: $36,000The Multimode SPM is a modular microscope set-up capable of supporting several differentimaging modes through a simple change in detectors. The fluid cell is essential for the biofilmcharacterization and G-wire kinetics to be able to characterize live samples in buffer media.

Silicon Nanoprobes and Silicon Nitride Probes (AFM tips: $7,500The AFM tips are essential for imaging samples and must be purchased commercially. DI is themost reliable manufacturer of AFM tips. Purchasing in wafers is the least expensive option.One wafer of each of these tips is expected to last several years. No STM tips are requestedsince we will cut our own from platinum-iridium wire purchased separately.

Video Only Microscope: $5850This “optical navigator”, capable of 1.5 µm resolution, will allow us to locate biofilm bacterialcolonies for imaging under the long-range scanner and for locating electrode contacts on themicrofabricated conductivity test structures for G-wire conductivity measurements.

Indirect costs: $8,040Charged to NSF at a negotiated rate of 66% on wages.

Amount requested from NSF $140,600

UNE’s cost-sharing is 30% above $100,000 or $12,180

The total (NSF request and UNE cost share) budget for two years is $152,780



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References: 1 http://www.uk.research.att.com/vnc/index.html2 D. Goldhaber-Gordon, M.S. Montemerlo, J.C. Love, G.J. Opiteck, & J.C. Ellenbogen, IEEEProc., April 1997.3 R. W. Keyes, Physics Today, 8, 42 (1992).4 D.L. Klein, R. Roth, A.K.L. Lim, A.P. Alivisatos, & P.L. McEuen, Nature 389, 699 (1997).5 C. Dekker, Physics Today 52, 22 (1999). andP.G. Collins & P. Avouris, Scientific American, 12, 62 (2000).6 T.J. Meade & J.F. Kayyem, Angew. Chem. Int. Engl. 34, 352 (1995).7 J. Jortner, Proc. Nat. Acad. Sci. USA, 95, 12759 (1998).8 R. Guckenberger, M. Heim, G. Cevc, H.F. Knapp, W. Wiegräbe, & A. Hillebrand, Science,266, 1538 (1994).9 T. Muir, E. Morales, J. Root, I. Kumar, B. Garcia, C. Vellandi, D. Jenigian, T. Marsh, E.Henderson, & J. Vesenka, J. Vac. Sci. Technol. A. 16, 1172 (1998).10 E. Braun, Y. Eichen, U. Sivan, & G Ben-Yoseph, Nature 391, 775 (1998).11 D. Sen & W. Gilbert, Biochem. 31: 65 (1992).12 T. Marsh, J. Vesenka, & E. Henderson, Nucleic Acids Res. 23, 696 (1995).13 D. Sen & W. Gilbert, Biochem. 31: 65 (1992).14 T. Muir, E. Morales, J. Root, I. Kumar, B. Garcia, C. Vellandi, D. Jenigian, T. Marsh, E.Henderson, & J. Vesenka, J. Vac. Sci. Technol. A. 16, 1172 (1998).15 Heim, M., R. Steigerwald, & R. Guckenberger, J. Structural Bio. 119, 212-221 (1997).16 Fritzsche, W., J M Kohler†, K J Bohm‡, E. Unger‡, T. Wagner, R. Kirsch, M Mertig and W.Pompe, Nanotechnology 10 331–335(1999).17 Fritzsche, W., K. J. Bohm, E. Unger, & J. M. Kohler, Applied Phys. Lett. 75 2854-2856(1999).18 The equipment, A Digital Instruments Nano E controller and Lateral Force Microscope werepurchased through a 1995 Research Corporation grant CC4204 to JV.19 J. Vesenka, R. Miller, & E. Henderson, Rev. Sci. Instr., 65; 7, 2249-2251 (1994).20 American Society for Microbiology website http://www.asmusa.org/edusrc/edu34i.htm21 Kroto, H.W.; Heath, J.R.; O'Brein, S.C.; Curl, R.E.; Smalley, R.E. Nature, 318, 162 (1985)22 Guo, B.C.; Kerns, K.P.; Castleman, A.W. Science, 255, 1411 (1992).23 Prinzbach, H.; Weiler, A.; Landenberger, P.; Wahl, F.; Wörth, J.; Scott, L. T.; Gelmont, M.;Olevano, D.; v. Issendorf, B. Nature, 407, 60 (2000).24 Dance, I. J. Am. Chem. Soc. 118, 6309 (1996).25 Paquette, L.A.; Ternansky, R.J.; Balogh, D.W.; Kentgen, G.; J. Am. Chem. Soc., 105, 5446(1983).26 Bursten, B.E. J. Am. Chem. Soc., 105, 121 (1983).27 Lee, S.H.; Gotts, N.G.; Vonhelden, G.; Bowers, M.T. Science, 267, 999 (1995).28 Yu, H.G.; Huber, M.G.; Froben, F.W. Applied Surface Science, 86, 74 (1995).29 Leanna C. Giancarlo, Hongbin Fang, Luis Avila, Leanard W. Fine, and George W. Flynn, J.Chem. Ed. 77:1, 66-71 (2000).30 H. Hansma, J. Vesenka, G. Kelderman, H. Morrett, R.L. Sinsheimer, V. Elings, C.Bustamante, & P.K. Hansma, Science, 256, 1180-1184 (1992).31 W.A. Rees, R.W. Keller, J.P. Vesenka, G. Yang, & C.J. Bustamante, Science, 260, 1646-1649(1993).



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