Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Marshall University Imaging Core
Director:
Michael Norton, PhD
Professor, Department of Chemistry, Marshall University
No human subjects
No vertebrate animals
PHS 398/2590 (Rev. 11/07)
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Marshall University Imaging Core (MUIC)
I.
Specific Aims of the MUIC in the Center for the Molecular Basis of Health
Disparities in Appalachia (CMBHDA)
Marshall’s COBRE application contains five projects, each of which addresses the role of dysregulation of
transport in disease susceptibility or treatment. Although the imaging requirements of each project are
different, each PI’s project will benefit from facilitation of their research by the Imaging Core. The primary aim
of the Marshall University Imaging Core (MUIC) is to enable these investigators to meet and surpass
the stated semi-quantitative and temporal imaging requirements of their proposed projects. Our
secondary aim is to enhance these projects, using new techniques, to enable these researchers to
meet their evolving research expectations. In order to achieve the first aim, the MUIC will provide the
following services, which support research from initiation to publication:
(1) Support in experimental design for imaging applications
(2) Support in biomolecular probe design and generation
(3) Collaborative Imaging support and training, including sample quality assessment
(4) Access to advanced imaging technologies
(5) Image analysis support and training
(6) Manuscript technique section preparation
(7) Assistance in preparation of post COBRE proposals and letters of project imaging support
(8) Inviting seminars/specialists to consult on complex and/or new methods relevant to individual projects.
Monitoring the distribution, redistribution and the temporal evolution of protein binding partners in selected
cellular populations and in selected subcellular regions of interest is of ever increasing value for quantitative
testing of hypotheses related to molecular signal transduction processes. Quantitative dysregulation of intrinsic
circuits are the essential elements of many disease states and the development of quantitative intracellular
probes is required to fully understand disease states and to evaluate the quantitative impact of molecular
therapies. As a result of the capability enhancement enabled by this award, we will assist in the design,
fabrication and testing of necessary intracellular probes specific for the conditions studied in several of the
projects which are part of this COBRE program. The Molecular and Biological Imaging Center (MBIC) at
Marshall has provided user access to a wide range of Optical, Electron and Scanned probe microscopy
systems and experiment design and expert services supporting the local research community. The
instrumentation which will support the current and immediate project objectives of five of the five PI’s is the
Leica SP5, a high speed, confocal/multiphoton 3D imaging system.
In pursuit of our second aim, To enhance these projects, using new techniques, to enable these
researchers to meet their evolving research expectations, we will develop, in parallel with traditional fixed
cell approaches, fluorescent protein methods to address quantitation and resolution objectives which are
enhancements, in some cases, over fixed cell imaging techniques. We anticipate that application of these
methods, which have been developed and found broad applicability in the biomedical sciences domain
elsewhere, will open new avenues of experimentation for addressing spatial and kinetic questions, for COBRE
and other investigators. This will have the effects of both promoting the growth of the MUIC and expanding the
application of these probes to biomedical research in general.
Funding of this Core will be an enabler to the investigators of this COBRE, making tools and services much
more available to them by enhancing the focused specialist talent dedicated to their projects, both now and as
they matriculate into RO1 holder status. This pre-existing Core imaging facility leverages funding from the
Army Research Office, the National Science Foundation, the Marshall Institute for Interdisciplinary Studies and
the State of West Virginia to deliver these state of the art capabilities to all researchers requiring such unique
capabilities. The following sections describe these unique resources, the qualifications of the MUIC staff, the
MUIC operational plan, and the potential impact of the MUIC on the cadre of current and future COBRE
researchers.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Research Strategy
II. Impact of the Marshall University Imaging Core (MUIC) on the Center for the Molecular Basis
of Health Disparities in Appalachia (CMBHDA)
Over the last 25 years, there has been a concerted effort to acquire sophisticated imaging equipment to serve
the increasingly complex needs of the Marshall research community. This collection of imaging systems was
integrated into a centralized Imaging Core Facility in the Fall of 2006 when construction of the Robert C Byrd
Biotechnology Science Center was completed, providing a suite comprised of six individual imaging
laboratories, two sample preparation labs and an office for the Imaging Specialist/Lab Manager. This building
also enabled the majority of biomedical science researchers to move from the Medical Education Building, a
distant (14 miles away) Medical Campus to join basic science researchers on the main university campus. The
purpose of the imaging facility has been to provide imaging services, micromanipulation, analytical tools and
training in the use of imaging modalities to all researchers in the region. Particularly of relevance for this
proposal, the facility is very well equipped for observing the localization of products of gene expression and
protein translocation. Imaging system capabilities and investigator requirements are constantly evolving. This
proposal requests resources which will be directed toward supporting studies carried out by the COBRE
investigators. The following listing indicates resources developed for our current user base which will be
expanded to meet the needs of the COBRE cadre.
The MUIC will provide support in 8 areas. These areas of support have as their primarily focus the
generation of data supporting the development of COBRE researcher programs, utilizing a Leica Confocal and
Multiphoton system for image acquisition. Particular description of the system and general applications are
detailed in section VC1a, while particular investigator experience with this system is reviewed in section VC1b
below. These eight support services to COBRE research projects and other investigators are: (1) Support in
experimental design; (2) support in biomolecular probe design and generation; (3) collaborative support and
training, including sample quality assessment; (4) access to advanced imaging technologies; (5) image
analysis support and training; (6) manuscript technique section preparation; (7) assistance in preparation of
post COBRE proposals; and (8) inviting relevant seminars/specialists to consult on complex and/or new
methods. While Imaging Service 2, probe generation and Imaging Service 8, Inviting specialists, are new
initiatives, only made possible by this COBRE program, each of the other services have been standard
practices for the imaging center. All services will be available to researchers at the start of the grant period.
There is substantial need for these services among the COBRE subproject investigators. Table 1 shown
below provides an overview of the current use, proposed use and MUIC predicted use of the Core capabilities
in fluorescence microscopy and fluorescent probe development. All five investigators/projects will require
optical imaging support. We also anticipate that several of the projects may spawn experiments which would
benefit from higher temporal resolution and fluorescent protein probe techniques, particularly as we
collaboratively develop appropriate protocols and preliminary data (summarized in Table 1).
Table 1. Proposed Marshall University Imaging Core Facility Supported COBRE Investigators
Project
Current MUIC Confocal /
User
Muiltiphoton
1) Maria Serrat
Yes
Y/Y
2) Maria Isabel Larre-Perez Yes
Y/Y
3) Yanling Yan
Yes
Y/Y
4) Travis Salisbury
Yes
Y/Y
5) Subha Arthur
Yes
Y/Y
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Fluorescent Probe
Development
Potential
Targeted
Targeted
Targeted
Potential
Projected Multiphoton
use (hrs)
50
30
20
50
20
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Program Director/Principal Investigator (Last, First):
III.
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Qualifications of the Imaging Core Staff
Dr. Norton has been a central resource for imaging since he was recruited to Marshall in 1991 and he has
been Director of the MBIC since its formal inception in 2006. While his research emphasis is in the area of
supramolecular chemistry, the design and characterization of assemblies of a small number of interacting
single macromolecules, this interest has driven the development of multiple instruments capable of meeting the
most critical of imaging requirements. As noted below, imaging requirements not only of researchers from on
campus but additionally from regional companies are addressed by the Center. Dr. Norton will oversee all
aspects of COBRE Faculty research support provided via the MUIC services. In addition to Dr. Norton, the
imaging center is currently staffed by Mr. David Neff, a Research Imaging Specialist who has been with the
laboratory since 2002, and earned his MS degree from Marshall’s Department of Biological Sciences in 2012
for his studies of the localization and development of the protein resulin, an important component of the fruit fly
flight mechanism. Mr. Neff’s current duties include new user training, assisting in user imaging, oversight and
performing experiments in addition to laboratory maintenance and supervision, Dr. Norton will also advise and
mentor an additional Imaging Applications Specialist, a PhD-level staff person to be recruited particularly for
this program. The addition of this new Imaging Applications Specialist is requested not only due to the
increased workload anticipated with this active group of researchers, but also as a “technology transfer”
opportunity, in which new ideas and new techniques can be brought into the Center. Currently at Marshall,
there are no faculty with experience in the production of custom protein reporters. The additional Imaging
Applications Specialist would have extensive experience in protein probe development and applications in
optical imaging and would bear major responsibility for new technique development and new probe design and
development in the lab. Dr. Norton, Mr. Neff and the Imaging Applications Specialist will work as a team to
develop protocols appropriate for each faculty imaging related Aim sub-project. A component of monthly
investigator meetings will be dedicated to the prioritization and scheduling of projects and progress reporting.
IV Instrumentation Available to all Researchers via the MUIC
IVA. Current Equipment: The major imaging instrumentation in the MUIC includes:
1) Leica Two Photon confocal fluorescence microscope (Leica TCS SP5 II AOBS). This optical imaging
system will provide the majority of imaging support for the COBRE researchers. It can be described as TCS
(true confocal scanner), SP5 (spectral imaging with up to 5 simultaneously active detectors), with a “MultiPhoton” laser tunable from 680nm-1080nm with a >3 Watts power at peak output, pulsed at 80MHz, with ~100
femtosecond pulses, which enable 2Photon events for fluorescence imaging. The system also has 405nm and
561nm diode lasers and Argon and HeNe gas lasers with multiple lines selectable (458, 476, 488, 496, 514
and 633nm), which together with the Ti-Sapphire (tunable) laser provides coverage for almost any selected
fluorophore. There are 6 non-descanned detectors, 4 descanned detectors enabling TLD (Transmitted Light
Detector, PMT) and standard epifluorescence detection. The system is prism/slit based, enabling full spectrum
tunable emission filtering. An AOTF (Acousto-Optical Tunable Filter) enables ROI (region of interest) specific
illumination with a high speed resonant scanner capable of illuminating 16,000 lines per second in the x/y
plane. Commercial Leica (LAS/AF) image processing software enables full 3D reconstruction and analysis.
With the Leica SP5 microscope (in non-MP mode) and optimal pinhole, resolution is as low as 200nm in the
XY and 350nm in the Z axis direction. The instrument is covered by a preventative maintenance agreement.
The cost of this contract fee is allocated among stakeholders according to use. The Leica system was
installed in December of 2010 and 9 publications (1-9) have issued from use of the system.
2) “Home built” widefield imaging system dedicated to single molecule fluorescence microscopy. Based on an
inverted, Nikon microscope, 521 nm Lasos diode pumped solid state 20mW laser TIRFM (total internal
reflection fluorescence microscope) and compatible with automated flow cell with temperature controller. A
Princeton 1 ProEM+ :512B camera with ~16% QE at 975nm enables image acquisition for most fluorophores.
3) Bruker “FASTScan” Atomic Force Microscopy (AFM) system with associated custom fabricated
fluorescence microscopy system. Fluid capable.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
4) Bruker MultiMode 8 Combined AFM/STM system with exceptional stability, fastscan mode and fluidic cell
compatibility.
5). Pacific Nanotechnology Nano-R AFM with programmable tip control for lithography.
6). Nanonics MV 1000 Near Field Scanning Optical Microscope with single photon detection (~20% detecton
efficiency at 975nm) and associated optical spectrometer. LN2 cooled Princeton Instruments Acton Spec10:400B with ~18% QE at 975nm.
7). JEOL 5310-LV (Low Vacuum) SEM with Backscattered Electron Detector, Cathodoluminescence Detector,
and Oxford Instruments Pentafet X-Ray Detector (EDS) with Isis Analysis Hardware/Software.
IVB. Future Equipment: The major imaging instrumentation roadmap for the MUIC includes:
1) Acquisition of a commercial super-resolution system would clearly benefit Marshall researchers. However
this field is rapidly evolving, and in recognition of the fact that the lifetime of such high end imaging systems at
Marshall is at least 8 years, we have decided to postpone acquisition while the field matures. Advances
stochastic optical reconstruction microscopy, allowing fast yet high resolution live image capture may be
rapidly adopted by competitive microscopy vendors, providing future cadres of COBRE investigators with
greater benefit than the systems now available would provide. While understanding that this COBRE provides
a powerful mechanism to update our current imaging systems, we recognize the global benefits of effective
budgeting, and an optical system upgrade is not included in this particular request.
2) As a component of the budget requested, we plan to craft a gas/humidity/temperature maintaining
incubation chamber for live cell studies.
3) A proposal to the NSF is currently being developed to acquire a field emission SEM to address the higher
resolution imaging requirements associated with growth of the Engineering disciplines at Marshall. Use would
not be restricted, and all of Marshall and local industries would be served.
V. Description of Core Operations
VA. Interaction with MUIC clients: Imaging services are provided to our clients in two different manners:
assisted or full service modes. In both of these modes, Dr. Norton, Mr. Neff and the investigator participate in a
preliminary consultation (planning the experiments to be performed and deciding the most appropriate mode
for the planned imaging studies), plan execution, intermediate assistance as required and final discussions at
the completion of each imaging experiment. Although initial contact is often made through Dr. Norton, David
Neff, our Imaging Specialist, whose office is in the core complex, provides detailed assistance, at any required
level, during the performance of any experimentation. We anticipate that Drs. Serrat and Arthur will require
mimimal supervision (assisted mode) while imaging experiments will be performed for Drs. Larre-Perez, Yan
and Salisbury (full service mode) until their technicians have been trained. While we will seek to inform
researchers of advances in imaging relevant to their needs/projects, we include in this proposal an
enhancement of this teaching by including a series of seminars by experts in COBRE investigator relevant
problem areas. This administrative core funded component should ensure continuing education for our
researchers and staff.
Prioritization of imaging requests: Each instrument in the facility has a web based sign-up sheet which
is used to schedule instrument time well in advance of anticipated use. In the event of oversubscription of an
instrument caused by high demand, in recognition of the support of the core facility, COBRE investigators will
be granted highest priority. Peaks in demand will be addressed by employing the following rules: (1) When
there is a backlog, Dr. Norton will move COBRE projects to the top of the queue. Priority within COBRE
requests will optimally be set via consensus; (2) Requests from all other investigators will be handled on a firstcome first-serve basis. The monthly COBRE investigators meeting will provide an excellent opportunity to
evaluate imaging needs and to schedule requests for instrument time.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Cost of services/Current Fee Structure: Charges for MUIC services are based on the cost of labor,
reagents, computing services and service contracts. However, the MUIC is not a self-supporting entity; the
University provides partial support for the facility in recognition of the importance of supporting preliminary
investigations by new investigators, including students. Our web advertised fee structure for sponsored
research by faculty has a rate for clock hours for the Leica SP5 of $55/hour for multi photon use. In
recognition of the role of COBRE in supporting the Imaging Core Laboratory and partial support of the service
contract, the costs for COBRE participants/.investigators will be reduced to $40 per hour for full service and
$20 per hour for assisted imaging. The facility has a five person internal oversight committee(Dr. Norton,
(Chemistry); Dr. Serrat (Anatomy and Physiology, MUSOM); Dr. Antonsen (Biology); Dr. Zill (Anatomy and
Physiology); and Dr. Harrison (Biology) which meet on an ad-hoc basis to address areas of budget and
infrastructure development for the center.?
VB. Imaging Core Personnel and Responsibilities
The Core will provide access to complex imaging systems, particularly the multiphoton and in the future,
scanning probe microscopes and will provide the training needed for understanding and interpreting the
obtained images. The staff, their responsibilities and their sources of support are summarized in Table 2.
Table 2. Proposed Key Personnel of the Marshall University Imaging Core Facility
Personnel/
Title
Percent
Responsibilities
Department
Effort/Source
Michael Norton, PhD,
MUIC Director
8% COBRE
MUIC Supervision of Protocol
Chemistry
8% NSF-RII*
Development, Project Management,
8% ARO*
76% State funds**
David Neff, MS,
Imaging Specialist /
50% NSF-RII
Imaging, training, analysis, design,
Biology
Facility Manager
50% MIIR*
fabrication, maintenance,
troubleshooting
To Be Recruited, PhD, Imaging Specialist
100% COBRE
50% Quantitative optical imaging for
Biochemistry &/or
COBRE Investigators and 50%
Microbiology
advanced technique/technology
development anticipating COBRE
* ARO Army Research Office and NSF grants provide funding for one month of summer research each; MIIR
Marshall Institute for Interdisciplinary Research.
**State funds support academic responsibilities including university teaching and service
VC. MUIC Imaging Core Systems
In the following section (VC1), we provide a detailed description of the primary imaging system that will be
used to support the COBRE investigators’ projects, the Multiphoton Fluorescence Microscope. All other
systems in the Center will also be available to these investigators. This section includes a description of the
instruments’ capabilities, current imaging support for the proposed COBRE investigators and anticipated
studies employing the microscope. A very short description of potential opportunities afforded by the
application of our newest system, a combined Fast AFM/wide field fluorescence microscope is provided in
section VC2.
VC1. Leica SP5 Confocal and Multiphoton Microscope
VC1A. SP5 General Utility and Capabilities Relevant to this COBRE
Multiphoton Microscopy (MPM): The Leica SP5 TCSII, paired with a Coherent Chameleon multiphoton
(MP) VisionII (IR) laser is capable of true spectral imaging with up to 5 detectors in the scanhead. The system
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
in the MUIC has 4 descanned detectors (DDs) and 6 non-descanned detectors (NDDs).
With the Leica SP5 in descanned mode, the emitted/reflected light passing through the detector pinhole is
diffracted by a prism and distributed to four detectors each with a slit allowing mapping of different wavelengths
with 1nm spectral resolution. There are a number of benefits associated with the use of the Leica SP5 confocal
microscope, including: A) Light rays from outside of the focal plane will not be recorded so unfocused light
does not create blurring. Leica LAS/AF (Leica software applications suite, advanced fluorescence) software
allows one to conveniently set the pinhole at one Airy unit allowing optimal x/y/z resolution to be obtained; B)
Scanning the object in the x/y-direction as well as in z-direction allows true, three-dimensional data sets of
voxels (volume elements) to be recorded. The MU Leica SP5 has a resonant scanner capable of 16,000 lines
per second in x/y plane; C) LAS/AF processing software enables full 3D reconstruction and analysis; and D)
Integrated electro-physiology software/hardware allows temporal coordination of imaging with physiological
measurements. With the Leica SP5 microscope (in non-MP mode) and optimal pinhole, resolution is as low as
200nm in XY and 350nm along the z axis. The power levels available from ancillary lasers associated with the
microscope well support techniques such as FRAP (fluorescence recovery after photobleaching) and FRET
(fluorescence resonant energy transfer, which are valuable for studies of transport and co-localization,
respectively.
Further benefits come with use of the Leica SP5 MP for microscopy: A) In MP mode the z-resolution is further
improved (making pinhole unnecessary) due to the superlinear absorption qualities of 2 photon absorption
(2PA). Only with very high laser power and very tight focus (high NA lens) will a 2PA event occur; this only
happens in a small volume (smaller than 1PA) surrounding the focal 'point' of the lens.
B) In MP mode although sample heating (IR induced) can be of some concern, bleaching is much reduced in
material surrounding the focal volume; and C) In MP mode, penetration depth is increased relative to standard
confocal imaging. In non-descanned mode (usually MP detection), the close proximity of the detector to the
sample increases detection efficiency.
The MUIC is very familiar with live cell techniques, and potential artifacts. Our samples are usually cultured
cells, grown on circular coverslips in multiwell plates. These cells can be maintained on the microscope stage
under buffer at variable temperatures in an open (currently no atmospheric control) micro-incubator. We plan to
modify the enclosure to enable humidity and gas composition control. Live cells are perturbed during transfer
and equilibration, leading to potential artifacts, which may be partially mitigated by use of a prolonged
equilibration period. For the studies as proposed here, this will not present a problem.
Microinjection: We currently have an Eppendorf microinjector that has an integrated compressor capable of
creating precisely controlled positive (outflow) or negative (for holding cells) pressure at the orifice of micro
capillary tubes. This functionality allows us to hold cells while simultaneously exposing them to a well
controlled chemical environment.
Manipulators: The scope has the capacity to hold actuators that allow micro-manipulation with sub-micron
precision. These mounted actuators are the finest available hydraulic manipulators and have been used at
Marshall to move and position cells and micro-pipettes to control the local extracellular environment as well as
in the non-biological task of mounting carbon nanotubes onto our atomic force microscopy tips.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
VC1B. Specific Use Cases and Anticipated Applications of Confocal/Multiphoton to COBRE
Investigators’ Subprojects (Drs. Serrat, Larre-Perez, Yan, Salisbury and Arthur)
Figure 1 Multiphoton System (left) and anaesthesia support station (right).
PI: Dr. Maria Serrat:
Dr. Serrat’s project originates from the observation that obese children have higher linear rates of bone growth
than children of average weight, leading to limb bowing, joint instability and fracture susceptibility. A major
thrust of Dr. Serrat’s COBRE proposal is the determination of the contribution of insulin-like growth factor
binding protein (IGFBP) concentration and distribution towards this enhanced bone growth rate. IGFBP
normally restricts bone growth by sequestering IGF, however IGFBP is reduced in obesity. Multiphoton
microscopy is an ideal technique for monitoring growth rate because it enables deep tissue live animal imaging
for the study of growth plate dynamics in vivo. Dr. Serrat, in collaboration with colleagues at Cornell University,
developed a platform for imaging intact skeletal growth
plates and to determine how systemic regulators arrive
and move within the cartilage matrix of the growth
plate under various experimental conditions. This
system, shown in Figure 1, provides a new mechanism
for observing the physiological regulation of bone
growth through dynamic changes in molecular
transport to the growth plate of a living animal. An
example image, taken in the Marshall imaging Core, is
presented in Figure 2.
Figure 2. In vivo multiphoton image of blood vessels in the plexus surrounding the tibial growth plate of a live,
anesthetized 5-week-old mouse. Vessels were visualized using a multiphoton microscope after an intravenous
injection of fluorescein. Plasma is red and blood cells appear as dark shadows within the vessels. The
collagen-rich perichondrium around the growth plate (green-gray pseudocolor) was visualized by second
harmonic generation (SHG), a robust signal from unstained collagen that is unique to multiphoton excitation.
SHG allows collagenous structures to be identified without injecting stains or dyes. Imaging was done by Maria
Serrat on a Leica TCS SP5 II Multiphoton Microscope housed in the Molecular and Biological Imaging Center
at Marshall University (image modified from Serrat, 2014 (2).
Among the experiments planned, transport and localization of labeled, biologically active IGF-1 will be
monitored in vivo, in the perichondrium and growth plate in real time, with and without saturation to block the
action of IGFBP. These experiments will be complementary to another set of macro-imaging (µCT)
experiments, in which IGFBP will be continuously pumped into the perichondrium on one side of growing
obese mice to test the hypothesis that growth factor transport is restricted by local binding proteins. Our
facility is ideal for supporting Dr. Serrat’s research thrusts. Five of Dr. Serrat’s recent publications employed
the multiphoton microscope at Marshall (1-5).
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Dr. Serrat has recently been awarded 5 research grants involving imaging using the Leica multiphoton system,
which are presented in Table 3. The imaging core provided a letter indicating the support of the Marshall’s
Molecular and Biological Imaging Center for the NIH R15 award (Entry Number 5 in Table 3).
Table 3. Dr. Serrat’s Recently Awarded Proposals Incorporating Use of MUIC Facility
1. NASA West Virginia Space Grant Consortium Research Initiation Grant; (no society grant number)
08/2011-8/2012; Imaging skeletal growth plates using in vivo multiphoton microscopy.
Role: PI; This project established a platform for live animal imaging using multiphoton microscopy to support
bone elongation research at Marshall University. NASA Technical Monitor: Dr. Jean Sibonga, Johnson
Space Center, Bone Mineral Lab; $20,000 (12 months)
2. CCTS University of Kentucky Pilot Grant Program (supported by NIH UL1TR000117)
(no society grant number) 06/2013-12/2014; Temperature enhanced bone elongation in growth plates.
Role: PI; This multidisciplinary project supports data collection for a new NIH R15 AREA submission. The
project uses in vivo multiphoton imaging and unilateral limb heating to study blood flow and molecular
transport at cartilage-vascular interfaces of murine tibial growth plates. The hypothesis is that heat localizes
delivery of systemic molecules into cartilage plates to promote bone lengthening. $25,000 (18 months)
3. American Society for Bone and Mineral Research GAP Award, (no society grant number) 04/201410/2015
Heat enhanced molecular delivery to growth plates for targeted bone lengthening.
Role: PI, This multidisciplinary project is based on a scored, but unfunded NSF proposal submitted August
2012. The purpose of the GAP program is to support continued research for future grant activity. $50,000
(18 months)
4. NASA West Virginia Space Grant Consortium Graduate Research Fellowship; (no society grant number)
2014-15 academic year; Unilateral heating to increase IGF-I uptake and bone length in mice.
Role: PI Mentor; This funded project provides a research stipend to Holly Tamski, a Marshall University
graduate student. $12,000 (12 months)
5. NIH R15AR067451-01; 09/19/14-08/31/2017; Heat enhanced molecular delivery to growth plates for
targeted bone lengthening.
Role: PI; This project uses in vivo multiphoton imaging and unilateral limb heating to study blood flow and
molecular transport at cartilage-vascular interfaces of murine tibial growth plates. The hypothesis is that heat
localizes delivery of systemic molecules into cartilage plates to promote bone lengthening. $383,064 (3
years)
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
PI: Dr. Maria Isabel Larre-Perez
In support of Aim 2 of Dr. Larre-Perez’s proposal, confocal Imaging will be used to determine differences in
the numbers and distribution of membrane bound Na/K-ATPase and Claudins (Claudin-2 and Claudin-4)
between proximal tubule cells from normal and obese animals (high fat diet). An example of the type of
relevant imaging which can be performed by the MUIC to assist/support Dr. Larre-Perez’s work is provided in
Figure 3 The objective of the experiment was to determine the effect of alpha subunit isoform expression on
tight junction composition, specifically Claudin-4
(CLDN4) localization. These confluent cultures of
LLC-PK1 were transfected with either alpha-1 or
alpha-2 isoforms of the alpha subunit of Na/K-ATPase.
Claudin-4 (CLDN4) protein is typically located in tight
junctions. The normal, apical localization of CLDN4 is
observed in the α-1 isoform case (Fig LP2 left),
however in the α-2 transfected cells, CLDN4 also
appears along the lateral domain (Fig LP2 right).
Figure 3 Top view (X,Y top) and Side view (X,Z)
bottom) fluorescence images of antibody labeled
Claudin-4 distribution in cultured LLC-PK1 cells
transfected with α-1 or α-2 subunits.
These sample types, cultured cells on suspended membranes, represent an ideal type of sample for
multiphoton microscopy because much less photobleaching is anticipated, and high resolution X-Z images can
be obtained. A strong case could be made that fluorescent protein labeling of the Na/K-ATPase and the
Claudins would enable studies of the dynamics of this system. Dr. Larre-Perez plans to extend these studies to
imaging of tissue mounts. Such studies will particularly benefit from the enhanced tissue penetration made
possible using the IR excitation (multiphoton) mode.
PI: Dr. Yanling Yan
Dr. Yan’s research involves the hypothesis that endocytosis of sodium pumps is a mechanism for modulation
of the population of sodium pumps in renal tubules, leading to blood pressure regulation. Therefore, studies to
be performed early during the COBRE project period will involve tracking protein entry, transport and export
from endosomes. The imaging core has worked with Dr. Yan to produce preliminary imaging studies which
have been performed to study the uptake of pNaKtide, an inhibitor of this endocytosis process. The images
presented in Figure 4 indicate rapid uptake and efficient distribution of a rhodamine labeled version of
pNaKtide, with maximal internal concentrations observed at 2 hours under the incubation/dosing conditions
employed.
Figure 4 Rhodamine-labeled pNaKtide
added to the culture medium is
efficiently distributed in 3T3L1 cells.
Similar cellular uptake levels were
observed in adipose tissue imaged at
the 12 hour time point after IP
injections of the same pNaKtide in
mice. Na/K-ATPase α-1 localization
and/or positional dysregulation will be observed using immunofluorescence, using fixed tissue probed with
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
primary monoclonal anti-α-1 antibody followed by tagging with Alexa-488 conjugated secondary antibody. The
spectral overlap of the fluorescence of these species is negligible and these species can be readily resolved
using the spectral filters of the SP5 confocal system. The images in Figure 4 were obtained using the Leica
system at Marshall and appear as Supplementary Figure S1 in a recently published article co-authored by Dr.
Yan. The paper, titled: “ pNaKtide inhibits Na/K-ATPase reactive oxygen species amplification and attenuates
adipogenesis”, was published in Sci Adv in 2015. (9)
Although the antibody based experiments Dr. Yan has proposed are certainly appropriate and will be fully
supported by the core, we believe that, while they will require creation by MUIC staff, the use of fluorescent
protein tags to follow the dynamics of protein transport in these systems would add significant value to the
imaging experiments by enabling live cell studies.
PI Dr. Travis Salisbury: Dr. Salisbury’s proposed efforts focus on identifying mediators and mechanisms
which link obesity to an increase in breast cancer risk, based on his observations that the application of media
conditioned by murine or human adipocytes increases the levels of L-Type Amino Acid Transporter 1 (LAT1)
protein and mTOR activity in estrogen receptor (ER) expressing MCF7 or T47D Breast Cancer Cells. LAT1 is
an amino acid transporter that mediates the uptake of leucine, among other amino acids, and leucine activates
mTOR, a kinase that is essential for cell growth. Particularly, Dr. Salisbury will determine which adipocytesecreted factors (ASF’s) increase LAT1 concentrations on the surface of lysosomes. In his proposed
Experiment 1B, ASF triggered trafficking of LAT1 to both the plasma and lysosome membranes will be
characterized. BCCs or MCF10A cells will be transfected with mCherry-LAT1 expressing plasmid for 24 h,
followed by treatment with vehicle control or ASF’s, singularly or in combination, at concentrations and time
points corresponding to those employed in Experiment 1A, which is a non-imaging protein localization study.
Both “live” imaging experiments and post-treatment imaging will be performed. “Live” experiments will provide
not only data on the dynamics of the process of transport to the plasma and lysosome membranes but also
may provide insight into the mechanism of transport (diffusional vs active transport). For fixed cells, methods
of organelle/protein labelling for identification will include for the nucleus (4,6-diamidino-2- phenylindole
(DAPI), cyan), for the lysosomes a lysosome specific protein (LAMP1, green), for the plasma membrane
(concanavalin A, blue), or LAT1 (mCherry, red) or by using specific antibodies. Co-localization of LAT1 with
LAMP1 (lysosome) or plasma membrane (concanavalin A) will be determined through image analysis. While
we have not performed the imaging studies described above, the laboratory has prior experience and has
performed studies of protein redistribution (10). Particularly for quantitative studies of protein distribution
between compartments and the membrane the newer, multiphoton system’s advantage is that it will diminish
the volume artifacts common to fluorescence measurements performed on cultured cells. Time sequential
images are readily obtained under full software control. 3-D images can be obtained through sequential
collection of 2-D images along the Z-axis at 0.35 micron steps.
The Leica multiphoton system can readily accommodate the multiple labels planned for these studies because
it has a tunable excitation source and the multiple variable bandpass “filters” can each be independently set to
obtain optimal contrast and spectral separation. Although the time constant for redistribution of LAT1 is yet to
be determined, the FRAP (fluorescence recovery after photobleaching) technique may be employed to
determine the kinetics if this is found to be a relatively rapid process.
PI Dr. Subha Arthur: An important regulator of enterohepatic bile acid recirculation is the intestinal apical
sodium bile acid transporter (ASBT). ASBT, which is the sole bile acid absorptive mechanism in the terminal
ileum, is localized at the brush border membrane of absorptive villus cells of the terminal ileum. ASBT requires
a favorable transcellular Na+ gradient for its optimal activity, which is provided by Na-K-ATPase present on the
basolateral membrane of villus cells. Dr. Arthur’s preliminary studies in the well-established in-vivo model of
obesity, specifically in Zucker rats (Obese vs. Leans), suggest that Na-bile acid co-transport is increased in
obesity. While it was expected that Na-K-ATPase might be enhanced at the cellular level to support this Nabile acid co-transport increase, interestingly, it was found to be in fact, reduced. Confocal microscopy will be
used to support Aim 2b of her proposed project, particularly to test the hypothesis that altered trafficking and
localization of α1 and β1 subunits of Na-K-ATPase is responsible for decreased Na-K-ATPase activity.
Immunohistochemical methods for labeling the relevant proteins have been employed by Dr. Arthur at her
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
previous institution and will be employed for studies of excised tissue. One hypothesis for the dysregulation is
that Ankyrin and spectrin, cytoskeletal proteins known to affect the trafficking of Na-K-ATPase may be
responsible for the lowered basal membrane activity, due to decreased localization at the membrane. While
the core has not previously studied this particular system, image analysis will be used to determine the relative
concentrations and localizations of the proteins of interest. If colocalization becomes valuable, antibody based
Fluorescence Resonant Energy Transfer (FRET) experiments can be performed to investigate spatial proximity
of cytoskeletal components and proteins of interest (11). Fluorescent protein based FRET experiments can
enable the dynamics of interactions of proteins to be studied if localization is signal dependent. The Leica
imaging system is ideal for the fixed tissue (immunohistochemistry) studies proposed here because
multiphoton excitation has a much longer effective penetration depth than single photon approaches. .
Fluorescent protein based labels which could be developed in this program could support future in vitro studies
which would enable the dynamics of this altered trafficking to be observed in model systems.
VC1C. Methods Training for Confocal and Multiphoton Image Acquisition and Analysis (Outreach)
COBRE project investigators and pilot grant awardees will require training in traditional confocal and multiphoton imaging. Speakers, including relevant experts, will be recruited for seminar presentations in order to
maintain an awareness among the investigators of current and emerging techniques in optical and if
warranted, scanning probe microscopy. However there is no substitute for hands on training and
experimentation. Although preliminary investigations may be performed for researchers by Core staff,
investigators are strongly encouraged to register their students/staff for training for all extended studies, such
as the projects proposed.
David Neff, our current Research Imaging Specialist, administers short (one week), personalized training
courses in Laser Scanning Confocal and Multiphoton Microscopy for new users on an ad-hoc basis. Although
the initial introduction session uses standard samples, the higher level training sessions utilize samples
provided by the researcher. Imaging results are reviewed and critiqued in collaboration with the
investigator/student and their PI. An Undergraduate and a Graduate level multidisciplinary introductory
microscopy course, covering electron, optical and scanning probe microscopy, are offered each year through
the College of Science at Marshall, taught by Dr. Norton, for those requiring a more in-depth understanding of
imaging techniques and imaging artifacts.
In recognition of the fact that the investigators supported by this COBRE program would benefit from a higher
level of support, the Imaging Center will recruit an Imaging Specialist to particularly support the COBRE
investigators. This new temporary position will allow the facility to provide dedicated support, including both
extensive student/faculty software and hardware training, “full service imaging”, and development of
fluorescent (protein) probes designed to support their evolving questions. The Core Director, our current
Research Imaging Specialist and the new Imaging Application Specialist will collaboratively assist
investigators, beginning with help in designing experiments which fully utilize the capabilities of the
instruments, through sample preparation, experiment execution, data collection and analysis, to in-depth
interpretation of the data and publication. Such custom support will assure immediate productivity for the
COBRE investigators. This Research Applications Specialist position should be attractive to future imaging
core directors both because it is created with a ready-made core of 5 COBRE collaborators, representing the
diversity of users expected of most facilities, but also because the Specialist will have the infrastructure
required in order to develop techniques, particularly those involving proteins as probes for fluorescence
microscopy,.. Researchers considering a career in Imaging Facility Direction/Management will find this
position to provide excellent experience and training as well as fertile ground for developing/maintaining a
strong publication record..
VC2 Potential Applications of combined fluorescence and atomic force microscopy
This core contains instrumentation present in very few Imaging Cores One particular example is the Bruker
Fast Scan AFM which we have combined with a single molecule widefield imaging system. The vesicle based
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
approaches to the study of membrane associated proteins developed by Drs. Laurre-Perez and Arthur appear
to be particularly amenable to conversion to planar formats which can be interrogated simultaneously using
light and AFM microscopy. While the use of AFM for quantitative membrane bound protein studies must be
developed and likely will remain an ex-vivo technique, the ability to observe surface dynamics and clustering,
with optical methods performing the role of confirming that the system demonstrates in vivo like characteristics
is tantalizing. Both fluorescent labeled proteins and immunofluorescence approaches can be applied, with the
antibody, alone or decorated with gold nanoparticles, acting as volumetric tags for AFM recognition. These
avenues of research extension will only be explored in cases in which they support the evolving research
requirements of the researchers.
VI. Coordination of MUIC Facility Expenses, Contingency Plans and Sustainability
Marshall University and the Marshall University Research Corporation have a history of support for
instrument repair and service contracts. Funding for our current Imaging Specialist, David Neff is derived
partially through grant support and partially through a Marshall University Research Fund (MIIR). These funds
are anticipated to remain stable over the course of the proposed COBRE performance period. Provision of
enhanced MUIC services to the COBRE investigators will require support from this grant application. The
majority of the budget request is for an Imaging Applications Specialist, dedicated to the COBRE projects,
particularly protein probe and protocol development. Our sustainability plan for support of this additional
position resides in the generation of sufficient number of proposals requiring imaging support that are funded
by the end of the performance period.
In addition to the researchers indicated above, the Imaging Center supports the requirements of researchers,
including faculty, undergraduates, graduates and postdocs, in Geology, Biology, Chemistry, Physics,
Anthropology, Forensics and Engineering as well as members of the School of Medicine and Biomedical
Science Program.
The Imaging Core at Marshall provides considerable expertise which is valued by the local industrial
community. Particularly, when companies request microanalytical support from the University, they are
referred to the Imaging Center. Three of the larger local employers are examples of such clients, Special
Metals, an alloy discovery and production facility, the Flint Group, a pigment manufacturing spinoff of BASF,
and Alcon, a major manufacturer of lens implants.
The Center’s capabilities will be enhanced, under a recently awarded NSF grant, to provide long distance
imaging support in the areas of Atomic Force and Fluorescence imaging. The Phase I award supports
development of local link-up capability which will enable projection of 3D images, at near acquisition speed, in
the Visualization Laboratory. The Vis Lab, part of the 3 year old Engineering Complex of the main Marshall
Campus, houses a 20 foot screen for 3D viewing using synchronized glasses. The high resolution 3D images
generated by our Leica Multiphoton system are best appreciated in such a venue, and will be available for
future researchers (including this COBRE group) and future students.
The Center requires continual rejuvenation. We anticipate submitting a Major Research Instrumentation
proposal to the NSF for acquisition of a Scanning Electron Microscope with high resolution and low voltage
imaging capability. In addition to the Norton group, researchers from Geology, Biology, Chemistry, Physics,
Forensics and Engineering will contribute to this proposal.
VII. MUIC Milestones and Assessment Plan
Two near-term goals of the MUIC are to support the research goals of the COBRE projects and to improve
the success rate of COBRE investigator initiated grant applications. Our milestones and assessment plan are
designed to evaluate our success in achieving these twin goals.
Milestone 1: We will demonstrate that the MUIC is supporting COBRE projects by contributing to two
publications per year in Years 02, 03, 04 and 05.
Milestone 2: We will contribute expertise and data generated in the MUIC to at least two grant applications
initiated by a COBRE faculty member per year in Y03, Y04, and Y05.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
VIII. Contributions and support from Marshall University
The University and the College of Science will continue their support of the services of the MUIC. Marshall
currently pays 50% of David Neff’s salary and 100% of Dr. Norton’s 9 month salary. The university provides
considerable furnished lab space in the Robert C. Byrd Biotechnology Science Center (1200 sq. ft.) at no cost
to the MUIC. Ms. Anna Thomas, COBRE Administrative Assistant, will place purchase orders for the Core. The
MU Research Corporation prepares monthly profit/loss financial statements and monitors compliance with
OMB Circular A-21. This support is adequate for the scope and volume of MUIC service requests.
IX. Literature Cited
1. Serrat MA, Efaw ML, Williams RM., Hindlimb heating increases vascular access of large molecules to
murine tibial growth plates measured by in vivo multiphoton imaging. J Appl Physiol,;116(4):425-38. doi:
10.1152/japplphysiol.01212.2013. Epub 2013 Dec 26. PMID: 24371019. 2014.
2. Serrat MA., Environmental temperature impact on bone and cartilage growth. Comprehensive Physiology.
4(2):621-55. PMID: 24715562. 2014
3. Serrat MA, Schlierf TJ, Efaw ML, Shuler FD, Godby J, Stanko LM, Tamski, HL., Unilateral heat accelerates
bone elongation and lengthens extremities of growing mice. Journal of Orthopaedic Research. 33(5):692-8
PMID: 25639189. 2015.
4. Serrat 2013
Serrat MA. Allen’s rule revisited: Temperature influences bone elongation during a critical
period of postnatal development. Anatomical Record. 296(10):1534-45. PMID: 23956063. 2013
5. Serrat 2010
Serrat MA, Williams RM, Farnum CE. Exercise mitigates the stunting effect of cold
temperature on limb elongation in mice by increasing solute delivery to the growth plate. Journal of Applied
Physiology. 109: 1869-1879. PMID: 20930127. PMCID: 3006398. 2010
6. Ritzmann 2013 Roy Ritzmann and Sasha N Zill, Neuroethology of Insect walking. Scholarpedia, 8 (9), pp
30879, 2013.
7. Zill 2013 Sasha N. Zill, Sumaiya Chaudhry, Ansgar Büschges and Josef Schmitz, Directional specificity and
encoding of muscle forces and loads by stick insect tibial campaniform sensilla, including receptors with round
cuticular caps, Arthropod Structure & Development, 42 (6), pp 455-67, 2013. doi: 10.1016/j.asd.2013.10.001.
8. Wu, T. C.; Rahman, M.; Norton, M. L., From nonfinite to finite 1D arrays of origami tiles. Accounts of
Chemical Research 2014, 47, 1750-1758.
9. Komal Sodhi, Kyle Maxwell, Yanling Yan, Jiang Liu, Muhammad A. Chaudhry, Morghan Getty, Zijian Xie,
Nader G. Abraham, Joseph I. Shapiro, pNaKtide inhibits Na/K-ATPase reactive oxygen species amplification
and attenuates adipogenesis, , Sci Adv 2015, 16, Oct. 2015. doi: 10.1126/sciadv.1500781.
10. Battistella-Patterson, A.S., Fultz, M.E., Li, C., Geng, W., Norton, M. & Wright, G.L. PKC translocation is
microtubule-dependent in passaged smooth muscle cells . Acta Physiologica Scandinavica 170 (2), 87-97,
2000.
11. A. C. Dykes, M. E. Fultz, M. L. Norton, and G. L. Wright, Microtubule-dependent PKC-α localization in
A7r5 smooth muscle cells, Am J Physiol Cell Physiol 285: C76–C87, 2003.
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
X. Protection of Human Subjects and Care of Vertebrate Animals
The MUIC itself is not proposing work involving human subjects or vertebrate animals as part of this proposal.
However, for example, Dr. Serrat will perform in-vivo imaging studies of mice. Investigators seeking to perform
such studies in the MUIC will be required to demonstrate appropriate Institutional Review Board or Animal
Care and Use Committee approval of their research plan in advance of any experimentation. The multiphoton
system is well instrumented for the use of gaseous anaesthesia agents, and protocols for cleanup and
decontamination of the system from potentially biohazardous materials after use are in place.
XII. Imaging Data and Resource Sharing Plan
Data generated by researchers utilizing resources of the MUIC are considered intellectual property of the PI.
Although limited physical backup of data is provided by the Core, the Core does not disseminate researcher
data. All MUIC services are available to participants in WV COBREs, the WV-INBRE program and all
biomedical researchers at WV universities and colleges.
Budget Year 1
Personnel
Faculty
Sal
8,499
Benefits
2,550
11,049
post doc
Sal
42,769
Benefits
12,831
55,600
graduate
stu
Extra Help
12%
Sal
Benefits
-
Sal
Ben
Total Salaries
51,268
Total Benefits
15,380
Personnel Total
66,649
Equipment
14,155
Travel
3,500
PHS 398/2590 (Rev. 11/07)
Continuation Format Page
Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Tuition
Supplies
14,269
Other Cost
15,500
Direct
Total
Year 1
114,064
Indirect
Total
Total
Request
41,966
156,030
IMAGING CORE BUDGET JUSTIFICATION
A.
Co- Investigator $ 11,049 (Salary + Benefits)
Michael Norton is a Professor within the Department of Chemistry at Marshall
University. Professor Norton will have primary responsibility for the direction of the Imaging Core
components of the project. He will also oversee the work performed by the Postdoctoral Researcher.
Professor Norton will devote ~50% effort during 2 summer months per year. The fringe benefits of
Professor Norton were calculated at the rate of 30%. This is the composite rate provided by our
Sponsored Projects Office for Academic summer salary. Anticipated cost of living increases are 3%
per annum.
B.
Imaging Applications Specialist 1 To be Named,$ 55,600 (Salary + Benefits)
This PhD level researcher will be primarily responsible for conducting the proposed experiments in
the domain of molecular and biomolecular probe development, preparation and imaging via multiple
methods, with chief responsibility for preparing samples for imaging studies. Substitution of
students/technician(s) for this position may be required while a suitable candidate is being recruited or
during any breaks in the continuity of the position, in order to provide continuous laboratory support
for the project. This person’s participation in the proposed studies will help them develop research
skills which will prepare them to contribute to development of imaging technologies during their
career. This person will devote 100% effort towards the project year round. The fringe benefits for this
researcher are calculated at 30%. Anticipated cost of living increases are 3% per annum.
C. EQUIPMENT: $ 14,155 Control Unit with analog gas flowmeter. Uses pre-mixed
gas tank INUF-UK-F1 Leica Part No: 8104479. This will enable longer time, non-perturbative imaging
of cells and tissue samples.
D. TRAVEL $3,500
Domestic Travel for the Co-Investigator and on a rotating basis, the postdoc, to attend national
conferences to present the results of the research. The total projected cost includes the cost of coach
airfare, ground transportation, lodging and meals. Domestic U.S. flag carriers will be utilized whenever
possible. The cost estimates are based on a survey of market costs and historical usage.
PHS 398/2590 (Rev. 11/07)
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
E. OTHER DIRECT COSTS $21,769
1. Materials & Supplies$21,769
Expendable supplies to be used in the research project: lab supplies including glassware,
chemicals, reagents, protein purification columns, clones, proteins, media for growth, buffers,
synthetic DNA, solvents, gasses for cell culture and incubator, project specific software, computing
machines to be used for instrument control and other miscellaneous supplies specifically and directly
beneficial to the project. Costs were estimated based on historical usage.
6. Other Costs: $15,500 ($8,000 + $7,500
$8,000 Custom molecular probe synthesis and Codon optimized plasmid synthesis acquisition cost.
$7,500 Component of annual service contract, prorated for a 50% Leica Multiphoton use rate in
support of COBRE researchers.
F. TOTAL DIRECT COST = $114,064
G. INDIRECT COSTS (F&A) $41,966
Indirect costs are calculated at 42% of modified direct costs. Modified direct costs are computed as
total costs less equipment. The 42% rate is Marshall University’s federally negotiated rate for on
campus research activity, with the US Department of Health and Human Services. The date of the
agreement with DHHS is 09/19/08. The indirect Cost Rate Period is July 1, 2008 to June 30, 2012.
F. TOTAL ANNUAL COST = $156,030
Annual budgets Years 2 – 5
Budget
Personnel
Faculty
Sal
8,499
Benefits
2,550
11,049
post doc
Sal
42,769
Benefits
12,831
55,600
graduate
stu
Extra Help
12%
Sal
Benefits
-
Sal
Ben
Total Salaries
51,268
Total Benefits
15,380
Personnel Total
66,649
PHS 398/2590 (Rev. 11/07)
Continuation Format Page
Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
Equipment
Travel
Tuition
3,500
Supplies
14,269
Other Cost
15,500
Direct
Total
99,918
Indirect
Total
Total
Request
41,966
141,884
IMAGING CORE BUDGET JUSTIFICATION
A.
Co- Investigator $ 11,049 (Salary + Benefits)
Michael Norton is a Professor within the Department of Chemistry at Marshall
University. Professor Norton will have primary responsibility for the direction of the Imaging Core
components of the project. He will also oversee the work performed by the Postdoctoral Researcher.
Professor Norton will devote ~50% effort during 2 summer months per year. The fringe benefits of
Professor Norton were calculated at the rate of 30%. This is the composite rate provided by our
Sponsored Projects Office for Academic summer salary. Anticipated cost of living increases are 3%
per annum.
B.
Imaging Applications Specialist 1 To be Named,$ 55,600 (Salary + Benefits)
This PhD level researcher will be primarily responsible for conducting the proposed experiments in
the domain of molecular and biomolecular probe development, preparation and imaging via multiple
methods, with chief responsibility for preparing samples for imaging studies. Substitution of
students/technician(s) for this position may be required while a suitable candidate is being recruited or
during any breaks in the continuity of the position, in order to provide continuous laboratory support
for the project. This person’s participation in the proposed studies will help them develop research
skills which will prepare them to contribute to development of imaging technologies during their
career. This person will devote 100% effort towards the project year round. The fringe benefits for this
researcher are calculated at 30%. Anticipated cost of living increases are 3% per annum.
C. EQUIPMENT: N/A
D. TRAVEL $3,500
Domestic Travel for the Co-Investigator and on a rotating basis, the postdoc, to attend national
conferences to present the results of the research. The total projected cost includes the cost of coach
airfare, ground transportation, lodging and meals. Domestic U.S. flag carriers will be utilized whenever
possible. The cost estimates are based on a survey of market costs and historical usage.
PHS 398/2590 (Rev. 11/07)
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Program Director/Principal Investigator (Last, First):
Sundaram, Uma/Norton, Michael L. (Imaging Core)
E. OTHER DIRECT COSTS $21,769
1. Materials & Supplies$21,769
Expendable supplies to be used in the research project: lab supplies including glassware,
chemicals, reagents, protein purification columns, clones, proteins, media for growth, buffers,
synthetic DNA, solvents, gasses for cell culture and incubator, project specific software, computing
machines to be used for instrument control and other miscellaneous supplies specifically and directly
beneficial to the project. Costs were estimated based on historical usage.
6. Other Costs: $15,500 ($8,000 + $7,500)
$8,000 Custom molecular probe synthesis and Codon optimized plasmid synthesis acquisition cost.
$7,500 Component of annual service contract, prorated for a 50% Leica Multiphoton use rate in
support of COBRE researchers.
F. TOTAL DIRECT COST = $99,918
G. INDIRECT COSTS (F&A) $41,966
Indirect costs are calculated at 42% of modified direct costs. Modified direct costs are computed as
total costs less equipment. The 42% rate is Marshall University’s federally negotiated rate for on
campus research activity, with the US Department of Health and Human Services. The date of the
agreement with DHHS is 09/19/08. The indirect Cost Rate Period is July 1, 2008 to June 30, 2012.
F. TOTAL ANNUAL COST = $141,884
PHS 398/2590 (Rev. 11/07)
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