A Networked Scanning Probe Microscope for Research Training

A Networked Scanning Probe Microscope for Research Training.



DMR-0116398



Non-Technical Abstract

The University of New England (UNE) requests NSF funds for the acquisition of a

“networked” scanning probe microscope (SPM) for undergraduate research training in the fields

of biophysics, biochemistry and microbiology. UNE is a non-Ph.D. granting institution that

seeks to provide its students undergraduate research experiences as a “capstone” part of the

educational experience. The core SPM facility will help to provide a multi-disciplinary research

facility that will help to bridge the artificial divide between life and physical sciences. We will

actively recruit diversity in student researchers from courses in advanced biology and chemistry

laboratories. Access by advanced life and physical science courses with large enrollments will

be through networked remote operations. Direct undergraduate use will be undertaken under the

guidance of UNE faculty in their respective research areas.



Technical Abstract

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

scanning probe microscope (SPM-Digital Instruments NanoIII) for undergraduate research

training in the analysis of quadruplex DNA, biofilms and metallocarbohedrenes. Optimal use of

the 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. Our

environmental microbiology Co-PI will examine the growth processes of biofilms responsible

for fouling surfaces exposed to running water. Our physical-inorganic chemist co-PI is interested

in the structure and electronic properties of metallocarbohedrenes. The PI’s interest focuses on

the growth kinetics and structure of a novel four-stranded “G-wire” DNA and research training.









University of New England SPM MRI, Page 1

A Networked Scanning Probe Microscope for 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 program

solicitation.

NSF Unit Consideration: Directorate for Biological Sciences OR Directorate for Mathematical

and Physics Sciences.: Division of Materials Research









University of New England SPM MRI, Page 2

A Networked Scanning Probe Microscope for Research Training.



Project Summary



Project Goals

The 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 the

fields of biophysics, biochemistry and microbiology. UNE is a non-Ph.D. granting institution

that seeks to provide its students undergraduate research experiences as a “capstone” part of the

educational experience. The core SPM facility will help to provide a multi-disciplinary research

facility that will help to bridge the artificial divide between life and physical sciences. We will

actively recruit diversity in student researchers from courses in advanced biology and chemistry

laboratories. The proposed instrumentation is part of a concerted effort to recruit women into

physical science majors.

To optimize instrumentation usage our effort is two pronged. First we will be providing

networked SPM access to larger class activities (between 10 and 20 students) and secondly we

are supporting intensive individual research projects. Students participating in advanced courses

can efficiently be trained on the SPM through the use of remote operations using from an

adjoining computer work station facility. The undergraduate majors in these courses range from

basic sciences to health related fields. These courses will culminate in class research projects

that emphasize research training tailored to each student’s need. Students participating in

individual biology, chemistry and physics research projects will run the SPM instrumentation

directly 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 biofilms

responsible for fouling surfaces exposed to running water. The time scale under which this

growth takes place (µm/min) is well suited to SPM examination. Our physical-inorganic chemist

co-PI is interested in the structure and electronic properties of metallocarbohedrenes. Both co-PIs

teach instrumentation as an integral part of their advanced laboratory courses. In these large

laboratory classes the Co-PIs plan to create a culminating class research project in which groups

of students are assigned tasks needed to complete the entire project. The PI’s interest focuses on

the 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, and

modeling of the self-assembly process. The G-wires also present themselves as an interesting

candidate for a biological “nanowire.” The advanced near-field detecting capabilities of the

proposed instrumentation are essential for the above proposed research. Providing our

undergraduates relevant research training in SPM technology is essential for making our students

competitive in Maine’s flourishing biotechnology and semiconductor industries.









University of New England SPM MRI, Page 3

A Networked Scanning Probe Microscope for Research Training.



a) Results from Prior NSF Support



DUE 9750942: Networked Scanning Probe Microscope

Brief Summary:

James Vesenka (JV) was awarded an NSF Instructional Laboratory Improvement award to

develop a networked scanning probe core facility at California State University, Fresno. The

facility was designed to serve large remote audiences, on and off campus, through broad band or

internet connections, and to serve as a research training tool for advanced undergraduates at

CSUF. 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 discipline

After 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 onto

the 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 operations

were limited to a commercial software package “PC Anywhere,” with limited platform abilities

in 1997. In addition, the commercial licensing fee of “PC Anywhere” made usage by the

intended audiences of faculty and students from other CSUs, community colleges, and local high

schools prohibitive. A free-ware package called virtual network computing (VNC1) by AT&T

lab associates was discovered on the internet in 1998. VNC allowed any trained user of the

SPMs to access the networked microscope from a remote site with a password. Though a

significant improvement over one-way transmission, the finite transfer rate (about 10 kb/sec) fell

far 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 a

prohibitive cost ($3000/month). Cable technology appeared to be the most cost-effective

solution, but cable standards were not in place by the time of grant completion. In sum, we

followed the available technology and tried to develop a strategy to secure the widest possible

and appropriate usage. One of the larger difficulties was securing adequate operational support.



Major findings

In 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 research

as well as molecular biology research. He was NOT provided any release time to support the

remote operation activities. JV submitted three grants to secure post-doctoral and/or technical

support over the life of the ILI grant. The first was submitted jointly to a California State

University technology development division with CSU-Hayward, the second to the NSF's

Engineering Experiences program and the last to the NSF MBRS program. All grants were

unsuccessful. Funding for staff support through CSUF was not possible because of staffing

freezes.

Although JV achieved the overall objective of developing a remotely operated

instrumentation core facility within the first two years of the grant, the ultimate objective of

creating a self-sustaining imaging facility was not realized during his tenure. In the spring of

2000 JV resigned from CSUF to help his family in New England, leaving the equipment in the

capable hands of his colleagues. Continuation of the project’s objectives are being undertaken

by one of the grant’s senior personnel, Dr. Alejandro Calderon-Urrea (CSUF Biology). He has







University of New England SPM MRI, Page 4

A Networked Scanning Probe Microscope for Research Training.



received MBRS funding and will be able to sustain a trained SPM operator to run the SPM

facility for the next four years.



What research and teaching skills and experience has the project helped provide to those

who worked on the project?

Over 20 students received research training on the SPM during the life of the grant. The

improvement in student research skills associated with the networked SPM was dramatic. Each

participant involved in research was required to present a paper at either a local or national

research symposium. In all but two cases, the research influenced or directly aided the student's

graduate school aspirations. In addition, three faculty members were trained in the operations of

the microscopes. These include a senior personnel at CSUF; a developmental biologist, Dr.

Calderon; an entomologist and microscopist, Dr. Fred Schreiber; and a biochemist, Dr. Dave

Chester 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 one

of two active research faculty in Biology at CSUF. Dr. Schreiber has since become the biology

department 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 grant

had originally proposed.



What outreach activities have you undertaken to increase public understanding of, and

participation 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 the

academic year, discussing progress in development of remote microscopy. An average of five

general 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 Dave

Andrews, was used as a forum to advertise the availability, and experiment with the utility of

remote microscopy. The integrated science program involved 160 teachers in the summer of

1998 and 1999 mostly from the Fresno Unified School District. Two of Paul Lake’s students

from the Clovis Unified School District, undertook part time summer research in the lab. Lastly

the web site received over 10,000 hits in the four years of operation at

http://maxwell.phys.csufresno.edu:8001/~csufspm/csufspm.html

The web site has since been closed due to JV’s departure from CSUF.









University of New England SPM MRI, Page 5

A Networked Scanning Probe Microscope for Research Training.



b) Research Activities

PI: James Vesenka (JV), University of New England biophysics, surface and material science:

Introduction:

Rapid advances in electronics miniaturization demands the development of a new generation

of micro-electronic circuitry capable of single electron delivery.2,3,4 The carbon nanotube

appears to be the strongest “nano-wire” candidate. However, nanotubes dramatically change

their electronic character when only a single carbon atom is out of place, or if the tubes are even

slightly 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 the

molecular 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 has

been established as the dominant form of electron migration in longer segments of DNA7. Under

hydrated conditions, LCSTM reveals that duplex DNA is a poor electron carrier compared to the

hydrated surface of mica8. Furthermore duplex DNA appears to collapse on the surface of mica

and other silicates (e.g. glassware). Such behavior would affect electron transfer along the

twisted backbone-shape9, raising continuity concerns. Biomolecular templates have been

employed as masks and scaffolding to create traditional miniature metalized conductors.

However, the large grain-size of the conductive metals make the resulting structures highly

irregular, and commercially unappetizing10. An ideal “nano-wire” would combine the flexibility

of 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 and

electronic structure of a novel four-stranded self-assembled DNA. These forms of nucleic acid

complexes have been discovered in the end regions (telomeres) of chromosomes. The hairpin

structures, 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 micrometer

lengths 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 grown

determined the type of caged metal cations (e.g. Mg, K, or Na), integrated into the structure13. In

comparison to double-stranded DNA, the integrated metal cations surrounded by the four-

stranded phosphate backbone plus extensive hydrogen bonding of G-wires might facilitate lateral

conductivity over their uniform 2.4 nm diameter and micrometer-lengths. The uninterrupted

nanometer-scale morphology of G-wire DNA, as characterized in my former lab at California

State 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 tunneling

currents above a picoampere at high humidity14. To our knowledge these observations were the

first 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 in

humid air, depending only upon the applied “bias” voltage15. Under low voltages, high-

resolution imaging is maintained by conduction through the hydration layer. At high voltages

ballistic tunneling takes place through the air gap into the hydration layer, at the expense of

resolution. What made our initial results interesting is the factor of 10 to 100 increase in







University of New England SPM MRI, Page 6

A Networked Scanning Probe Microscope for Research Training.



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 of

the 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, have

developed microfabricated substrates for biopolymer conductivity measurements16. The

biopolymers need to be several micrometers in length in order to span the gold contacts, or to be

attached to the contacts through electron beam deposition17. After establishing an SPM

laboratory at the University of New England,18 my lab set about growing G-wires from a

commercially available, purified oligo sequence. Two undergraduates undertook preliminary

studies this past summer to establish the time needed for the growth of long G-wires for

simplified macroscopic electronic characterization. Several problems were immediately evident.

The zero time “pure” G4T2G4 oligo sequence was contaminated with self-assembled G-wires

averaging 30 nm in length (Figure 2). Approximately 40 oligos are bound up in this length of

molecule. The pure oligo should have a length of about 3.4-nm, below the 5-nm lateral

resolution limit of the best contact AFM imaging. In principle pre-melting (heating the G-wires

briefly to near boiling temperatures) can reduce partially assembled G-wires into their oligo

building blocks. This is non-trivial matter, since heating the oligo can also lead to irreversible

dissociation. 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 micrometer

lengths published by Marsh, Vesenka, and Henderson12. Figure 3 is a plot from preliminary data

relating the “reduced” mean length of the G-wires, measured by the AFM, as a function of

growth time. The experimental fit to the data leads to a power law with the general equation:

(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 in

the original studies with Marsh et al.9, where we grew micrometer length G-wires in a few

months time at Iowa State University, there is clearly an error somewhere in our sample

preparation at UNE. After we independently confirmed that purity of the oligomer was not in

question, obvious areas of scrutiny were the growth cocktail media and SPM sample preparation.

Additionally, we undertook a literature review of molecular self-assembly growth

characterization to help define baselines but found no discussion applicable to our system.

We propose a two-year undergraduate research project to establish the conditions for

conductivity 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 scales

needed to grow practical size (micrometer length) G-wires. In particular, an answer to the first

question will provide us with better samples to address the issues of conductivity. All four

questions 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-resolution

images of the G-wires both in dry air and liquids. These measurements are essential to establish







University of New England SPM MRI, Page 7

A Networked Scanning Probe Microscope for Research Training.



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 the

lateral features of biological samples due to the finite tip geometry19. One of the enormous

advantages of imaging in buffered media is the ability to use G-wire cocktails at their full

concentrations. G-wire dilutions of about a factor of 1000 are needed, in PURE WATER, for

imaging in air due to the artifact-induced contamination from residual dried buffer ions. Such

dilutions are likely to be deleterious to the G-wires because of their entropically driven growth

processes. Dilution drives disassembly of the molecules into their constituent building blocks,

the guanine oligonucleotide tetramer. However, imaging in the G-wire growth medium and

concentrations assures mean G-wire lengths reflective of current equilibrium conditions.

Imaging G-wires in their growth medium is NOT speculative. I have confirmed this capability

during a preliminary study on the same equipment we are proposing. Details are provided in

section 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. We

will compare our length-time studies above with numerical computations. The nature of the

problem can be seen in Figure 4. The total equilibrium constant is the product of individual

constants 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 the

G4T2G4 oligo into hairpin or looped structures with the telomeric Guanine-quartet is kinetically

more favorable than remaining in the oligo state. This system of n-linear equations requires

numerical solutions. With the proposed instrumentation, we will have the means to take accurate

growth measurements. Co-PI, Computational Chemist, Dr. Clinton Nash, will assist in the

numerical simulations. An important issue at stake is how to deal with the time dependency of

each individual equilibrium constant of G-wires with different chain lengths. At a minimum, we

will 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 grid

position for macroscopic current measurements. Even if long G-wires are not fortuitously

connected between contacts, we can employ electron beam deposition (the contamination residue

from repeated line tracing with an electron microscope) with the UNE scanning electron

microscope to complete a biopolymer circuit7. Control experiments include the comparison of

the current through G-wires, duplex DNA, and electron beam deposited (EBD) scanning electron

microscope carbon contamination traces using a transistor-type electrode set-up (Figure 5) with

the LCSTM. Conductivity will likely be a function of ambient relative humidity and residual

buffer ions. Humidity/ion control is extremely important not only to limit SPM-induced sample

damage 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. limited

movement) ions may be responsible for transverse and lateral conductivity, these experiments

could provide fundamental information surrounding G-wire structure and its relation to electron

transfer through four stranded DNA.







University of New England SPM MRI, Page 8

A Networked Scanning Probe Microscope for Research Training.



4. Future question: Conduction mechanism.

The caged metal cations (Figure 4) integrated into a hydrated G-wire molecule may have some

mobility. In addition, the metal cations are sufficiently close to each other (estimated between

0.3-0.7 nm) to support electron tunneling. Lastly, the p-bonding of adjacent Guanine-quartets

may overlap enough to enhance electron mobility. All of these mechanisms can contribute to the

lateral conductivity we are trying to characterize. The experiments mentioned above cannot

distinguish 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 mobile

metal cations, or 2) enhancing p-bonding along the length of the G-wire chain through the

addition 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 national

meetings of my professional organizations, Council of Undergraduate Research and the

Biophysical Society meetings. These results will then be converted into web site presentations

and appended under the “student research” link at my UNE faculty web site

(faculty.une.edu/cas/jvesenka/DrVIndex.HTM). Suitably publishable results will be submitted

either 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 SPM

to help establish optimal G-wire growth conditions. Undergraduate research stipends are

independently provided by UNE.

Academic Year 2001-2: Train students in powerful techniques on proposed MRI SPM

operation and recruit potential summer research participants from microbiology (BIO226) and/or

physical chemistry (CHE327).

Summer 2002: Fluid-cell AC imaging of G-wires for rigorous characterization of growth

rates. Undertake preliminary conductivity assays.

AY 2002-3: Continue conductivity assays with select honors and or independent research

students. Repeat training and recruiting of undergraduates from advanced courses.

Summer 2003: Undertake full-scale conductivity characterization. Estimated 2-4

undergraduate research assistants, again all supported by UNE stipends.



a b

V

pA



DNA w/ moisture layer

Dielectric substrate

Figure 1: a) Cross-section schematic of electron tunneling path between tip and sample, likely to

be through a thin aqueous layer containing residual buffer salts and G-wires. b) Top view

schematic 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 at

82% relative humidity, -7V bias voltage and 3.0 pA tunneling current. Vertical height is 0 - 5

nm from dark to light color. Scale marker is 100 nm long.









University of New England SPM MRI, Page 9

A Networked Scanning Probe Microscope for Research Training.



Figure 2: The “pure” solution of ten nucleotide oligomer was imaged

by the atomic force microscope at t = 0 (i.e. at the instant they were

plunged into the growth cocktail). Imaged at about 10 nN of constant

force and 10 % relative humidity. The G-wires had an average length

of 28-nm. The vertical color scale indicates the G-wire rise an

average of 2.0 nm above the mica substrate, whereas the lateral

resolution is only good at best 5 nm because of the finite tip

geometry. In this image the G-wires are broadened an average of 20

nm, seriously affecting our length measurements. The scale marker

is 100 nm long.





G-wire Growth Study Figure 3: Preliminary

120

G-wire growth study.

Raw data (round

100 circles) with error bars

Mean Length (nm)









(standard deviation of

80 mean length) plotted

versus days. N=100 for

60 each data point.

Diamonds correspond to

40

“reduced length”,

20 achieved by subtracting

the t = 0 mean length

0 from the raw data. An

0 5 10 15 20 exponential fit to the

Days reduced length data

reveals:

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

Though this is a typical function for the simple molecular equilibrium process, note the errors

bars are so large that a variety of other function (e.g. linear) could also be used to fit the data.

GGGGTTGGGG

GGGGTTGGGG + GGGGTTGGGG L1

GGGGTTGGGG

K1 = [L1]/[GGGGTTGGGG]2

= caged metal cation of Mg2+ or K+

GGGGTTGGGG

L 1 + L1 GGGGTTGGGGGGGGTTGGGG L2

GGGGTTGGGG

2

K2 = [L2]/[L1] L3

L 1 + L2



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 equilibrium

constant for the growth of the G-wires. A system of “n” simultaneous first order linear equations







University of New England SPM MRI, Page 10

A Networked Scanning Probe Microscope for Research Training.



is generated, requiring numerical analysis. One of the assumptions is that the addition of L1 to a

G-wire chain is the most dominant process. A hidden complication is the equilibrium constants

being time dependent because the mean concentrations of the ladder components change in time,

as seen in Figure 3.

pA









V

pA



Figure 5: Side view of conductivity measurements of G-wires (brown) made through two

electrodes (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, will

enable us to characterize any non-ohmic behavior of the G-wires. Two pico-ammeters are

required, 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 a

primary grazing surface for moluscan and arthropod grazers which themselves subsequently

provide food for higher organisms. Much study has been done on the role that algae and

cyanobacteria play in stream biofilms. However, the structuring of such communities depends

upon the earliest colonists of a newly cleared surface, which are often the bacteria. Research has

been difficult in determining how contact and colonization proceeds because the resolution of







University of New England SPM MRI, Page 11

A Networked Scanning Probe Microscope for Research Training.



light microscopy is inadequate to see this stage, and various electron microscopic approaches to

the 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 bacterial

colonization of painted, plastic, teflon, metal, and other surfaces which form the submersed

portions of boat hulls. Understanding how biofouling of boats, ships, and other man-made

surfaces occurs is an important area for research and of particular interest to the commercial boat

manufacturing industry in the State of Maine. Biofilms also form inside the human (and other

animal) bodies; on teeth for example. Contact and colonization occurs well before the origin of

dental 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 to

this range of scale, and provides the possibility of imaging live organisms and relatively

undisturbed mucopolysaccharides.

Class Projects:

At the University of New England I teach courses in microbial ecology and independent

research among others. Since I require that students do actual research in these courses, they

lend themselves to students using scanning probe microscopes (SPM) to find out how biofilms

are generated. As a starting point for dealing with the formation of these structures, I propose

that 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 in

proximity to an appropriate surface where colonization can be observed and recorded with the

SPM in fluid environments. We can even simulate low flow rates in the SPM imaging chamber

through input/output ports of the fluid cell. Low flow rates are necessary to avoid disturbing the

imaging tip.

Not all bacteria are the same. Neither are surfaces. Selecting appropriate bacteria and

surfaces for testing will be critical in defining the value of the studies. Imaging rough surfaces is

difficult given the limitations of the vertical range (10 µm) of the equipment's scanner. Most

protozoa and algae are not suitable for the same reason. However, there are some surfaces and

microorganisms 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 the

standard substrata for use in SPM because it provides such a smooth surface. Glass surfaces

coated with fine grades of paint may also be possible, and would provide a model system for

marine fouling communities. Many bacteria found in natural communities do not grow in

culture, so in addition to specific isolates of bacteria from various habitats, whole samples of

mixed natural communities will also be used.

Many bacteria require other species in their close proximity to survive or thrive. These

symbiotic groups of bacteria are called consortia. One of the questions that we wish to address is

the differential efficacy of colonization by individual species of bacteria – versus consortia of

bacteria working together. Developmental rates of mucopolysaccharide will be determined by

time course studies using the SPM. Spatial arrangements of several bacterial morphotypes will

provide an indication of which system is quicker in producing a “colonizable” film, for second

stage biofilm development. Additional comparisons of mucopolysaccharide development under

various nutrient and dissolved carbon enrichments will also be done on the same basis to

determine 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 a

holding medium for compounds that provide a competitive advantage to the initial colonists.







University of New England SPM MRI, Page 12

A Networked Scanning Probe Microscope for Research Training.



Pulsed experiments, where one species is added first followed by a second after the first has laid

down an initial mucopolysaccharide layer – contrasted with systems where both organisms gain

access 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-fouling

agents, but also to inform stream, lake, and ocean ecologists of the relevance of successional

models to this type of micro-habitat development.

The variety of possible research projects using this equipment makes it ideal for a focus in

microbial ecology classes. Several teams can use the same instrument to focus on different

aspects or questions. The various projects allow for students to follow their own interests while

at the same time, linking or coordinating the knowledge they gain with others focusing on

different aspects of the same system. Thus, in a way the student researchers get an introduction

to 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 now

identified as the fullerenes,21 there has been a great deal of effort invested and success attained in

the characterization and understanding of the structure-property relationships inherent in these

cluster molecules.

Fullerenes have been defined as cluster species consisting of closed cages of carbon atoms

that conform to the so-called isolated pentagon rule. This rule is a statement that special stability

is 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 prototypical

fullerene 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 that

fullerenes be classified more generally by only the presence of the twelve pentagonal faces

required to close the cluster surface upon itself. The isolation of pentagons ensures that no single

atom resides in any two pentagonal faces and results in maximal aromatic stabilization as the

fusion of five-membered rings results in a Hückel antiaromatic cycle of eight.

Although others are known, a particularly interesting exception to the isolated pentagon rule

is 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 These

clusters were originally proposed to adopt a cage structure of Th symmetry analogous to the

unstable fullerene C20 that has recently been identified in the gas phase.23 The proposed structure

of this species exemplifies an extreme case of violation of the isolated pentagon rule as every

atom is included in the maximum possible number (three) of five-membered rings. Recent

quantum 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 is

the presence of energetically accessible metallic d valence orbitals that must in some way

enhance the "aromaticity" of the molecule. Support of this notion is provided by the fact that

dodecahedrane, C20H20, the fully hydrogenated derivative of the twenty-vertex fullerene is a







University of New England SPM MRI, Page 13

A Networked Scanning Probe Microscope for Research Training.



known compound adopting the idealized icosahedral geometry of the dehydrogenated

compound.25 As a saturated compound there can be no antiaromatic destabilization for

dodecahedrane. It must be concluded that the ephemeral existence of C20 is a result of

unfavorable p-orbital interactions rather than any sort of carbon-carbon bond angle or ring-strain

effects, because these should be roughly the same in both compounds. When metals are

substituted for certain carbon atoms in these structures, the unfavorable effects are reduced or

eliminated thereby leading to the stability seen in Castleman's and others' results. This is a

situation reminiscent of the phenomenon of metalloaromaticity which leads, for example, to the

existence of metallocyclobutadienes, whereas no four-carbon conjugated ring is known to be

other than transiently stable.26 Unfortunately, there is not yet available direct information

concerning the structure of Ti8C12 or any other metallocarbohedrene. The limited and indirect

information that is available comes from mass spectrometric ion mobility analysis of laser-

ablated titanium carbide soots. However, this has demonstrated that indeed the molecule does

adopt 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 by

using titanium-carbon electrodes in an 'arc-welding' apparatus of the type frequently used to

produce C60 and higher fullerenes. We have access to an Edwards evaporative coating apparatus

that has the control of temperature and gas needed to generate monolayer Ti/C soots evaporated

directly only highly oriented pyrolitic graphite substrates. The Ti/C soots produced in such a

way will contain both the cage-like molecules Ti8C12 and Ti8C13 (the latter a met-car containing

an endohedral carbon atom) as well as titanium carbide nanocrystals of rock-salt like

stoichiometry, Ti12C13. A recent transmission electron microscopy investigation Ti/C soot by Yu

and Huber was unable to distinguish met-cars from the nanocrystals.28 We propose to use both

AFM and STM to examine Ti/C soots and thereby directly characterize the structures of the

titanium metallocarbohedrene. As a necessary first step in this process, we will conduct similar

studies on quantites of air-stable dodecahedrane that can be relatively easily obtained in gram

quantities. As mentioned earlier, C20H20 has a similar size and analogous structure to those of

the 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 our

understanding of the electronic and structural characteristics of non-carbon fullerene-like

structures thereby expanding their potential inclusion among the palate of elements to be used in

fullerene science and technology.

An undertaking such as this will serve as both a capstone in our development of a Physical

Chemistry laboratory curriculum for our major students and an engaging vehicle for

undergraduate research. The project itself represents an important investigation into the nature

of this particular class of cluster compounds and provides a number of achievable milestones

along the way. The very nature of project involving repeated sample comparisons is well suited

for a large class project. The University of New England is fairly unique among primarily

undergraduate colleges in that we currently have a capacity to perform work having at its center

the tools of AFM. The enhancement of this capacity to include STM will allow us to orient the

physical chemistry laboratory experience around research training, as has been successfully

achieved elsewhere29. At the same time we seek to provide opportunities for our students to

work with powerful, cutting edge tools with which to do promising science.







University of New England SPM MRI, Page 14

A Networked Scanning Probe Microscope for Research Training.



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 Digital

Instruments Nanoscope IIIa controller and MultiMode™ scanning probe microscope is exactly

this tool. The Nanoscope operates on a Windows NT platform, providing a hardy operating

system capable of withstanding lots of accidental abuse. NT is supported by a remote operations

software, Virtual Network Computing, essential for large-scale research training. Training

students for independent operation is facilitated by a user-friendly software interface and

sensibly 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 the

same manufacturer. A detailed quote from Digital Instruments is included in the Supplementary

Documents section.

Mark Johnson, biofilms: Imaging large surfaces for bacterial colonization requires 100 µm

scan 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 dislodging

the bacterial growth. Since adhesion of the bacteria to the surface is a central question in MJ’s

research, being able to record phase differences (measurements of adhesion properties) hidden in

the intermittent contact signal and force-position measurements (Young’s modulus

characterization) are essential. The proposed equipment includes a vertical engage “J” scanner

for long range imaging and fast approach to quickly monitor numerous positions over a biofilm

surface, and a phase box accessory to extract adhesion information. Our current configuration, a

Nanoscope E controller with lateral force microscope and mid range scanner cannot be used for

undertaking any of the above proposed experiments.

Clinton Nash: metallocarbohedrenes: High resolution mapping of metal-substituted soot

particles 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 to

switch operations from one near field imaging technique to the (e.g. AFM to STM). The

important piezoelectric scanners, lying underneath the detectors, can be used in any imaging

mode. We currently have 1.0 µm and 10 µm scanners from our old lateral force microscope that

can also be used on the proposed SPM, at a savings of several thousand dollars to the proposal

budget. These scan ranges will be used to establish specimen purity and high-resolution

imaging. The LCSTM TipView detector, requiring the Phase Extender to operate, is included in

the MRI proposal.

James Vesenka – Four stranded DNA characterization: Our existing SPM has the ability to

monitor “contact” topography, lateral force, deflection (error signal) and make tip-sample force

measurements. We have been measuring G-wire lengths as a function of growth time to help

predict the incubation times needed to construct long G-wires. These contact AFM images are

very hard on the biological samples, which must be thoroughly dried to avoid degradation during

imaging. No images of G-wires have been successfully captured in buffered media or other

liquids in contact imaging mode. Though fluid cell imaging can greatly reduce imaging forces

on DNA, these forces (≈ 1.0 - 0.1 nN) are still high enough to sweep the G-wires off the surface

in 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 be

done. JV has successfully and routinely imaged G-wires in a fluid cell using DI’s

TappingMode™ technology. These observations were made during visits to a colleague at IBM

Almaden while JV was an assistant professor of physics at California State University, Fresno.







University of New England SPM MRI, Page 15

A Networked Scanning Probe Microscope for Research Training.



Another imaging mode that was forfeited during JV’s cross-country move was low current

scanning tunneling microscopy (Figure 1). The hardware for this instrumentation was secured

through a NSF ILI grant to CSUF (cf. Section “a”-Results from prior NSF support.) and

remained at CSUF after his move. The proposed instrumentation includes LCSTM, fluid cell

AC imaging technology, and Phase Box Extender essential for the proposed research. These

hardware configurations overlap with the other two proposals such that there are no

instrumentation requests that are unique to a single researcher. Both silicon tapping tips (for

high resolution imaging in air) and silicon nitride tips (for high resolution imaging in liquid) are

also 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 to

leverage an upgrade path ($30,000 non-cost sharing credit) towards securing the above described

equipment. 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, allowing

multiple 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 and

training 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 Leitz

metallographic inverted microscope for the examination of geological specimens, and a Leica

differential interference microscope for large biological samples. These are used by the Co-PI

for his geology and microbiology courses. The other Co-PI (CN) is a theoretician who is

securing 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 generate

soot 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 with

Gilson upgrades Single-wavelength UV-Vis and Spectronic 20 Vis Spectrophotometers,

Aminco-Bowman Series 2 and Jasco Scanning Spectrofluorometers, Instrumentation

Laboratories 251 and Perkin Elmer 3110 Atomic Absorption/Emission (flame), BioRad FTS-7

Fourier-transform Infrared, Varian T-60 Nuclear Magnetic Resonance

Electrochemistry holdings: BioAnalytical Systems CV-50W Multipurpose Electrochemical

Analyzer, Misc ISEs; computer-interfaced pH/ion mV meters Potentiometry, Electrogravimetric

Analyzer

Chromatography holdings: Hewlett-Packard GCD GC-Mass Spectrometer and Hewlett-

Packard 5890 GC with FID and TCD Capillary Gas Chromatography, Perkin-Elmer

AutoSystem, GC Gow-Mac Model 350 dual column (2 units) Gow-Mac Model 550 Packed-

column Gas Chromatography, : Waters pump & injector w/Varian variable wavelength UV

detector, Perkin-Elmer w/PE variable wavelength UV and refractive index detectors, Beckman

pump & Waters conductivity detector (ion chromatography) High Performance Liquid

Chromatography









University of New England SPM MRI, Page 16

A Networked Scanning Probe Microscope for Research Training.



d) Impact of Infrastructure Projects

The 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 this

typically consists of a research project that ties together the different threads of their educational

training. The chemistry/physics department was recently (since this past year) split off from the

Department of Life Sciences at UNE. The department’s instrumentation holdings consist of

standard older model undergraduate laboratory tools. We have little in the way high-resolution

analytical equipment. Any sophisticated instrumentation analysis is typically sent off camps.

For example, all high resolution NMR imaging is farmed out to large research institutions, the

closest being the University of New Hampshire over 90 minutes away.

Our program proposes to integrate the proposed scanning probe microscope into the capstone

experiences in two ways. The traditional approach involves individual, faculty-guided integrated

research projects. These projects would start in the fall of 2001, involving life and health science

majors in a microbiology course (BIO226). A class project, involving the real-time analysis of

biofilm growth, will examine a range of practical problems, such as the fouling of boat hulls to

the construction of organic matrices needed to support life on aquatic surfaces. In the spring of

2002 our biochemistry majors will examine metallocarbohedrenes as part of their physical

chemistry (CHE327) class project. They will use high resolution scanning tunneling microscopy

under inert environments to see the differences in atomic structure induced by metal

substitutions. During the summer the above two projects are expected to continue with highly

motivated undergraduate research assistants recruited from these courses. In addition,

undergraduates will have research opportunities in self-assembled DNA as well as examining

four stranded DNA electronic properties. The proposed SPM would dramatically increase the

opportunities 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 a

variety of “near-field” detection schemes. SPM technology is routinely used in the research and

commercial enterprises ranging from the measurement of individual rupture forces of molecules

to microfabricated wafer characterization. When JV arrived at UNE he was able to bring his

older Nanoscope E controller and lateral force microscope. Maine is home to a growing number

of silicon and biotechnology enterprises. However, in the state of Maine UNE is one of only a

few universities that have any SPM tools. Securing a late-model SPM with non-invasive

imaging abilities and a broad range of near-field detection mechanisms will dramatically expand

the 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 school

and improve our graduates’ marketability in Maine’s growing high technology industries. We

anticipate improving the representation of women in chemistry through the recruitment efforts in

these advanced courses. Taught by all three senior personnel the top students from our general

service courses at UNE are dominated by women who tend to shy away from the biochemistry

major. We are particularly eager to recruit women from the pool of health and life science

majors (60% women) into biochemistry.









University of New England SPM MRI, Page 17

A Networked Scanning Probe Microscope for Research Training.



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 major

requirements 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 at

four universities (cf. vita) make him an obvious choice for staffing the proposed equipment. The

PI has the expertise in computer hardware and operating systems to trouble shoot most problems

on 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 be

responsible 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-month

summer research period providing JV two thirds of his summer time to pursue his own

undergraduate research projects. The cost of supporting the maintenance and operations of the

SPM by the PI is less than a half time staff position. The level of support will continue after the

conclusion of the grant (cf. letter of support from the Dean). No manufacturer’s maintenance

contracts are needed because of the outstanding support Digital Instruments provides.

Instrument time during the academic year will be prioritized first towards research training

for large classes and secondly toward individual undergraduate research projects. The latter have

the flexibility of using the equipment during off-hours. Furthermore, individualized advanced

user training will be undertaken during the summer and shared equally amongst the research

groups. Large class training sessions will be facilitated by Virtual Network Computing (VNC)1

software, enabling each class member to monitor and/or operate the networked SPM from their

own individual workstation located in an adjacent computer laboratory. This laboratory also has

a large wet lab bench space for sample preparation purposes. Individual student instruction will

be undertaken directly in the SPM lab. In deference to the minimal resources our research

community has, no user fees will be charged. We will optimize outreach through recruitment of

students and faculty in advanced courses, dissemination during our yearly on-campus research

symposia, and through weekend professional development workshops supported our dean. We

seek 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 year

and 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 Multimode

Scanning Probe Microscope. Training of one Co-PI (MJ) and his BIO226 students in the SPM

operations. 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 summer

research projects. Estimated involvement of 2-3 faculty and 6-9 undergraduate students. JV

recruits one more UNE faculty in SPM research as part of capstone and/or research educational

experiences. 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 of

growing SPM usage and training in SPM operations. Completion of any unfinished capstone

research experiences. Co-PIs will consult with JV as necessary in SPM use but are expected to

operate equipment autonomously. Estimated involvement of 1 new faculty and 24-36

undergraduate students.









University of New England SPM MRI, Page 18

A Networked Scanning Probe Microscope for Research Training.



Biographical Sketches: Mark Johnson



Biological Sciences (Mark Johnson – MJ): Mark undertakes research in locations ranging

from the swamps in west Alabama to the pack ice of Antarctica. The unifying theme to all of his

research 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 of

the least understood realms in ecology. The assays Mark performs on the microorganisms in his

research involve a basic understanding of photosynthesis and the motion of these systems

through 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 who

form a complex community in the minute fissures in pack ice filled with highly saline brine. In

the Alabama wetlands he tracked the populations of ciliates, flagellates, amoebas, algae, and

bacteria and their productivity through various seasonal changes to get one of the most complete

pictures of microbial populations ever done. An unexpected finding there was that small

flagellate protists increased in numbers during the winter when just about every other measure of

life dimmed. In addition, Mark has worked on the development of a technique to determine

activity levels in bacteria. Putting these two factors together is something worth investigating

here in Maine. Currently, Mark is setting up incubation chambers to do controlled lab studies of

the effects of temperature on the behavior of ciliates from our local ponds and wetlands; as well

as to try rehydrating naturally freeze-dried cyanobacterial mat communities from Bratina Island,

Antarctica. He has undertaken extensive research characterizing macroscopic affects of biofilm

growth in riverine systems. He is pursuing a better understanding of the microscopic processes

involved in the development of biofilms.









University of New England SPM MRI, Page 19

A Networked Scanning Probe Microscope for Research Training.



Biographical Sketches: Clinton Nash



Physical/Inorganic Chemistry (Clinton Nash - CN): His primary research interests involve

the theoretical chemistry of heavy elements, particularly those in which relativistic effects are

manifest such as the actinides and transactinides. CN’s Ph.D. work focused on elucidating the

periodic relationships among these transactinides and their familial congeners as well as the

source of disruptions in this periodic behavior. Although he still consider this “general

chemistry 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 more

immediate environmental interest such as aqueous actinide chemistry. More generally, he is

interested in the application of computational methods to all branches of chemistry, including

biochemistry, 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 biochemistry

majors advanced courses in inorganic and physical chemistry. CN intends to take advantage of

the small departmental structure of the University of New England to break down the artificial

barriers that can often be erected between the physical sciences. As it currently stands, there is

no laboratory component to the physical chemistry course at UNE. One opportunity for

integration that presents itself is to use our local capacity to do scanning probe microscopy to

examine the structures of surfaces. Addressing questions surrounding the electronic structures of

materials are fundamental to both CN’s research and to the approach he takes to lectures. To be

able to take students into the physics laboratory and demonstrate the nature of electron tunneling

and illuminate, in a very concrete way, the consequences of atomic and molecular structure is a

very exciting prospect. An example of this will include the AFM and STM examination of

fullerenes, materials in which the molecular and electronic structures are intimately intertwined.

Students would perform the experiments to determine an approximate molecular structure for

these materials and then justify their structures in terms of molecular orbital theory (e.g.

Giancarlo et al.29.









University of New England SPM MRI, Page 20

A Networked Scanning Probe Microscope for Research Training.



Biographical Sketches: James Vesenka



Biophysics, Material Sciences (James Vesenka – JV): JV teaches general physics and

undertakes research in molecular self-assembly and physics education. He has 11 years of

expertise in the area of scanning probe microscopy (SPM), including the successful development

of a networked SPM core facility at California State University, Fresno (see section “a” on

Results from Prior NSF Support.) JV received post-doctoral training at the University of Oregon

at the Institute for Molecular Biology where he developed tools for imaging duplex DNA with

the AFM in the laboratory of Carlos Bustamante (now at UC Berkeley). The power of SPM

technology lay in its ability to image biological specimens without coatings or freezing, i.e. in

the DNA’s native state. JV had two papers published in Science which detailed the procedure

for imaging DNA in liquid30 and measuring DNA melting-angles during RNA polymerase

transcription31.

He continued his post-doctoral research as part of the Signal Transduction Training Group at

Iowa State University in the laboratory of Eric Henderson. At ISU JV developed vertical

calibration standards32 and image reconstruction software with Professor Richard Miller from the

Department of Mathematics33. The two papers combined to provide reliable recreation of

surface topography artificially broadened by the finite geometry of the SPM imaging tip. JV also

assisted in high resolution imaging of nucleo-proteins, discovering the conditions under which

detailed 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 an

assistant professor position of Physics at California State University Fresno (CSUF).

At CSUF JV concentrated his effort on using the SPM as an undergraduate research training

tool, publishing several articles with his students that used the G-wires as a target system. As

part of a CSU system-wide effort, JV secured NSF ILI funds for a network scanning probe

microscope anchored at CSUF. Access to the equipment was provided on campus to individual

users and to larger audiences through remote operations using virtual network computing. In

addition to his own professional research interests, JV has maintained and operated the SPM’s

ever since his first exposure to scanning probe technology over 11 years ago. He has trained

almost 50 graduate and undergraduate students in SPM operations and helped them develop

research skills that helped them to move on to professional school or to secure Ph.D. in science

fields ranging from Molecular Biology and Materials Science.

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

end.









University of New England SPM MRI, Page 21

A Networked Scanning Probe Microscope for Research Training.



James Philip Vesenka



University of New England

Department of Chemistry and Physics

11 Hills Beach Road

Biddeford, ME 04005

Office: (207) 283-0170 Ext. 2560, Fax: (207) 282-6279

Email: jvesenka@mailbox.une.edu

Homepage: faculty.une.edu/cas/jvesenka/DrVIndex.HTM



Education & Research Experience

Post-graduate Researcher, Iowa State University, 8/92-6/95 P.I.: Prof. Eric Henderson

Post-graduate Researcher, University of Oregon, 10/90-6/92. P.I.: Prof. Carlos Bustamante

Post-graduate Researcher, University of California, 10/89-9/90. P.I.: Prof. Yin Yeh

Ph.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 Experience

Assistant 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 Maintenance

Digital 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. Molecular

Imaging (Tempe, AZ) Picoscope controller and SPM. Digital Instruments Nanoscope E w/ Lateral Force

Microscope. Responsible for the maintenance of all SPM and training of 6 graduate students and 14 undergraduates

in the operation at California State University Fresno (8/95 – 6/99).

Digital Instruments Nanoscope III w/ Bioscope biological Atomic Force Microscope. Digital Instruments

Nanoscope II atomic force microscope and scanning tunneling microscope. Responsible for the maintenance of the

two machines in addition to training four graduate students and two undergraduates in their operation at Iowa State

University (9/92-6/95).

Digital Instruments Nanoscope II w/ atomic force microscope. Brief experience with Digital Instruments

Nanoscope I scanning tunneling microscope. Responsible for the maintenance of the SPMs and trained five

graduate students and one post-doc in their operations at the University of Oregon (9/89-8/92).



Professional Affiliations

American Association of Physics Teachers

Council on Undergraduate Research

Sigma-Xi, undergraduate research society

Project 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).







University of New England SPM MRI, Page 22

A Networked Scanning Probe Microscope for Research Training.





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 a

session 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’ DNA

investigated by Low Current Scanning Tunneling Microscopy,” 44th American Vacuum Society meeting, San Jose,

CA (11/97).



Last 10 Publications from 38 Lifetime: “*” = Student Participant

38. 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 DNA

measured 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 of

DNA 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 of

Ribosomal Subunits and rDNA Chromatin Imaged by Scanning Force Microscopy”, J. Vac. Sci. Technol. B

14, (1996).



31. J. Vesenka, T. Marsh, R. Miller, & E. Henderson, "High Resolution Atomic Force Microscopy Reconstruction

of 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 to

Procedures 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).



Patents

E. 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).









University of New England SPM MRI, Page 23

A Networked Scanning Probe Microscope for Research Training.



Revised Budget Impact Statement



Item NSF-Request

Summer staffing wages and benefits $14,860

The PI has 11 years of experience with Digital Instruments SPMs. He anticipates training 10

students/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. The

Dean’s Office from the College of Arts and Science at UNE is dedicated to the long-term support

of this equipment (see attached letter of support).

125 µm Vertical engage scanner $9000

The scanner is essential for biofilm analysis to help characterize the differences between

bacterial “clustering” versus uniform surface covering. The long-range scanner will also be

necessary for the characterization of long G-wires for conductivity measurements.

Extender electronics (phase box): $8,000

The extender electronics are essential for monitoring the adhesion characteristics of the bacterial

colonies for biofilm growth.

Low current STM detectors $6,350

The extender electronics is also essential for extracting low current STM signal and electronic

characterization of the G-wires and metallocarbohedrenes.

Nanoscope E to Nanoscope IIIa Control Station Upgrade: $45,000

Upgrade 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 is

needed to be able to handle the variety of imaging modes essential to the proposed research (AC

methods such as intermittent contact and low current STM).

Nanoscope Multimode SPM with fluid cell: $36,000

The Multimode SPM is a modular microscope set-up capable of supporting several different

imaging modes through a simple change in detectors. The fluid cell is essential for the biofilm

characterization and G-wire kinetics to be able to characterize live samples in buffer media.

Silicon Nanoprobes and Silicon Nitride Probes (AFM tips: $7,500

The AFM tips are essential for imaging samples and must be purchased commercially. DI is the

most 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 requested

since we will cut our own from platinum-iridium wire purchased separately.

Video Only Microscope: $5850

This “optical navigator”, capable of 1.5 µm resolution, will allow us to locate biofilm bacterial

colonies for imaging under the long-range scanner and for locating electrode contacts on the

microfabricated conductivity test structures for G-wire conductivity measurements.

Indirect costs: $8,040

Charged 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







University of New England SPM MRI, Page 24

A Networked Scanning Probe Microscope for Research Training.







-









University of New England SPM MRI, Page 25

A Networked Scanning Probe Microscope for Research Training.









University of New England SPM MRI, Page 26

A Networked Scanning Probe Microscope for Research Training.



References:



1

http://www.uk.research.att.com/vnc/index.html

2

D. Goldhaber-Gordon, M.S. Montemerlo, J.C. Love, G.J. Opiteck, & J.C. Ellenbogen, IEEE

Proc., 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). and

P.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 were

purchased 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.htm

21

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).





University of New England SPM MRI, Page 27

A Networked Scanning Probe Microscope for Research Training.





32

J. Vesenka, S. Manne, R. Giberson, T. Marsh, & E. Henderson, Biophysical J., 65, 992-997

(1993).

33

J. Vesenka, R. Miller, & E. Henderson, Rev. Sci. Instr., 65; 7, 2249-2251 (1994).

34

L. Martin, J. Vesenka, E. Henderson, & D.D. Larson, Biochemistry, 34; 14, 4610-4616 (1995).









University of New England SPM MRI, Page 28