July 2013 - Mary Babb Randolph Cancer Center

The Imaging Facility Newsletter
Volume 2, Issue 2
July 2013
The Microscope Imaging Facility (MIF) currently has eight microscopes, as
well as an offline imaging workstation, to support all of your imaging research
projects. In addition, the Animal Models and Imaging Facility (AMIF) has
two systems for small animal imaging, an offline data analysis workstation and
technical services to support your animal research needs.
Featured Researcher: Xueping Zhou, PhD
Inside this issue:
FRAP
2
Featured Microscope
3
Tech Tips
4
New Services and
Promotions
5
NSG Mice Available
6
Recent Publications
7
New Rates
8
Upcoming Events
9
Contact us
10
I am a post-doc in Dr. Mustafa’s lab. My research project is trying to elucidate the
underlying cellular and molecular mechanisms of adenosine-mediated coronary
flow regulation, in particular, focusing on the role of adenosine-induced reactive
oxygen species (ROS) production and its downstream cell signaling in modulating
ischemic and metabolic coronary vasodilation. Combined targeted adenosine
receptor knockout mice, as well as pharmacological reagents are applied to
identify the specific adenosine receptors responsible for the ROS production and
coronary flow regulation.
Adenosine A1 and A2A AR expression on C57 mouse aorta
Adenosine-induced H2O2 production in isolated coronary arteries of A1 KO mice
Featured Microscope Technique: FRAP
Page 2
Fluorescence Recovery after Photobleaching (FRAP) is a technique used to obtain quantitative kinetic
measurements of several cellular processes:
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Protein/protein interactions
Changes in protein conformation
The mobility of a specific protein into a confined space
The lateral diffusion of membrane - associated proteins
In FRAP, a fluorescent molecule is
irreversibly bleached by a high-power
focused laser beam, resulting in an area that
is devoid of fluorescent signal. As nonbleached fluorescent molecules repopulate
the bleached area, the diffusion constant,
or the rate of protein movement in the
absence of flow or active transport, can be
measured. All proteins display this type of
movement if they are not immobilized or
undergoing active transport. The two main
parameters that can be deduced from FRAP
are the mobile fraction of fluorescent
molecules and the rate of mobility, which is
related to the characteristic diffusion time.
Below is a comparison of a fluorescently labeled lipid bilayer and a monolayer. The left FRAP curve
illustrates the initial fluorescent intensity, the sudden loss of signal, and then an increase in fluorescent
intensity as other fluorescent lipids move back into the bleached area. The image on the right
demonstrates a lack of lateral lipid mobility within the monolayer, with the labeled lipids unable to
move into the bleached area. This results in a flat line, indicating a lack of fluorescent recovery after
bleaching.
Fluorescent signal is recovered after bleaching
http://microscopy.duke.edu/gallery.html
Fluorescent signal is not recovered after bleaching
Page 3
Featured Microscopes:
LSM Violet Confocal
Features:
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Upright Zeiss Axioimager Z1 Microscope
Objectives:
10x/0.3 EC Plan-Neofluar
20x/0.8 Plan Apochromat
40x/1.3 Oil EC Plan-Neofluar
63x/1.4 Oil Plan Apochromat
Image Blue, Cyan, Green, Yellow, Red &
Far Red Fluorophores
Lasers:
Argon/2
458, 477, 488, 514 nm
HeNe 543
543 nm
HeNe 633
633 nm
Diode 405-30 405 nm
Zeiss AIM software, Rel. 4.2
Applications:

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Confocality in up to 4 channels
Water dipping lens for direct contact with
the specimen—no coverslip needed!
LSM 510 Confocal
Features:
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Inverted Zeiss Axiovert 100M Microscope
Objectives:
10x/0.5 Fluar
20x/0.75 Fluar
40x/0.75 Plan-Neofluar
63x/1.2 Water C-Apochromat
100x/1.3 Oil Plan-Neofluar
Image Cyan, Green, Red & Far Red
Fluorophores
Lasers:
Argon 458/488 nm
HeNe 543 nm
HeNe 633 nm
Zeiss AIM software, Version 3.2
Applications:

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Live Cell Imaging
FRAP
Nanoparticle detection
Remember, if you don’t need to see things in 3D, we also have the Zeiss
AxioImager Upright Epifluorescent Microscope!
Volume 2, Issue 2
Page 4
Page 4
Imaging Pitfalls—What does colocalization really mean?
One common use of fluorescence microscopy is to determine the spatial localization patterns of two
fluorescently labeled molecules. Using different tags, this approach can be used to determine
protein/protein localization, understanding intracellular transport, or determining signaling pathways
within the cells. Traditional colocalization analysis has involved the use of green and red-tagged
proteins, and wherever they overlap, you get yellow. However, the proper determination of
colocalization is much more complex than Red + Green = Yellow.

The colocalization of fluorescent signals in an image cannot be used to show molecular
interactions between proteins. Rather, it can only show whether two molecules associate with
the same structure or occupy the same pixel space.

The presence of yellow in the merged image is normally taken to show colocalization between
green and red pixels. However, you will only get a yellow color if both of the fluorophores
occupying the same pixel are of equal intensities. This method of showing colocalization is
strictly qualitative and 2D, and does not consider the 3D nature of the sample or the probe.

In order to quantitate and analyze colocalization, a scatterplot of individual pixels from both the
red image and the green image is plotted. The scatterplot displays the intensity range of the red
and green pixels , as well as the degree of colocalization. The dimmer pixels are located near the
axis (0,0 origin), while the brighter, more intense pixels are located further out.
Analysis of the difference in colocalization using a scatterplot (from Olympusmicro.com).
A. No colocalization between the red and green signal results in a scatterplot with very little yellow overlap.
B. Partial colocalization between the red and green signal results in a band of yellow in the scatterplot.
C. A high degree of colocalization between the red and green signal results in a scatterplot that is mostly yellow .
*Adapted from: Seeing is believing: A beginner’s guide to practical pitfalls in image acquisition. AJ North.
* Adapted from http://www.svi.nl/ColocalizationCoefficients
2006. JCB.
Page 5
Self-Service Coming Soon
The AMIF is transitioning from full-service only to a choice of full-service or selfservice. We are now opening the facility up for users to run the imaging
systems for their own experiments. This includes both the IVIS Lumina II (for
optical imaging) and the Vevo2100 (for micro-ultrasound imaging). Training by
AMIF personnel is required, so please plan appropriately for timing purposes.
Full-service imaging will continue to be available.
In addition, Sarah will be going on maternity leave in August. Some of our
injections and procedure type services may not be offered while she is out, so if
you need any training for injections or other procedures she typically performs
for your lab, please let us know as soon as possible.
Promotions on Contrast Reagents
Perkin Elmer
Save up to 50% on in vivo optimized fluorescent and bioluminescent reagents
Bone metastasis imaged
by bioluminescence (top)
and fluorescence—
RediJect 2-DG 750 probe
(bottom).
Fluorescent Agents – Buy 1 Get 1 Free
ProSense, MMPSense, IntegriSense, AngioSense and others
Contact us for more information.
Genotyping Service Available
West Virginia University and Transnetyx, Inc. have partnered together for a trial
period to provide an 'Institutional Testing Program', where fully automated
mouse, rat and zebrafish genotyping will be offered to all investigators. Lab
Animal Resources at WVU has negotiated a 35% discount for services with
Transnetyx, Inc. Transnetyx has already established its Institutional Testing
program at over 70 non-profit research institutes worldwide. Lab Animal
Resources will charge a small labor fee for mailing samples.
Transnetyx provides researchers with automated genotyping for mutant mouse
lines. Our service clears the way for investigators to begin and concentrate on
what is most important – their research. Transnetyx ensures accurate, reliable
and cost-effective testing with fast results utilizing qPCR. With more than 37
million successful reactions and 99.97% accuracy, Transnetyx eliminates the
tedious process of extracting and testing DNA and guarantees (or your order is
free!) a turnaround time of 24 or 72 hours. Utilizing this automated process
not only replaces the need for supplies and reagents, but also eliminates the
opportunity for human error and contamination.
To find out more about how Transnetyx automated genotyping can save you
both time and money, please contact Tim Mayer, Northeast Sales Manager, at
617-283-0414 / [email protected]
NSGPage
Mouse
Colony at WVU
6
Page 6
We have recently started a breeding colony for NSG mice at WVU. Our goal is
to produce 50-60 pups each month. These mice will be for sale at our cost of
production, which is significantly discounted from the commercial price. Price is
$35.00 up to 6 weeks/mouse and $2.75 for each additional week.
NSG mice are severely immunocompromised and engraft the widest range of
normal and diseased human cells and tissues of any immunodeficient strain.
 No mature T or B cells
 Lack functional NK cells
 IL-2R gamma chain deficiency
 Long lifespan (>16 months)
 Higher level of engraftment of human cells
 Capable of hosting humanized immune system
Common applications include:
 Engraftment of cancer cell lines, tissues and primary tumors
 Therapeutic antibody testing
 Model for infectious disease
 Cell replacement therapy for type 1 diabetes
For more information on the NSG mouse, please visit the strain information
page through The Jackson Laboratory.
To request mice for use in your experiments, you must fill out the
reservation form on our website. Mice requests will be met according to
demand and availability.
Cardiovascular Workshop
The Imaging Facilities are committed to continuing education for our staff to
acquire new expertise to ensure the best possible service for our customers.
As part of this program, Sarah recently attended the Workshop on
Comprehensive Approaches to the in vivo Assessment of
Cardiovascular Function in Mice at The Jackson Laboratory.
A variety of cardiovascular topics an techniques were covered. The course
started with basic cardiac physiology. The second day focused on
echocardiography, using the ultrasound system that we have available in the
AMIF. Day three was all about telemetry. Lectures discussed the basic
principles of telemetry, which consists of implanting a device into a mouse and
then being able to measure various parameters (HR, BP, etc.) 24 hours a day.
This is beneficial to allow researchers to obtain true baseline resting
measurements, as well as, measurements during exercise or stressful situations.
Participants observed a demonstration of implanting the device, and then,
participants were able to practice the surgery. The final day of the course
covered PV loops. Participants observed the procedure and worked through
the analysis of some of the data that was obtained.
Please let us know if any of these techniques can benefit your lab’s research.
Human Heart
Facts
*The average heart rate
is 72 beats per minute;
100,000 times a day; 3.6
million times a year; and
2.6 billion times during a
lifetime of 70 years.
*Average heart weight is
11 ounces and can
pump 2,000 gallons of
blood through 60,000
miles of blood vessels
per day.
*Every day, the heart
generates
enough
energy to drive a truck
for 20 miles. In a lifetime, that is equivalent
to driving to the moon
and back.
Page 7
Please let us know
when you publish
a manuscript with
data from the MIF
or AMIF so that we
may acknowledge
your achievement
in our newsletter!
Recent Publications
Evans JV, Ammer AG, Jett JE, Bolcato CA, Breaux JC, Martin KH, Culp MV, Gannett
PM, Weed SA. Src binds cortactin through a SH2 domain cystine-mediated linkage.
J Cell Sci. 2012. Dec 15. PMID: 23097045.
Ice RJ, McLaughlin SL, Livengood RH, Culp MV, Eddy ER,
Ivanov AV, Pugacheva EN. NEDD9 depletion destabilizes
Aurora A kinase and heightens the efficacy of Aurora A
inhibitors: implications for treatment of metastatic solid
tumors. Cancer Res. 2013 May 15. PMID: 23539442.
Arnold KM, Goeckeler ZM, Wysolmerski RB. Loss of
focal adhesion kinase enhances endothelial barrier
function and increases focal adhesions. Microcirculation.
2013 Apr 19. PMID: 23600470.
Baseler WA, Dabkowski ER, Jagannathan R, Thapa D,
Nichols CE, Shepherd DL, Croston TL, Powell M,
Razunguzwa TT, Lewis SE, Schnell DM, Hollander JM.
Reversal of mitochondrial proteomic loss in Type 1 Ice et al.
diabetic heart with overexpression of phospholipid hydroperoxide glutathione
peroxidase. Am J Physiol Regul Integr Comp Physiol. 2013 Apr 1. PMID: 23408027.
Luanpitpong S, Chanvorachote P, Stehlik C, Tse W, Callery PS, Wang L, Rojanasakul
Y. Regulation of apoptosis by Bcl-2 cysteine oxidation in human lung epithelial cells.
Mol Biol Cell. 2013; 24(6): 858-69. PMID: 23363601.
Evans et al.
Luanpitpong et al.
Please Remember to Acknowledge Us!
AMIF: “Small animal imaging and image analysis were performed in the West
Virginia University Animal Models & Imaging Facility, which has been
supported by the Mary Babb Randolph Cancer Center and NIH grants P20
RR016440, P30 RR032138/GM103488 and S10 RR026378.”
Arnold et al.
MIF: “Imaging experiments and image analysis were performed in the West
Virginia University Microscope Imaging Facility, which has been supported by
the Mary Babb Randolph Cancer Center and NIH grants P20 RR016440, P30
RR032138/GM103488 and P20 RR016477.”
Volume 2, Issue 2
Page 8
New Rates Effective July 15
A new fee schedule for the imaging facilities will go into effect July 15. These changes reflect actual usage
and expenses, based on a cost analysis of the past fiscal year. In addition, the new rate schedule will be
compliant with federal cost principles applicable to NIH grants. These fees have been reviewed and
approved by the director of core resources and the core oversight committee.
The imaging cores are receiving significant financial support to subsidize our operating budgets resulting in
more affordable charge-back rates. In the table below, you can compare the new rates to the actual costs
of operations.
Let us know if you have questions or concerns. We will work with your lab to select the best imaging
technologies to fit your budget.
Actual Cost
(per Hour)
Subsidized Rate
(per Hour)
AMIF IVIS Lumina II
AMIF Vevo2100
AMIF Procedures
$259.08
$468.52
$211.72
$40.00
$60.00
$30.00
MIF Nikon Live Cell*
MIF Nikon Swept Field*
MIF Olympus Histology
MIF Zeiss Confocal
MIF Zeiss Fluorescent
MIF Zeiss PALM
MIF Zeiss Tissue Culture
MIF Zeiss Violet Confocal
MIF Workstation #1
$68.08
$68.08
$68.08
$68.08
$68.08
$68.08
$68.08
$86.36
$68.08
$15.00
$15.00
$15.00
$15.00
$15.00
$15.00
$15.00
$30.00
$15.00
Service
* For experiments lasting >8 hours, the rate decreases to $10.00 after 4 hours.
Upcoming Webinars - AMIF
Presented by the Jackson Laboratory
July Webinar Series—Mouse Models for Diabetes and Obesity Research
11 July—Modeling Human Disease with Diet-Induced Obesity (DIO)
18 July—Beyond Leptin: Emerging Mouse Models of Type 2 Diabetes
August Webinar Series—Spotlight on the Immune System and Humanized Mice
8 August—Mouse Models of Autoimmune and Inflammatory Disease
22 August—Humanized NSG Mice: Revolutionary Models of Human Infectious
Disease
September Webinar Series—Foundations of Mouse Research
17 July—12PM—In vivo Imaging—Nuno Sacadura
Presented by VisualSonics
Page 9
Upcoming Events
Picture Perfect: Quick Tips to Optimize Imaging
July 16—2 pm
2nd Floor Conference Room BMRC
Image Intensity Instruction
Can I make my images
brighter for publication?
How can I compare
intensities between
images?
How can I quantitate
intensity data?
What is saturation and
why should I care?
Join us for some practical imaging
solutions. More will be coming in the
future, so please let us know if there is a
topic you would like us to cover.
New Animal Imaging Course Offered This Fall
A&VS 591 Advanced Topics: Animal Imaging
The goal of this introductory course is to help students who anticipate
using animals in their research improve their understanding of non-invasive
imaging options. Topics include:
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Digital Radiography (DR)
Computed Tomography (CT)
Ultrasonography
Optical Imaging
Intra-vital imaging
Dual-energy x-ray absorptiometry (DEXA)
Magnetic resonance imaging (MR)
Positron emission tomography/computed tomography (PET/CT)
Contact Dr. Jeryl Jones or more information.
Cool Images from around the HSC
Today’s image is from Dan Vanderbilt in Dr. Mike Ruppert’s lab.
We have titled it “Ghost of Graduate Students Past!”
Contact Us!
1 Medical Center Drive
Erma Byrd BMRC
West Virginia University
Morgantown WV 26506
Karen Martin
[email protected]
Phone: 304-293-6965
Fax: 304-293-4667
Mandy Ammer
[email protected]
Phone: 304-293-0942
Fax: 304-293-4667
Sarah McLaughlin
[email protected]
Phone: 304-293-0518
Fax: 304-293-4667
anatomy.hsc.wvu.edu/mif
wvucancer.org/Cores/AMIF
If you have a strange, interesting, or totally awesome image that
you would like to put in our newsletter, please send them to us!
About this Newsletter
The purpose of this newsletter is to inform researchers about the AMIF and
MIF.
We want all investigators, graduate students and staff to be
knowledgeable about the equipment and resources that are available. The staff
are always glad to discuss upcoming studies with investigators to best utilize the
core resources available. To learn more about our facilities, please check out
our websites (to the left) or contact us to speak directly with AMIF or MIF
staff.
If you have something you would like to include or feature in the next
newsletter, such as a recent publication, a new technique or a beautiful image,
please contact Mandy Ammer at [email protected].