1. Participant information (Major student or tech users in your labs should be
listed here as well):
a. Approximate number of hours worked on the machine, including training or
administrative:
Beginning 7/1/2011 a technician (David Neff) was hired and began duties in support of the Leica
SP5 at 20 hour/week. Funds from MIIR are currently paying the for technician’s salary through
November 2013.
Startup/shutdown procedures as well as data sheets and diagrams describing the hardware
(spectral configuration, obectives available) have been prepared by the technician with revisions
ongoing. These resources are also available at the SP5 webpage
(http://www.marshall.edu/mbic/instrumentation/LEICA%20SP5%20TCSII%20MP/LEICASP5.html )
As well as training users and maintaining the system, the technician has performed imaging for
Zill lab, Claudio lab, Kmeic lab, and others and for biology, chem, and biomedial science
department course work. We have trained approximately 12-15 researchers, students and
technicians to use the microscope independently.
The first entry in the logbook reads 11/4/2010, since this date, ~633 hours have
been logged with about 1/3 of these hours including the use of the multi-photon
laser. To encourage new users, we had a 6 month year grace period regarding billing scope
users for beam time. Since we began to send out invoices in May of 2011, we have billed users
for 592 hours of microscope time. During the second half of 2012 (in keeping with the MRI
requirement that funds resulting from usage of instrumentation must be spent within the grant
period), we used the full balance of these funds for improvements to the system. With these
funds we have added significant function to the system which are described below.
Maintenance and repairs;
STAGE: During year one of operation, the large motorized stage that is part of our
configuration (new to Leica) as supplied to MU was made fully functional via
reprogramming and hardware upgrades. Limit switches and one motor were
replaced by Mike’s Machine shop. Cost for this disassembly and repair was covered
by Mike’s machine and was carried out via phone conversations between technician
and the shop.
In an effort to identify sources of drift and develop corrective measures to reduce
sample/image drift we performed stage/sample drift experiments using
temperature probes located on the stage (blue plots) as well as outside the
immediate microscope area to measure room air temperature. We did not fully
separate the image drift effects caused by lens/scope body changes vs stage
expansion/contraction but we were able to produce worst (3um in xy and 5um in z)
and best (less than <1um in z and <1um in xy) case scenarios while continually
imaging over a 30 minute period. Thus, we were able to establish room and scope
conditions that minimized image/sample drift allowing us to resume live cell time
course work. The extremes are summarized below with images shown from
beginning and end of each time course.
Best (bottom) and worst case (top) scenarios correlating temperature on
stage and in room air with image drift in x, y, and z.
LASERS: Coherent laser has installed a total of 3 MP lasers on our system during the
2.5 year life of the system. The original MP laser supplied with the SP5 exhibited
persistent wavelength tuning difficulties so an interim laser was installed in
12/2011 (Ultra II without pre-compensation optics). This laser was replaced by
our current MP laser, a Chameleon VisionII, in 5/2012. This laser is an upgraded
design that includes a one piece chassis that apparently is now used by Coherent in
all VisionII systems; this laser has performed very well since installation.
ALIGNMENTS: Two full laser/scanner alignments have been performed by Leica
technicians since installation of the SP5.
MISC: During year 2 The MP EOM (electro optical module) driver was recalled by
the manufacturer for safety reasons, as of 2/12/2013 we are on a waiting list to
receive a rebuilt controller. The electro optical module/driver has functioned
normally during this time. During inspection of the controller, Leica service
noticed an opacity in the EOM crystal itself – we have been promised by Leica a new
crystal whenever the controller replacement is scheduled.
In addition, working with Leica technical has allowed us to clarify
some specific aspects of instrument configuration including spectral detector setup
and control of polarization both at sample and in the pre-detector emission light
path. Also we have resolved a couple of hardware/software communications bugs
including a scope controller work around that will no longer be necessary when the
scope touchpad interface is replaced (also promised by Leica) with one having
updated firmware.
UPGRADES TO SYSTEM: ,Using funds raised by invoicing for SP5 beam time we
were able to upgrade the SP5 in 2 significant ways. As of 12/2012 we have the
optical and mechanical hardware needed to arrange our SP5 in inverted
configuration (see image below). We purchased an optical inverter tube(white
arrow) from LSM Tech that is threaded to fit 7 of our 8 objectives. We also had a
stage plate machined and purchased all necessary hardware to attach the ‘inverter’
stage (red arrow) to the main stage. This setup allows us to use motorized focus
and stage controls in inverted configuration. Partly in response to user requests,
we also purchased a TE cooled color ccd camera from Leica which has been
seamlessly integrated into the SP5 system (blue arrow) with no changes in function
or appearance of the software (except a camera tab which allows users to select
light path and camera settings). This is also shown in the image below.
Camera and stage upgrades made possible by SP5 user fees.
b. A brief description of your contribution (note, I can fill most of this in for those of
you on this email, please do this only if you have something you want to be
highlighted, or something you feel I might not know about):
2. Organizational Partners (educational institutions or business partners you
are working with, research or educational) - with 1-2 sentence description:
Parabon nanolabs – can’t remember the project details
3. Other collaborators or contacts (include personal research or educational
collaborations with individuals here) - with 1-2 sentence description:
External collaboration with Concord enabled by the SP5 system
A collaboration with the objective of determining optimal conditions for enhancing the
fluorescence of quantum dots via metal nanostructures has been initiated by Dr. Timothy D.
Corrigan, who is an Assistant Professor of Physics at Concord University and his undergraduate
research student Nguyet Le. The approach, which utilizes a combinatorial library of metal
nanostructures is perfectly matched to the capabilities of the Leica system.
In metal-enhanced fluorescence (MEF), the fluorescence molecules separated from a metal
surface by several nanometers can be enhanced. The fluorescent enhancement is dependent on
the size and spacing of the nanoparticles, as has been shown previously for a number of
fluorophore molecules. Fluorescence from quantum dots is of particular interest because the
quantum dots do not lose fluorescence ability with prolonged exposed to light and they have
higher intensity of fluorescence than most organic fluorophores. The purpose of this study is to
determine the effect of size and spacing on fluorescence intensity when coupling gold nanoparticles with quantum dots. In the combinatorial studies, gold particles ranging in diameter
from 30 nm to 130 nm with varied spacings are fabricated in arrays on the substrate, followed by
a protein spacer-layer and quantum dots. The fluorescence signal from the metal enhanced
quantum dots are then determined using the confocal microscope. Two aspects of the
microscope are particularly relevant to this study. First, the system affords multiple high power
lines suitable for excitation of quantum dots, which can be excited over a wide range of
wavelengths. The availability of these lines enables selection of excitation energies which
minimize background signals. The second pertinent capability is the ability to select the emission
wavelength center and bandwidth to match precisely the emission properties of the quantum
dots. This emission tunability also enables fascile scanning in order to determine sample
emission spectra to be obtained. In complex systems, such as these nanofabricated systems,
unanticipated impurity signals often arise. For small scale samples, such as these, these signals
can overwhelm the “target” signal, producing an unacceptable background, which would not be
identified without spectral scanning capability. Fortunately, an unexpected photoresist
fluorescence signal was identified and, because quantum dots can be purchased with a wide
range of emission wavelengths, quantum dots with emissions outside the envelope of the
interference were identified. Without the spectral scanning capability of this microscope, it is
possible that these artifacts would have interfered with the study, without our knowledge. The
system has enabled us to produce a much higher level study.
The results of these studies will be presented at the next APS conference, which will be held in
Maryland from March 18 - 22nd. The title of the presentation is “Combinatorial Approach to
Studying Metal Enhanced Fluorescence from Quantum Dots”.
Reflected light image of gold
nanoparticle array used for
navigation and context during
spectral imaging.
Microbiology with high school senior. As part of a pre-undergrad semi-formal research project,
Alex Gain a local high school graduate used the SP5 to image and show differential staining of
bacteria and fungi (both mold and yeast forms ) using calcofluor white fluorescent dye. He
brought in active cultures of 3 different microbes and we stained and imaged them live.
Aspergillus brasiliensis mold form
stained with calcofluor white and
imaged by high schooler Alex Gain
during a 2 week long pre-undergrad
research experience. Field of view
is about 250um x 250um. Candida
albicans and bacterial rods were
also stained and imaged.
On 2 separate occasions in 2011-2012 Dr. Marcia Harrison hosted a visiting
scholar from Glenville State, Sara Sawyer
-can’t remember this project-
4. Activities and Findings:
a. Research and education activities (brief description of your research use of the
machine, courses taught with images from this machine with number of
grad/undergrad participants, grad or undergrad research projects in your labs that
use this machine, anything else that seems appropriate):
b. Findings (brief description of your data and implications):
Kmeic lab: Imaging for the Kmiec lab (associated with MIIR) has included relatively low
resolution imaging of comet assays for DNA damage. Deep penetration of sample via optical
sectioning allowed us to visualize many more cells in less time than their standard procedure
(standard epi-fluor scope). We also have been tapping the potential of the SP5 to generate high
resolution 3 channel images of cultured cells. In these experiments, co-localization of nuclear
proteins indicating DNA damage was made possible through use of the high power glycerol
objective, UV laser, and fine spectral separation available with the SP5 4PMT configuration. The
comet assays are currently being incorporated into manuscripts. The high resolution images of
cultured cells will guide a series of experiments that were not possible at MU before the
acquisition of the SP5. Unfortunately Dr. Kmiec left MU for Delaware in 2012 so at that time, this
line of experimentation came to an end.
Since leaving MU, the Kmiec group has published (listed below) and with data from
the SP5 were able to support their prediction that gene corrected cells attached onto
nanofibers and proliferated robustly.
Norton nanoscale chemistry lab: Ongoing imaging expts. with simple 2 color qdot
samples (quantum dot spin coated on glass and imaged as single particles) have
demonstrated apparent limitations of the confocal scanning method when applied
to these single blinking nanoparticles (confirmed with our older MRC1024 confocal).
On site and remote aid from Leica technicians in this imaging challenge yielded
confocal images that appear much less clear than those of the same sample imaged
with ccd cameras. We are continuing these investigations. In addition we have
done some preliminary work using the SP5 to image fluorescence from dna
conjugated carbon nanotubes. Early results were not entirely discouraging but we
likely will need an additional IR sensitive detector to pursue that work.
c. Training and Development (any specialized training you or your students have
done, Mike and David's microscopy course, etc):
Imaging course:
The two photon system has been an integral part of the Imaging Course since before it was
installed. During the evaluation period, the scope ( and the presentations provided by potential
vendors) were used in the course to introduce students to the wider range of experimental
parameters they would be able to use once the system was installed. Because the course covers
a wide range of microscopic image capture systems, including atomic force microscopy and
electron microscopy, the two photon and confocal portion of the course represents about 1/3 rd of
the material presented. About 2/3 of the students participating in the course, during the period
of the grant, have based their course projects on use of the confocal system and more generally
fluorescence microscopy. It is worth noting that the Imaging Course, which is offered to
students with a need for deeper understanding of imaging, and which has included students from
Archaeology, Geology, Biology, Biomedical Sciences as well as Chemistry, had its genesis in an
NSF MRI granted to Marshall for the acquisition of a Scanning Electron Microscope in 1996. This
instrument is still the EM workhorse at Marshall and is used in the spring imaging course and in
other research and teaching (primarily forensics, biology and geology) with logged hours
averaging 400/year. Marshall has a strong interest in teaching students, including
undergraduate students, about the most recent imaging methods, and encouraging their use of
such systems through a thorough system of training. This formal mechanism for introducing the
two photon system is anticipated to extend far beyond the end of the award period, as will our
less formal, partially self-guided training mechanism and our program of technician aided imaging
for those requiring preliminary data in support of applications to the NSF for funding. Student
imaging course projects with data generated largely with the SP5 during the past 2 1/2 years
include:
-Monitoring the effect of bradykinin and cadmium chloride on endothelial cell nitric
oxide production via fluorescence microscopy by Irfan a. Khan 2012
-The localization of the effector protein fritz by Lori Coyner 2012
-Localization of Chmp1 protein in Drosophila melanogaster by Meagan Valentine
2010 (used SP5 in 2010 during Leica demonstration and has used the installed SP5
frequently since)
Undergraduate student Lori Coyner used the SP5 to image her NIH 3T3
cells. Actin is stained red, Fritz effector protein is green and the cell’s
nuclear material blue.
d. Outreach activities (anything you have done to spread awareness of the
microscope and its capabilities):
UNI101:
A course required for all incoming freshmen here at MU is called UNI 101. Studentls
in this course are multi-disciplinary (including non-science). Terry Shank from
biology dept. focused the attention of his UNI 101 students onto quantum mechanics
seen in everyday experience. He brought this class of both science and non-science
students to the imaging lab in small groups and we were able to use the SP5 to
dramatically demonstrate the quantum phenomena of laser induced fluorescence
using stained cultured cells. A few of the group stayed late asking good questions
and appearing quite interested in the system.
Interfacing the Engineering Complex’s 3D projection system and the Two Photon Microscope
Marshall invested in a visualization laboratory, the “Viz Lab” as a cornerstone of its new
engineering complex built in 2008. Central to this laboratory is a 17 ft x 9 ft display system
which is capable of providing true (for each eye) 8.85 MP (4096*2160) 3D stereo resolution.
Several students have been introduced to the use of this system for display of the 3D data
acquired in the form of image “stacks”. For some projects, such large scale display enables
insights not easily grasped using 2D or “pseudo” 3D display mechanisms often employed with
confocal images. Necessary software packages for converting formats and enabling suitable
stitching to provide relatively seamless visual presentations have been acquired, installed and
utilized. 3D images acquired by researchers are so compelling that they have become standard
“example images” used by the Engineering Department in presentations to the public including
recruiting tours. A current expansion of the engineering complex, which will essentially unite the
Biotechnology Complex (where the SP5 multiphoton system is housed) and the engineering
laboratories, is anticipated to also encourage greater use, and fusion, of the microscopy and Viz
Lab capabilities.
We have used the SP5 in a small number (4-6) of demonstrations for visiting
high school students.
The imaging lab is normally one of the stops made by visiting faculty or
prospective students on recruiting visits to biology, chemistry, or bms
departments. We do have this sort of visitor just about weekly.
5. Journal Publications (please highlight grad or undergrad authors):
Borjigin M, Strouse B, Niamat RA, Bialk P, Eskridge C. Xie J, Kmiec, EB.
Proliferation of genetically modified human cells on electrospun nanofiber
scaffolds. Mol Ther Nucleic Acids. 2012;1:e59.
6. Books or other one time publications:
BURSICON, METAMORPHOSIS AND DEVELOPMENTOF RESILIN IN THE FRUIT FLY
A Thesis submitted to the Graduate College of Marshall University
by David Paul Anthony Neff
Approved by Simon Collier, Ph.D., Committee Chairperson
Sasha Zill, Ph.D. Victor Fet, Ph.D. Michael Norton, Ph.D.
May 2012
7. Web/Internet sites:
http://www.marshall.edu/mbic/instrumentation/LEICA%20SP5%20TCSII%2
0MP/LEICASP5.html
http://science.marshall.edu/dneff/ (imaging course webpage)
8. Other Specific Products not listed in 5-7:
9. Contributions (I can likely take care of this one, but if you have anything
specific that you would like to bring to my attention, please do so, such as
specific contributions within your research discipline, contributions relating
to programs you oversee like TREK or NASA):
a. Contributions within Discipline:
b. Contributions to Other Disciplines:
c. Contributions to Human Resource Development:
d. Contributions to Resources for Research and Education:
e. Contributions Beyond Science and Engineering:
10. Conference Proceedings (please highlight grad or undergrad authors):
Zill, S, Chaudhry S, Büschges, A, Schmitz, J (2011) Discrete sensory encoding of force increases
or decreases in the stick insect leg. Society for Neuroscience Abstracts 37, program no. 182.1.
Zill lab confocal reconstructions of stick
insect sensilla groups from 2011 Society for
Neuroscience meeting.
APS 2013 spring meeting (Maryland, March 2013)
by Nguyet Le and Tim Corrigan, Concord U.
Abstract:Combinatorial Approach to Studying Metal Enhanced Fluorescence from Quantum Dots
Fluorescence is extensively used in biochemistry for determining the concentration or purity of molecules in a biological
environment. In metal-enhanced fluorescence (MEF), the fluorescence molecules separated from a metal surface by several
nanometers can be enhanced. The fluorescent enhancement is dependent on the size and spacing of the nanoparticles, as has
been shown previously for a number of fluorophore molecules. Fluorescence from quantum dots is of particular interest
because the quantum dots do not lose fluorescence ability when exposed to light and they have higher intensity of
fluorescence. The purpose of this study is to determine the effect of size and spacing on fluorescence intensity when coupling
gold nano-particles with quantum dots. We employ a combinatorial approach, depositing gold particles ranging in diameter
from 30 nm to 130 nm with varied spacings onto the substrate, followed by a protein spacer-layer and quantum dots. The
fluorescence signal from the metal enhanced quantum dots were determined by confocal microscopy.
Timothy D. Corrigan
Assistant Professor of Physics
Concord University
11. Special Requirements (things like IACUC approvals):