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Cartel Land
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Nick Allen picks one of his favorite Roger reviews.
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Page 1
In Vivo Suppression of HIV by Antigen Specific T Cells
Derived from Engineered Hematopoietic Stem Cells
Scott G. Kitchen1*, Bernard R. Levin1, Gregory Bristol1, Valerie Rezek1, Sohn Kim1, Christian Aguilera-
Sandoval2,3, Arumugam Balamurugan4, Otto O. Yang2,3,4, Jerome A. Zack1,2,3
1Division of Hematology-Oncology, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 2Department of Microbiology,
Immunology, and Molecular Genetics, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 3The UCLA AIDS Institute, The
David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 4Division of Infectious Diseases, Department of Medicine, The David Geffen
School of Medicine at UCLA, Los Angeles, California, United States of America
The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but
ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the
HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically
manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected
cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets
an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then
introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and
were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the
mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in
vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of
the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the
transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant
suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control,
indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These
results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral
infections and provide a foundation towards the development of this type of strategy.
Citation: Kitchen SG, Levin BR, Bristol G, Rezek V, Kim S, et al. (2012) In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered
Hematopoietic Stem Cells. PLoS Pathog 8(4): e1002649. doi:10.1371/journal.ppat.1002649
Editor: Daniel C. Douek, NIH/NIAID, United States of America
Received November 22, 2011; Accepted March 2, 2012; Published April 12, 2012
Copyright: ? 2012 Kitchen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by grants from the NIH (number R01AI078806 (S.G.K), number R01AI043203 (O.O.Y), and number R01AI070010, (J.A.Z.)), the
California HIV/AIDS Research Program (CHRP) (number 163893) (S.G.K.), the California Institute for Regenerative Medicine (CIRM) (number RC1-00149-1) (J.A.Z.),
the UC Multicampus Research Program and Initiatives from the California Center for Antiviral Drug Discovery (number MRPI-143226), and from funding by the
UCLA Center for AIDS Research (CFAR) (number P30 AI28697). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Human hematopoietic stem cells (HSCs), through development
in the thymus, are capable of producing progeny T cells that
generally display one of a vast repertoire of T cell receptors
(TCRs). In the case of many non-persistent viral infections, T cells
bearing TCRs specific to viral antigens mediate a potent antiviral
response that results in the clearance of the virus from the body.
Even in the presence of most persistent viral infections, a potent T
cell response is mounted; however it often fails to clear the virus
from the body. A critical component of the T cell antiviral
response is the CD8+ cytotoxic T lymphocyte (CTL), whose
primary function is to recognize viral antigens (in the context of
human leukocyte antigen class I (HLA I)) and kill virus-infected
cells. In HIV infection, the potent antiviral CTL response is
critical for establishing relative control of viral replication during
the acute and chronic infection stages of the disease [1–6].
However, unlike what is observed in most non-persistent viral
infections, the CTL response fails to clear HIV from the body. The
magnitude, breadth, functional quality, and kinetics of the
antiviral CTL response all are critical in controlling ongoing viral
replication; however, the reasons for the failure to rid the body of
virus are not completely understood [7,8]. Ongoing viral
replication and viral evolution in the infected host is one
important, although highly confounding, factor in the persistence
of HIV in chronic infection [4,5]. Even under effective
antiretroviral therapy (ART), the virus is not cleared from the
body and the level of HIV specific CTLs declines, likely due to
lower levels of antigen to stimulate the persistence/generation of
these cells [9,10]. Due to the importance of T cell responses in
controlling and eliminating viral infection there exists a great need
to explore ways to enhance antiviral T cell immune responses.
Recently, much of attention in HIV research has focused on
ways to enhance or correct the defects in HIV-specific CTL
responses. Gene therapy-based approaches that augment immu-
nity towards viral antigens represent unique, yet largely unex-
PLoS Pathogens | www.plospathogens.org1 April 2012 | Volume 8 | Issue 4 | e1002649
Page 2
plored, strategies towards the treatment of HIV disease. We have
previously examined the feasibility of a stem cell-based gene
therapy approach to enhance cell-mediated immunity towards
chronic HIV infection. In these studies, we demonstrated that
human HSCs genetically modified with genes encoding a human
HIV-specific TCR can produce mature, fully functional T cells in
human thymus implants in severe combined immunodeficient
(SCID) mice. The resulting genetically directed CD8+ T cells are
capable of killing HIV antigen-expressing cells ex vivo [11].
Further, we showed that the appropriate restricting human
leukocyte antigen (HLA) class I molecule is required for proper
development of transgenic TCR-containing CTLs. In all, our
earlier studies demonstrated that TCR-modified human HSCs can
be directed to develop into mature CTLs in a human thymus
environment in the context of the proper HLA type. However, as
the SCID-hu mouse model demonstrated poor peripheral
reconstitution and function of human immune cells, these studies
did not address the ability of these cells to suppress HIV
replication in vivo.
In the present studies, we examined the ability of genetically
modified T cells derived from HSC transduced with a single HIV-
specific TCR to suppress viral replication in vivo. We utilized a
modified version of a newly established humanized mouse model,
the non-obese diabetic (NOD)-SCID, common gamma chain
knockout (cc2/2), humanized bone marrow, fetal liver and
thymus (the NSG-BLT) mouse model, which allows the generation
of peripheral human immune responses, and serves as an effective
model for HIV infection and pathogenesis [12–14] (see Figure 1A).
These humanized mice display multilineage human hematopoiesis
and systemic engraftment of peripheral organs with human blood
cell types including T lineage cells, B lineage cells, myeloid lineage
cells, NK cells, as well as cells from other lineages [12] (and see
Figure 1B). We modified human hematopoietic stem cells in this
model with molecularly cloned genes corresponding to a TCR
specific to the HIV-1 Gag 77–85 SLYNTVATL (SL9) epitope to
allow the production of mature HIV-specific CTLs in multiple
organs of these reconstituted mice. We determined that human T
cells that expressed the HIV-specific TCR were capable of
suppressing HIV replication in vivo and preventing or slowing viral
damage to the engrafted human immune system. These studies
establish a system to examine ''genetic vaccination'' approaches
that target chronic viral infection and to more closely examine
mechanisms of human antiviral immunity in vivo.
Genetic modification and multilineage human
hematopoiesis in vivo
We have previously demonstrated that human hematopoietic
stem cells can be genetically modified by delivering a gene for an
HIV-specific TCR, and develop into mature T cells in an HLA-
restricted fashion in the human thymus of SCID-hu mice [11].
These newly produced, SL9 gag antigen-specific, naive T cells
were determined to be capable of producing IFN-c in response to
peptide stimulation and were found to be lytic to SL9 peptide
loaded target cells. However, it was not known whether these
genetically modified HIV-specific CTLs could traffic to relevant
organs in the mice and whether they were capable of killing HIV
infected cells in vivo. To address this question, we established an
improved model, based on the NSG-BLT model previously shown
to allow HIV replication [15,16], as a surrogate system to assess
the antiviral efficacy of engineered, HIV-specific T cells in vivo.
NSG mice were implanted with human fetal liver-derived CD34+
HSCs that had been modified with a lentiviral vector containing
the genes for a TCR targeting the HIV Gag SL9 epitope, or as a
control, with HSCs modified with a lentiviral vector containing a
non-HIV-specific TCR with unknown specificity. In addition,
these mice received implantation of human fetal Thymus and
Liver under the kidney capsule to facilitate human T cell
development. Hence, we term this the NSG-CTL model
(Figure 1A).
As genetic manipulation of HSCs is required in this model, we
initially determined the effects on this type of lentiviral
transduction on multilineage hematopoietic potential of HSCs in
the humanized mice. Phenotypic markers of human hematopoiesis
were examined by flow cytometry in mice within 6 weeks following
implantation of human tissues. One hundred percent of the mice
receiving human tissue had human cells in the peripheral blood,
including myeloid, natural killer (NK), T cell, and B cell lineages
(Figure 1B). In these mice, the average percentage of human
CD45+ cells in the peripheral blood was 53% of the total cells
(with a standard deviation of 29% and a range of 19%–80%,
n=12). We more closely examined the bone marrow in these mice
for the presence of human cell engraftment, particularly human
HSC engraftment. We found a significant population of human
CD34+ HSCs in the bone marrow (Figure 1C). The majority of
these cells coexpressed the CD45 molecule, which is indicative of
cells with lymphopoietic potential [17]. In addition, there were
significant populations of both CD3 expressing T cells and CD19
expressing B cells in the bone marrow of these mice. This
indicated that multilineage human hematopoiesis occurs in these
mice and provides evidence that, in addition to T cells, other
components of the human immune system are present. These data
demonstrated that our modification of the NSG-BLT humanized
mouse utilizing genetically modified human hematopoietic stem
cells does not negatively affect human hematopoiesis.
We then examined the animals for the presence of cells
expressing the transgenic, HIV specific TCR by MHC tetramer
staining. We found CD3+ T cells expressing the transgenic TCR
in all organs assessed, including the bone marrow, thymus, spleen,
liver, and peripheral blood of the mice receiving transduced
human hematopoietic stem cells (Figure 1D). Thus, we have
Author Summary
There is a desperate need for the development of new
therapeutic strategies to eradicate HIV infection. HIV
actively subverts the potent natural immune responses
against it, particularly cellular cytotoxic T lymphocyte (CTL)
responses. The development of a therapy that allows long-
lived immune self-containment of HIV and restoration of
these CTL responses by the host would be ideal. Through
genetic manipulation of human blood-forming stem cells,
we introduced a molecule– an HIV-targeting T cell receptor
(TCR)–that allowed the generation of functional HIV-
specific CTLs following differentiation within human
tissues in a humanized mouse model. To assess if these
newly developed, HIV-specific CTLs can allow active
suppression of HIV replication, we infected these mice
with HIV. We found that the development of genetically
modified, HIV-specific CTLs in these mice results in the
presence of a functional antiviral CTL response in vivo that
significantly lowers viral replication following HIV infection.
These results have strong implications for the use of this
technology to engineer the human immune response to
combat viral infections and suggest that genetic engi-
neering via HSCs may allow tailoring of the immune
response to target and eradicate HIV.
Engineering Antiviral Immunity from Stem Cells
PLoS Pathogens | www.plospathogens.org2 April 2012 | Volume 8 | Issue 4 | e1002649
Page 3
Engineering Antiviral Immunity from Stem Cells
PLoS Pathogens | www.plospathogens.org3 April 2012 | Volume 8 | Issue 4 | e1002649
Page 4
observed long-term, multilineage human immune reconstitution
and the development of mature T cells that express the transgenic,
HIV-specific TCR in multiple organs in the NSG-CTL mouse.
Suppression of HIV replication and CD4 cell depletion in
To assess if peripheral cells resultant from human hematopoietic
stem cells that expressed the recombinant SL9-specific TCR were
capable of suppressing HIV replication in vivo, NSG-CTL mice
containing the HIV specific TCR or a control TCR were infected
with HIV-1NL-HSA-HA. HIV-1NL-HSA-HAis an engineered variant
of HIV-1NL4-3that contains the murine heat stable antigen (HSA)
reporter gene modified to contain an Influenza hemagglutinin
(HA) antibody epitope, which is cloned into the open reading
frame of the vpr gene to allow detection of HIV-infected cells by
cell surface detection of HA expression using flow cytometry [18].
Peripheral blood was assessed for the level of productively infected
cells two and six weeks post infection. Within 2 weeks post
infection, we observed a reduced level of productively infected cells
in mice containing the HIV-specific TCR versus mice containing
the control TCR (Figure 2A). In addition, there was less initial
CD4 depletion in mice containing the HIV-specific TCR versus
mice containing the control TCR. Within six weeks post infection,
while there was an overall increase in virus-expressing cells from
the earlier time point, we observed a marked reduction in
productively infected cells in mice containing the HIV specific
TCR versus the control TCR, indicating suppression of viral
replication over time (Figure 2B). At this time point, mice
containing cells expressing the HIV-specific TCR had a greater
preservation of CD4+ T cells and higher CD4 to CD8 T cell ratios
when compared to mice expressing the control TCR. Amongst all
mice in the experiment, there was no statistically significant
difference 2 weeks following infection with either CD4 cell count
or with the percentage of cells expressing HIV, however there was
a trend towards better preservation in CD4+ cell numbers as well
as lower levels of virus-expressing cells in mice containing the
HIV-specific TCR (Figure 3). However, by 6 weeks post-infection,
there was a statistically significant difference in CD4 cell numbers
and levels of infected cells between mice with cells expressing the
HIV-specific TCR and mice with cells expressing the control
TCR. Thus, genetic modification of HSCs with a single HIV-
specific TCR produces peripheral T cells capable of suppressing
cellular HIV expression and CD4 depletion in vivo.
HIV-specific TCR suppression of plasma viremia in vivo
We next sought to determine if cells modified with an HIV-
specific TCR could suppress virus levels in peripheral blood
plasma. However, quantitating plasma viremia in the mouse
model is difficult due to the amount of plasma obtained per blood
draw (typically ,50 microliters), the limit of detection obtainable
with this amount of blood, and the high cost associated with
commercial assays. Therefore to measure viremia in this system,
we developed a novel quantitative PCR-based technique for HIV
in mouse plasma. Based on the recently elucidated secondary
structure of the HIV genome [19], primers were designed to
specifically target relatively ''open'' regions of the RNA genome
that contain minimal secondary structure to attempt to allow
increased sensitivity to detect viral RNA. Utilizing this technique,
which has a reliable sensitivity of 5 copies of HIV RNA per
sample, we determined that the viral load 2 weeks and 6 weeks
post infection was significantly lowered in mice receiving the HIV-
specific TCR versus mice receiving cells transduced with the
control TCR (Figure 4A). This suggested systemic suppression of
HIV replication in vivo. Surprisingly, analysis of the viral RNA for
mutations in the SL9 epitope did not reveal the presence of any
mutations in this epitope in the majority quasispecies, which was
identical in comparison to the sequence of the input virus and the
virus of infected mice containing the non-specific TCR control
(Figure 4B). This suggested that in this period of time, viral escape
to the selective pressure of the SL9 specific TCR had not occurred
in the blood of these mice, possibly due to limited viral replication
in this model. Thus, there was significant suppression of viral
replication in vivo in mice expressing the HIV-specific TCR versus
the control TCR and this suppression did not result in significant
viral escape within 6 weeks following infection.
TCR-engineered suppression of HIV in multiple lymphoid
As illustrated in Figure 1, T cells expressing transgenic HIV-
specific TCRs were found in multiple organs in mice receiving
genetically modified HSCs. Based on this, we next addressed
suppression of HIV in multiple organs in the lymphoid
compartment in mice containing cells expressing the HIV-specific
TCR. NSG-CTL mice that had received HSCs transduced with
the HIV SL9-specific TCR or, separately, the non-specific control
TCR were infected with HIV-1NL-HSA-HA. Sets of infected animals
were then assessed 2 weeks and 6 weeks post infection for the
quantity of HIV proviral DNA sequences in human cells in the
spleen, bone marrow, and human thymus implant (Figure 5). We
observed significant suppression of HIV replication in human cells
in these organs as early as 2 weeks post infection (in the bone
marrow) in mice receiving HSC containing the HIV-specific
TCR. 6 weeks post-infection, HIV levels were significantly lower
in the spleen, bone marrow, and human thymus implant in
animals receiving the HIV-specific TCR as compared to mice
Figure 1. Construction and multilineage reconstitution of NSG-CTL mice. A. Schematic illustrating the construction of NSG-CTL mice:
CD34+ cells are isolated from fetal liver by cell sorting (1). then are transduced with lentiviral vector containing the SL9-specific TCR (2). A fraction of
these cells are then implanted under the kidney capsule in NSG mice flowing combination with fetal liver stromal elements and fetal thymus in
matrigel (3a). Another fraction of these transduced cells are viably frozen in liquid nitrogen (LN2) (3b). Three weeks following implantation, the
engrafted mice are then sublethally irradiated (3 Gy) and previously frozen cells are thawed and injected intravenously into these mice, where the
cells home to and engraft in the bone marrow (4). 6–12 weeks following injection of cells, TCR expression was analyzed and mice were infected with
HIV (5). Mouse blood is then assessed for HIV infection 2 and 6 weeks following infection (6). B. Multilineage hematopoietic reconstitution of NSG
mice receiving genetically modified HSCs. Peripheral blood from these mice were assessed by flow cytometry and gated for CD45+ human leukocytes
(top panel). These cells were assessed for the denoted cell surface marker expression including HLA-DR+CD11c+myeloid cells, CD3-CD56+ NK cells,
CD3+ T cell, CD19+ B cells. CD3+ cells were gated (lower left panel) and assessed for CD4 and CD8 expression (lower right panel). The numbers
indicate the percentage of each population of cells in the mouse peripheral blood. C. Repopulation of HSCs in mouse bone marrow. Mouse bone
marrow was assessed for the presence of CD34+ human HSCs (left panel) and CD3+ T cell and CD19+ B cell engraftment 6 weeks following CD34+ cell
injection by flow cytometry. D. Reconstitution of NSG-CTL mice with cells expressing the HIV-specific TCR transgene. Cells were isolated from the
indicated organ in NSG-CTL mice 6 weeks following CD34+ cell injection and analyzed by flow cytometry for CD3+ T cells binding SL9-containing
tetramers. The numbers indicate the percentage of T cells within the indicated organ expressing the transgenic TCR. The data are representative of
mice receiving human tissue and HIV-specific TCR transduced CD34+ cells in the same experiment identified above (n=12).
Engineering Antiviral Immunity from Stem Cells
PLoS Pathogens | www.plospathogens.org4April 2012 | Volume 8 | Issue 4 | e1002649
Page 5
Figure 2. Suppression of viral replication in cells in the peripheral blood. Mice receiving the HIV Gag SL-9 specific TCR (left and right panels)
or the non-specific control TCR (middle panels) were either left uninfected (left panels) or were infected with HIV-1NL-HSA-HA(right and middle panels)
and assessed for HIV (HSA-HA) expressing, human CD45+ cells (top panels) and CD4 and CD8 expression by gated CD3+ T cells (bottom panels). Mice
were assessed by flow cytometry for these cell surface markers A. 2 weeks, and B. 6 weeks post infection. The numbers indicate the percentages of
Engineering Antiviral Immunity from Stem Cells
PLoS Pathogens | www.plospathogens.org5 April 2012 | Volume 8 | Issue 4 | e1002649


text:
Posts filtered on Category
Spurs 112, New Orleans 93: And the beatdowns go on
Summing it up Winning NBA games isn't generally easy, but the Spurs are certainly making it look that way. They've now won 11 in a row, with the past eight coming by nine points or more. Their latest victim had won three straight, entering Monday's contest with the seventh-most efficient offense in the league. Modest […] [Read More]
Final: Spurs 112, New Orleans 93
SAN ANTONIO — Another game, another conquest for the streaking Spurs, who pummeled New Orleans at the AT&T Center on Monday to keep pace with Indiana for the league's best record at 13-1. They've now won 11 straight games, the past eight coming by nine points or more. Barring a modest push in the first […] [Read More]
The week ahead
SAN ANTONIO — Two more victories will give the Spurs the best start in franchise history through 15 games. As things stand they're 12-1, winners of 10 straight and tied with Indiana for the league's best record. They're trouncing their opponents by more than 11 points per game, also No. 1. And they've done it without […] [Read More]
Blowout win = major tomfoolery
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SAN ANTONIO — This play was so good I hated to bury it in the postgame recap. So here's a separate post for Manu Ginobili's half-court bounce pass to adopted son Marco Belinelli in Friday's victory over the Grizzlies. He's made a career out of this kind of off-the-cuff creativity, but it never fails to […] [Read More]
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Final: A late 12-2 run by Boston took a little luster off what was otherwise a dominant victory for the Spurs, who pulled away with a pair of big surges in the third and fourth quarters. The Spurs have now won eight in a row, keeping pace with Indiana for the league's best record at […] [Read More]
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Why Read This ReportWe surveyed 141 executives from large North American firms to find out about their customer experience endeavors. It turns out that 90% of respondents think that customer experience is very important for their companies and 80% are trying to use it as an area of differentiation. While the lack of funding was the top problem last year, the lack of a clear strategy has emerged as this year's No. 1 obstacle. There's a lot of activity underway: 62% of companies have a voice of the customer (VoC) program, and nearly half have an executive in charge of their overall customer experience efforts. Our data shows that companies with this type of leadership have fewer obstacles and are more mature in their customer experience efforts.
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Ac/Climate Control Burnt out?
I took apart ,y ac/heater control unit because The lights are burnt out. I was under the impression, like everyone else, that you can't replace them. My dad told me that he read somewhere that you could, so I did. I took it apart and pulled out 3 little bulbs just like are in the speed o meter. Also, to get off track; I just replaced all 10 bulbs on my instrument panel and it looks so much better! Highly recommended! But i was just wondering if anyone knew you could remove the bulbs and replace them. The trouble is, just like the instrument panel bulbs, I couldn't fond them anywhere accept the dealer. I went to Kregens autozone and napa. news is they were $1 a piece. Anyways, Does anyone know that the part number is on these climate control bulbs?
Without the exact year of your truck I cant be sure, but try 05013815AA.
Oh sorry, forgot i don't have a Sig on here. its an 03
thanks! Ill try that number.
I tried that part number on moparpartsamerica.com and it didn't show as a part number
Well, it shows up in DealerConnect so it's probably a good number unless it's been superceeded.
thanks for all the help! I'm a former dodgeforum guy, found out about the fourmz and Im on Caliberfourmz and this one. still a new to this site, its much better than my last tho. Anyways I was trying to get that part online. is it possible to do that? My dealer is 30+ miles away. Thanks
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Remorse Code: Memorable Celebrity Apologies
By Kat Giantis
On Nov. 9, Ashton Kutcher found himself on the receiving end of an 8 million-strong backlash after posting an indignant tweet about the firing of Penn State football coach Joe Paterno. Seems the "Two and a Half Men" star hadn't taken the extra 30 seconds to find out why Paterno had been sacked (child sex-abuse scandal).
"How do you fire Jo Pa?" Kutcher railed. "#insult #noclass as a hawkeye fan I find it in poor taste." What made his observation even worse: The actor, along with wife Demi Moore, fronts a charity to stop child exploitation.
Ashton's damage control was swift and severe. He handed over his Twitter account to his managers and issued a mea culpa. "My sincere apologies to anyone who I have offended," he said. "It was a mistake that will not happen again."
But Kutcher is hardly the first star to backpedal after an embarrassing incident. Click through to see some other mistake-making celebs who have been forced to chow down publicly on a big slice of humble pie ...
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GlowBash 5K: Race Entry
Kaka'ako Makai Gateway Park • Honolulu, HI
Saturday, February 22, 2014
What you get
- $39 ($85 Value) for One GlowBash 5K Entry
- Includes T-Shirt, Race Bib, Finisher's Medal, Glow Swag & Sponsor Goodies
- Solve Ten Clues to Arrive at the Right Location & Win the Race!
- Please Register Your Voucher Online After Purchase
A combination 5K and scavenger hunt, GlowBash is an adventure race that lights up the night across a 3.1-stretch of Honolulu. Form your team (two or more runners) and then dig into the ten clues you'll receive. Solve each clue and get to the right locations to finish the race. And know that you're glowing for a great cause -- proceeds benefit Wounded Warrior charities.
$39 ($85 value) for one entry, including:
• Custom GlowBash 5K T-shirt
• Glow swag
• Race bib
• Finisher's medal
what to bring with you
City map
Pens and pencils
Fine printREFUND POLICY: This event is nonrefundable. No full or partial refunds are given to customers who miss the event. In the event of a cancellation, your voucher will be fully refunded • Each voucher is good for 1 person • Must be 21+ to drink alcohol • The deal is valid only for tickets on the date of the event as listed and may not be exchanged or redeemed for any other dates • Groups wishing to sit together must purchase their vouchers together; seating is fulfilled with no specific priority • Purchaser may change the name of the individual retrieving tickets up to 24 hours in advance of event via the LivingSocial website event details section; no name-changes or alternate pick-ups permitted without notice at venue • Entire value must be used in a single visit
GlowBash 5K: Race Entry
times for
More upcoming events
Nearby deals
Kailua Pilates
Pilates or Pulsed ElectroMagnetic Field Therapy
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Acupuncture Package with Tui Na or Cupping
Jose Dynamite & Friends
Two or Four Tickets to a Comedy Show
Tradewind Charters
Private Reef Fishing Adventure Charter
Ocean Legends
PADI Certification
Cavi-Lipo Hawaii at 'Ohana Chiropractic Center
Ultrasonic Cavi-LipoTreatment
Way Gone Laser Tattoo Removal
Two Tattoo-Removal Sessions
Chef Chai
Four-Course Meal for Two, Three, or Four People


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The Top 10 In Action Sports
01Brick By Brick: Camping Lego Style
02Booze and Bikes: 14 Tours That Let Combine Two of the Best Things in the World
03Who Needs Hollywood? These Incredible Videos Were All Filmed on the Simplest Cameras
04Sam Pilgrim vs. The World: The Champion Aiming To Reclaim His Crown In A New Era Of Biking
05Eccentric Adventures Episode 2: Britain's Most Brutal Illegal Event is All About Cheese
06A Journey Into Darkness: We Went Mountain Biking in the Abandoned Mines of Slovenia
07After the Apocalypse: Inside The World's Most Breathtaking Ghost Towns
08Debate: Let's Stop Hating On Parents Who Want To Raise Adventurous Kids
09Guns, Roses, And Eating Insects: Adventure Cycling in Vietnam
10From Intense Trails to Intensive Care: Dicing with Death In a Mountain Biking Paradise
Benny Fairfax - 'Day In The City 2' (2002)
A Skateboarding video by Sidewalk - 16,892 views
Benny Fairfax taken from the second Day In The City DVD, filmed in Leeds and Manchester.
Camera duty and editing by Kevin Parrott.
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WOBURN, Mass., Nov. 3 /PRNewswire-FirstCall/ -- Culbert Healthcare Solutions, a fast-growing health information technology and operations professional services firm, announced today that Norm Botsford has joined the company as Principal Consultant in the Strategic Services practice.
Mr. Botsford recently served as President and Chief Executive Officer at University Physicians Healthcare (UPH), the health system and academic medical practice of the University of Arizona College of Medicine faculty. The practice consisted of approximately 375 full-time academic physicians, 14 clinical departments, 22 clinic facilities, and a freestanding ambulatory surgery center. The corporation operated a fully accredited 197-bed acute care hospital, a Medicaid risk contract providing managed care services to 48,000 members, and a small-business health plan covering 8,000 members. UPH employed a staff of 2,100, comprised of physicians and staff working in a range of settings, including clinics, hospital, central billing office, health plan, corporate, and practice administration.
During his 15-year tenure, Mr. Botsford led UPH through significant change, and was responsible for strategic planning, organizational development, and practice operations, as well as oversight of physician compensation, Medicaid managed care plans, and the risk management/malpractice insurance corporation. Mr. Botsford also led UPH's acquisition of a county-owned community hospital (formerly Kino Community Hospital, now University Physicians Hospital) which resulted in an innovative partnership between UPH, the University, and county government. Under his leadership, the hospital began to reestablish itself as a full-service hospital and outpatient treatment center.
As a member of the CHS Strategic Services practice, Mr. Botsford will
advise the senior leaders of academic medical centers on growth and new
business planning, including:
-- Strategic Planning
-- Or
|SOURCE Culbert Healthcare Solutions
Copyright©2008 PR Newswire.
All rights reserved


text:
Beata Moon: Finding Her Own Voice
Evan Chambers: You Must Change Your Life
In conversation, Evan Chambers conveys his ideas using words in a strikingly similar fashion to how he delivers them in music: honestly, intelligently, with neither fear of open emotion nor of making a sharper point than his laid-back demeanor might at first lead you to expect. As he speaks about his familial roots in folk music, his love of poetry, and the responsibility he feels as an artist to acknowledge broader social, political, and environmental challenges, a portrait of the composer emerges that reveals again how incompletely shorthand genre descriptors and professional biographies capture art and artist.
Nat Evans: Outward Bound
Over the past two years in cities throughout the U.S., groups of people have been gathering, digital music players in hand and headphones in place, to watch the sun rise or set. It must be an odd sight for anyone stumbling across these scenes—25 to 50 people all "plugged in," intently facing in the direction of the sun. They are all listening to the same music by composer Nat Evans.
Glenn Branca: Where My Ears Want To Go
Glenn Branca has had a deep and lasting impact on several music scenes, but he was never really a part of any of them. With Theoretical Girls, he created a new kind of punk rock music that came to be known as No Wave. Later on, he redefined what a symphony could be. Making music that was more visceral and louder than anything in the new music scene, he even frightened John Cage. Thirty years later, he's still making waves.
Joseph C. Phillips Jr.: Balancing Act
While Joseph C. Phillips Jr.'s music sometimes incorporates improvisation and his ensemble features several prominent jazz musicians, he does not consider himself a jazz composer. An adept multitasker who balances creating music for film, dance, symphonic bands, and his own 25-piece ensemble with teaching music to kindergartners, Phillips creates very clearly 21st-century music—incorporating a broad range of styles while being ultimately beholden to none.
Sebastian Currier: Reversible Time
While the myriad details that are crammed into Sebastian Currier's scores are reminiscent of the elaborate layers found in the Romantic music of the 19th century, and his detailed conceptualizations for pieces seem as thoroughly plotted as those of a post-War total serialist, Currier writes music that very much belongs to our own less certain times.
Lembit Beecher: To Tell a Tale, To Sing a Story
When Lembit Beecher was named composer-in-residence with the Opera Company of Philadelphia (in collaboration with Gotham Chamber Opera and Music-Theatre Group of New York) in 2011, he didn't bring a large portfolio of operatic work with him to the brand new three-year program. An instinct and affection for storytelling, however, already infused his compositions.


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You can now invite anyone to meet at your favorite Starbucks.
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40 year old man | Los Angeles, CA, USA | Seeking women 25-40 within 60 miles
Active within 1 week
Looking for someone who is willing to grow...
Looking for someone who is willing to grow towards a long lasting meaningful relationship.. Must have God as the center of our lives. The sport of basketball has taken me to different parts of the world. And I am hoping to find someone who likes to explore and travel. A bit about me: I have a deep desire to help today's youth.. I run a non profit youth program geared towards using the sport of basketball to develop life skills, and as a vehicle for education advancement.. I am very passionate about what I do and forever looking for new ways to become better at my craft!!! I'm also a certified personal trainer.. I've worked with clients from entertainment industry, professional sports, to the everyday joe! I love love love helping people... Im loving, loyal, honest, adventurous, generous, unselfish,, and looking for a counterpart whos here to build something substantial. I am looking for someone with a huge heart and sense of humor. I believe laughter is so important for longevity and leading a healthy life. That's all for now... If want to know more email me. :)
Dining out , Movies/Videos , Music and concerts , Playing sports
Basketball, Weights / Machines
I exercise 5 or more times per week.
Middle of the Road
Belize of course.. :)
University of California, Riverside, Riverside, CA
LIve Music.. RnB, Jazz, Reggae, Gospel. Love dining and attending movies.
I enjoy attending concerts, dining, and going to movies.. Live Music is the best... Drives up the coast. Quick get aways.
Makes Me Want To Holla The Kite Runner John Grisham book series.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'3" (190cms)
|5'7" (170cms) to 6'6" (198cms)
|Body type:
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Post college I played a little basketball in south America and Belize. I currently run a youth sports program and professional training company.
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Born in Belize City, Belize..
|Christian / Catholic
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|Bachelors degree
|Bachelors degree, Graduate degree, PhD / Post Doctoral


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Wednesday, July 01, 2015
Marie Beaulieu Obituary
1920 - 2005
Marie Antoinette Beaulieu, 84, died Sunday, April 24, 2005 at Wentworth-Douglass Hospital in Dover.
She was born in Gonic on May 28, 1920, the daughter of Michel Beaulieu and Cecile Croteau, and had been a communicant of Our Lady of the Holy Rosary Church in Rochester.
Miss Beaulieu had lived with and was cared for by David and Sandra Hall of Berwick, Maine for approximately nine years. The Office of Public Guardian in Concord also assisted with her care for over 19 years.
There are no known surviving family members.
A private memorial gathering will be held, and following cremation, her remains will be scattered by her friends.
H.J. Grondin & Son Funeral Home, 177 North Main Street, Rochester is assisting her friends with arrangements. Please visit www.GrondinFuneralHome.com to sign the online guestbook.
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text:
Rockbitch. Obvious.
This great product in white from our department Women and T-Shirt category can be printed just for you in the size and style you like. The design Warlock Rockbitch was created by Gupolo and uploaded to Spreadshirt for you to have printed on a T-Shirt. Next to a host of other great designs, you can find this Warlock Rockbitch design in Spreadshirt's Rock & Metal category. If you are interested in topics like Rockbitch or Shredder, then you'll love Warlock Rockbitch designs by Gupolo. Spreadshirt also offers a huge selection of personalised T-shirts, hoodies and accessories for the category Rock & Metal as well as top Spikes clothing. Gupolo's designs have already impressed many of our customers, so we are sure that we've got right stuff for you! Spreadshirt is your shop for great designs of all kinds – whether it's Rockbitch, Shredder or Spikes. We are sure that you will find something nice in our shop, there are many amazing personalised Rock & Metal T-shirts, jumpers or accessories for you to find! Tags: Design ID: 14527322
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still to fill


text:
The Neel House, the former home of Mr. and Mrs. Jopseph N. Neel, Sr., was designed in 1910 by Georgia's
famed architect, Neel Reid.
The historic home, located at 730 College Street, is available for rental for weddings, receptions, parties and other special events. The house is also available for group tours for a nominal fee by appointment.
Our Gift Shop offers unique gifts at bargin prices.
The Neel House is also Headquarters of the Federated Garden Clubs of Macon, Inc., organized in 1934, and is used for their
meetings and programs. The Federated Garden Clubs of Macon, Inc., is a non-profit council of garden clubs whose purpose is to promote civic
beautification, gardening education, and environmental conservation.
The Federated Garden Clubs of Macon, Inc., is a member of the Garden Club of Georgia, Inc., and the National Council of Garden Clubs, Inc.


text:
New Photos From Scream Queens Reveal That Lea Michele Can and Will Play Another Nerd
Lea Michele is a nerd.By Lindsey Weber
Lea Michele is a nerd.By Lindsey Weber
Inspired by Marie Kondo.By Maggie Lange
A "horror-comedy."By Lindsey Weber
They filled social media with tributes.By Véronique Hyland
Her first regular TV role since 1989.By Halle Kiefer
Special guest star: Jamie Lee Curtis.By Danielle Henderson
They survived the movie, and we salute them.By Abraham Riesman
Called The Final Girls.By Zach Dionne
Wait, Future can see the future, and this is what he sees?By Lindsey Weber
Please fall in love, you two!By Eliot Glazer
It's not Schmidt, or Nick, or Winston.By Andre Tartar
Jessica has finally revealed the baby bump everyone knew she wasn't hiding very well.By Amy Odell
Meanwhile, Taylor Swift gives thanks to Ismail Mohan at RockSugar.By Hadley Tomicki
Meanwhile, Taylor Swift gives thanks to Ismail Mohan at RockSugar.By Hadley Tomicki
He's got stories about the Christopher Guest movies, 'Salem's Lot,' and mistakenly turning down 'Airplane!'By Gwynne Watkins
Warning signs!By Willa Paskin
The producers certainly hope so.By Willa Paskin
Another movie about women being petty at a wedding.
Plus: "Steven Spielberg is pursuing Noah Wyle."By Amos Barshad
They let the couple sneak off to their Hamptons place. Plus, Cameron Diaz thinks something lives in the TV, and other spooky, weird tidbits in the Halloween gossip roundup.By Tim Murphy


text:
It's gotta be fresh
Easy Way Food Stores is as much of a Memphis tradition as FedEx, the Liberty Bowl and Graceland, but unlike those other Memphis staples, Easy Way has made its way under the radar screen.
The company, started in 1932 by Pate Carter, opened its first store at the corner of Jefferson and Main in Downtown Memphis and currently has nine stores in the city, with plans for a tenth store, Easy Way's first in Mississippi, later this year.
As the times have changed around Easy Way, the company has managed to make changes in order to survive, with one of the most recent changes being the company's 54,000-square-foot warehouse and distribution center, which opened in 2001 and serves as the company's headquarters.
David Carter is secretary/treasurer of the company and part of the fourth generation of the Carter family to run the company along with his brother, Barry, the president. He says one of the biggest secrets to Easy Way's success is the eating habits of their customers.
"With any diet, you're told to eat fresh fruit and vegetables," he says. "People eating healthy helps our business and with the attention being paid to getting kids to eat better, that's been a real boost for our business."
Another reason for the long-term success of Easy Way is the first rule in business -- the customer is always right. Carter says Easy Way has constantly listened to suggestions from customers that resulted in the company's survival in competition with giants like Kroger and Wal-Mart.
While the Downtown store is a complete grocery store, the company's other stores stock produce, milk, eggs and other items that customers might need on the way home, but don't want to fight with the human and automobile traffic of a big store.
"Our customers have said that if we had more things in stock, they could stop in on their way home and get what they need," he says. "They can get in and out of here before they can even get in a Kroger store."
Carter says everything from the product carried to the location of the stores is calculated by the company heads, who felt the need to locate all of their stores in neighborhoods.
With such a volatile product as produce, freshness is the most important issue. Products like onions and potatoes may have a three-week shelf life, but the average shelf life of other produce is 3-5 days, if that long.
"You have to have low inventory and keep product moving all the time," he says. "We get product on the shelf the same day we get it in. That's something the big chains can't give you. They may have the low prices, but since it's coming from another state, it may be two or three days old when they get it. We come from Memphis to Memphis stores."
That dedication to freshness and a couple of innovations has resulted in one of the biggest turnabouts in company history: The competition now buys from Easy Way.
Easy Way's wholesale division was formed at the suggestion of a few customers who owned restaurants. The company began selling bulk to customers like Tops Bar-B-Q, Lenny's, Quiznos and 20-30 small local restaurants. On a whim, Easy Way began courting Wal-Mart and other competition, who now buy product from Easy Way when supplies run low.
"We started slowly with that and expanded it. Now it's a vital part of our business," Carter says.
Today, Easy Way has six trucks that make morning and afternoon runs to customers. Because of rising fuel costs, the orders have to be at a minimum of $50 or there is a delivery charge.
In addition to natural growth and growing different areas of the business, Easy Way has enlisted Sossaman & Associates to create ad messages.
Brooke Tweddell, vice president of development for Sossaman, says the new campaign will center around driving business to the Downtown store and getting the company's story out.
"Our goal is to help Easy Way develop a more convenient and neighborly produce market Downtown, similar to those found in larger cities," she says. "With the growing number of businesses and residents moving into the Downtown area, we think they'll appreciate the new approach."
Carter says the most rewarding part of the company is the longevity and growth with employees. some of whom have 30 years with the company. Also, with a family business, there is not the red tape found in large corporations.
"If there are any decisions to be made, it goes to either my brother or me. There aren't 19 different people you have to talk to," he says. "This is all we've ever done. Three of my sons and one of my brother's sons is here. Businesses usually don't make it to the fourth generation, but here we are. It has been really good to us."
email@example.com | 259-1722


text:
[Illinois] Advanced Materials Characterization Workshop 2012: Tutorial 5: Secondary Ion Mass Spectrometry/Rutherford Backscattering
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4 months ago
I know who I was when I woke up this morning, but I feel as if I've changed several times since then.
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This is a terrific film; I watched it three times in a row and enjoyed the plot -- honest and believable. Even the awkward parts. Read morePublished 1 month ago by Marcy S. Getz
This is definitely an original type film and in multiple ways, very funny! You just have to have that weird sense of humor, that seems to be getting more rare nowadays... Read morePublished 2 months ago by CN
This movie stinks on ice. I kept waiting for something...anything to happen, but nothing happens. Man, what a bummer. It's a shame too, because this is a fantastic cast. Read morePublished 5 months ago by Melisa Matkin
Why 4 stars for a 3 star movie? Because it is worth it. I have not written a movie review before. I know, it shows. I usually go strait to the 3 star reviews here on Amazon. Read morePublished 6 months ago by J. King


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text:
Hello Arnoud,
I installed Mathematica yesterday and wanted to connect it to my Raspberry Pi.
I also have the same problem as described in the above posts:
From my Mac I can ssh to my RasPi and launch there Wolfram, and make it add 1+1.
But I can't figure out how to get a connection to the RasPi from Mathematica on my Mac using the RDK.
In the setup mask of the RDK (Configure Pi Connection) I entered
Network machine of Pi: IP address of the WLAN connection of my RasPi (as said, this one works for connecting via ssh)
Login of Pi: pi
I my point of view there is not much that the user can do there wrong, it should work out of the box.
When I try to connect to my RasP and evaluate the Pi Test & Example notebook I am asked to enter my password for my pi login, I have to wait then quite a while, then I get the following two errors:
The child process 594 reports: sh: Private: command not found
The kernel Raspberry Pi failed to connect to the front end. (Error = MLECONNECT). You should try running the kernel connection outside the front end.
When I enter Options[$FrontEnd, EvaluatorNames] on my local desktop I get
In[2]:= Options[$FrontEnd, EvaluatorNames]
Out[2]= {EvaluatorNames -> {"Local" -> {"AutoStartOnLaunch" -> True},
"Raspberry Pi" -> {"RemoteMachine" -> True,
"TranslateReturns" -> True, "AutoStartOnLaunch" -> False,
"Executable" -> "/usr/bin/wolfram",
"HostName" -> "192.168.178.22", "RemoteLogin" -> "pi"}}}
I googled the problem but couldn't find anything that would solve my connection problem.
I would be very happy if you could help me with this issue.
Kind Regards
OK, now I have a Raspberry Pi 2. Still no luck with the RDK. I can't even get the RDK to successfully install... the CDF installer reports that it succeeds, but the RDK palette never appears in the Palettes menu.
I am running Raspbian, installed from the NOOBS image:
pi@raspberrypi ~ $ uname -r
pi@raspberrypi ~ $ wolfram
Wolfram Language (Raspberry Pi Pilot Release)
Copyright 1988-2015 Wolfram Research
Information & help: wolfram.com/raspi
In[1]:= $Version
Out[1]= 10.0 for Linux ARM (32-bit) (February 3, 2015)
Taking yet another run at this a year or so later.
Raspberry Pi 2
Fresh NOOBS 1.4.0 (2015-02-18) SD card
Macbook Pro (Retina late 2012) OSX 10.10.2
Mathematica 10.0.2.0
Network ethernet cable
Purpose - Run Mma on the RPi kernel from a front end running on the Mac
This was a new RPi, and the network had been reshuffled, so the first problem was getting a functional ssh from Mac Terminal to RPi. Resolution (after a day and a half of googling) was to find the .ssh/knownhosts from the Mac file system. I tried several edits suggested in the forums but could make none work. Removal of the knownhosts file was the only thing that fixed it. That file was a half-encrypted text file that included recognizable host ip addresses from past configurations, but why the simple persistence of old data causes fatal termination with such alarming security warnings, I have no idea.
Now, I evidently have a resysthesized known_hosts file, a functioning ssh from the Mac Terminal, and a functioning wolfram program on the PRi.
Next I tried hooking the Mac to the RPi using the Mac's Mma RDK. First, I verified that I only had the single (Local) kernel installed in Evaluation>Kernel Configuration Options, then I clicked on the Configure Pi Connection on the Remote Development Control Panel, filling in the RPi's ip address (from running ifconfig on the RPi after I first set it up). I used the palette to open a Pi Test & Example Notebook. Executing $MachineName got me nothing. Options[$FrontEnd,EvaluatorNames] gets me nothing. As a matter of fact, 2+2 gets me nothing. Evaluation>Notebook's Kernel shows Raspberry Pi checked.
I clicked on the Raspberry Pi in the Evaluation>Kernel Configuration Options panel and found an entry in the Basic Options > Remote Host: e$738, and that looked suspiciously like a Module variable that somehow didn't get resolved, so I edited it to the RPi's ip address, opened a fresh Pi test notebook. $MachineName gets me a Message "The child process 2095 reports:" and "The kernel Raspberry Pi failed to connect to the front end. (Error = MLECONNECT). You should try running the kernel connection outside the front end."
I need a hint.
Here is what worked.
In the Pi Test notebook, I set the Evaluation>Notebook's Kernel to Local, then
Options[$FrontEnd, EvaluatorNames]
{EvaluatorNames -> {"Local" -> {"AutoStartOnLaunch" -> True},
"Raspberry Pi" -> {"RemoteMachine" -> True,
"TranslateReturns" -> True, "AutoStartOnLaunch" -> False,
"Executable" -> "/usr/bin/wolfram",
"HostName" ->
"RemoteLogin" -> "pi"}}}
, EvaluatorNames ->
"Local" -> {"AutoStartOnLaunch" -> True}
, "Raspberry Pi" ->
"RemoteMachine" -> True
, "TranslateReturns" -> True
, "AutoStartOnLaunch" -> False
, "Executable" -> "/usr/bin/wolfram"
, "HostName" -> "10.0.1.18", "RemoteLogin" -> "pi"
Then set the Evaluation>Notebook's Kernel to Raspberry Pi and execute
It seems clear (to me anyway) that this is a buggy Remote Development Kit failing to resolve the variable reference in RemoteDevelopmentKit`Private`
This worked for me:
(Using Mac OS X Yosemite)
SSHing into the Raspberry Pi threw an RSA key error.
So I went to the Mac Terminal and Typed: open /Users/yourusernamehere/.ssh. I navigated to the "knownhosts" file and I found the key associated with my Pi's IP address and deleted it.
open /Users/yourusernamehere/.ssh
I tried to login into the Pi again and it asked my to verify the key.
After I logged into the Pi I went to Wolfram(local machine not pi) and changed my notebook type to from (Local) to (Raspberry Pi). I then typed $MachineName and the Output was "raspberrypi".
For me at least I am guessing that the RSA key was the issue.
I just thought I would post this in case anyone else had the same Problem.


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Image Overlapping to Im age Overlapping to Improve Micro PIV ...
8TH INTERNATIONAL SYMPOSIUM ON PARTICLE IMAGE VELOCIMETRY - PIV09 28, Melbourne, Victoria, Australia, August 25-28, 2009 Image Overlapping to Improve Micro PIV Accuracy 1 2 1 C.V Nguyen , A. Fouras and J. Carberry C.V. 1 Dept. of Mechanical & Aeronautical Eng., Monash University 3800, Australia email@example.com 2 Division of Biological Eng., Faculty of Eng., Monash University, 3800, Australia ABSTRACT A number of techniques have been developed to improve the Micro PIV uses volume illumination; therefore the velocity racy out-of-focus accuracy of micro PIV by reducing the effect of out measured at the focal plane is a weighted average of the particles. Gui et al [1] proposed a band band-pass filtering velocities within the measurement volume. The contribution technique to reduce both single pixel random noise and low of out-of-focus particles to the PIV correlation can generate frequency background noise. By adjusting the filter sizes, s significant measurement errors particularly in near wall pass in-focus particles band-pass filtering can selectively pass the in regions. We present a new application of image overlapping out-of-focus particles. while suppressing the noise and the out which is shown to very effective in improving measurement accuracy by effectively reducing the measurement depth. The Micro PIV data typically suffers from low seeding densities performance of image overlapping and correlation averaging and low illumination levels and consequently averaging were studied using synthetic and experimental images of techniques are required. Whilst currently correlation averaging , micro channel flow, both with and without image pre pre- image [3], is the method of choice for micro PIV imag processing, processing. The results show that for flows without particle correlation averaging does not address the errors generated by clumping, image overlapping provides the best measurement out of focus particles. The image overlapping technique was accuracy without any need for image pre pre-processing. For originally proposed by Wereley et al [6] to increase the flows with particle clumping, image overlapping combcombined seeding density in recorded PIV images. In this paper we with band-pass filtering provides the best measurement propose a new application of image overlapping to reduce the accuracy. effect of out-of-focus particles. 1. INTRODUCTION Micro particle image velocimetry (micro PIV) is an extension of PIV to measure flows at the micro scale, [5][1]. Due to the small measurement region, it is impossible to create a laser sheet to capture a slice of the flow, as traditionally done in macro PIV. Instead the laser beam illuminates all particles focus within the viewing volume and both in-focus and out out-of-focus particles appear in the image. Thus in micro PIV the depth over which the measurement is taken is the distance over which the particles, both in focus and out out-of-focus, significantly affect the measurement. The dep depth of measurement is characterized by "the depth of correlation" of the imaging system [6][4], where the depth of correlation δzcorr , is defined as the depth over which the correlation signal of particles significantly contribute to the correlation function. A threshold ε is chosen below which the particles correlation signal is considered negligible, typically ε is 1% of the focus maximum correlation provided by the in-focus particles [4]). The change of the particles and their correlation s signals with direction respect to the depth or z-direction is illustrated in Figure 1. Figure 1. Schematic showing the optical setup for micro elocity PIV of channel flow. The velocity profile is shown as a The velocity measured in micro PIV is the weighted average parabolic curve. The focal plane is located at the centre volume, of the velocity within the measurement volume where both of the channel. Particles further from the focal plane the shape and location of the PIV correlation peaks contain appear larger and dimmer a and the strength of the contributions from both in-focus and out-of of-focus particles. In correlation corresponding peak of auto-correlation decreases. The the majority of cases the contribution from the out out-of-focus depth of correlation, δzcorr, is defined as the distance particles is unwanted. In regions where the contributions of auto-correlation is above 1% over which the peak of the auto focus the out-of-focus particles either side of the focal plane are not correlation of the auto-correlation at the focal plane. Due to the equal and opposite a bias error results. The implications of this variation of particle correlation signal over δzcorr, the f can be demonstrated by considering the channel flow in Figure measured velocity is a weighted sum of velocity variation 1: when the focal plane is at the channel centre all the out-of- within the depth of correlation focus particles in the PIV image will be travelling slower than d the particles at the focal plane and the velocity measured will 2. IMAGE OVERLAPPING TE TECHNIQUE be lower than the velocity at the channel centre. Similarly, when the focal plane is at the channel wall the micro PIV Image overlapping is applied to a set of images, Ik by taking measurements will overestimate the velocity. location, the maximum pixel intensity at each pixel location producing a single overlapped image, Imax.: (1) When applied to a set of micro PIV data consisting of Npairs processing each image was divided into regions of 64×64 image pairs, a pair of overlapped images is produced. The first pixels and the velocity calculated within each region. The image is derived from the set of Npairs first exposures whilst measured velocity is the average of these velocities. The the second corresponds to the second exposures. Cross standard deviation of the measurements was calculated using correlation is then applied to this overlapped image pair to the velocities for each region for each of the Npairs.subsets. obtain a time-averaged velocity field As shown in Figure 1, particle intensity varies across the focal plane. The brightest Results are presented for a measurement plane near the wall, z particles are in-focus, close to the microscope focal plane. = 0.9R. This location was chosen instead of z = R as at the Further from the focal plane, particles are dimmer and out-of- wall the correct velocity is zero making it difficult to evaluate focus. By collecting maximum pixel intensities, image the accuracy of the PIV techniques. The results are presented overlapping collects the brightest particles while excluding ∗ . using a normalized measured velocity, . dimmer particles at the same pixel locations. In this way . image overlapping collects the particles closest to the focal plane, effectively reducing the depth of measurement. Image 3.1 Experimental Method overlapping requires much less computational time than A syringe pump (Harvard PHD 2000) with a 5cc syringe was correlation averaging. In PIV calculations the correlation used to perfuse glycerol solution through a rectangular calculation is the most time-consuming operation. To produce channel with a nominal cross section of 2000x200µm2. The an average velocity vector, image overlapping requires only a flow rate of 0.02ml/min gives a maximum velocity of single correlation calculation whereas correlation averaging 1.364mm/s at the channel centre, corresponding to a maximum requires Npairs correlation calculations. The time consumed by displacement of 19.5 pixels in the recorded images. The actual image overlapping, is very small compared to that by glycerol solution was made from a mixture of 2ml glycerin, image correlation and therefore image overlapping is nearly 8ml water and 1ml fluorescent particle suspension (1% solid). Npairs times faster than correlation averaging. The glycerol solution had density of 1.05 g/cm3, and viscosity 1.76 centipoise. The Reynolds number based on mean velocity Both image overlapping and correlation averaging can be and channel height is 0.11 and therefore the flow can be combined with image pre-processing techniques such as band- considered as a Poiseuille flow. The flow was seeded with pass filtering to further improve the measurement accuracy. fluorescent particles, 3µm diameter, density 1.05 g/cm3 and When applied with correlation averaging band-pass filtering emission wavelength of 612nm. The average particle seeding will act to reduce the effect of out-of-focus particles. density in the glycerol solution was 6.43 x 10-5 particles/µm3. However, when applied to an overlapped image the main benefit of band-pass filtering is the removal of particle clumps The refractive index of the glycerol solution causes the 200µm which are often present in experimental data. high channel to have an apparent height of 147.06µm when viewed with the objective through air. The channel was 3. METHODS displaced relative to the microscope objective using a In this paper the performance of image overlapping is computer-controlled 3D stage with sub-micron accuracy. The compared to that of correlation averaging for measurement of scanning step of 2.5µm generated a 3.4µm displacement of the velocity in a micro channel. Results are presented first for focal plane inside the glycerol filled channel and 59 steps were synthetic images, where the true velocity is known and required to transverse the channel height. subsequently for real experimental data. Images were captured in the centre of the channel width, using a 10× objective lens with NA=0.30 and a working distance of 3.1 Synthetic Data 10mm. Volume illumination was provided by a continuous A total of 2048 independent image pairs of size 1024 pixels diode pumped solid state laser (Melles Griot) emitting at ×1024 pixels were generated in 8-bit greyscale. No artificial 532nm and transmitted to the microscope through a liquid noise was added into the images and Brownian motion was light guide (Leica). A MotionPro X5 intensified camera (IDT) not considered. The uniform parabolic displacement profile in captured 1024 image pairs, 512×512 pixels in size at each z direction generates a maximum displacement of 27.03 pixels scanning location throughout the channel height. To between image pairs in the channel centre. The velocity in the implement the overlapping technique the time between image x and y directions remains constant. Particles of 1µm diameter pairs was selected such that in the slowest regions the particles are uniformly distributed within the viewing region with move more than one particle diameter between image pairs. particle density of 1.74 x 10-4 particles/µm3, equivalent to 10,000 particles per image. Particle image formation follows For PIV processing each image was divided into regions of the model employed by Olsen et al [4], with a simulated 64×64 pixels. The velocities within these regions were microscope lens of 10X magnification, 0.30 numerical averaged to yield the velocity at each z location. Correlation aperture and 10mm working distance. The fluorescent averaging and image overlapping without/with image pre- wavelength emitted from the particles is 560nm. processing were performed on each data set and velocity measurements are reported in terms of pixel displacements δx. For quantitative comparison between the velocity To study the effect of varying the number of image pairs measurements and the theoretical profile, the norm of the difference, ℓ (Npairs) used in the overlapping and correlation averaging , was calculated for each measurement technique, where ℓ techniques, an image set containing 2048 pairs was split into 2048/ Npairs image subsets. The measurement techniques were is defined as: applied to each subset to produce an in-plane velocity field. To − (2) make a fair comparison, each result must contain data from the same total number of image pairs, which is 2048. Vector averaging was applied to velocity measurements from the 2048/ Npairs image subsets to provide the final result. When Npairs= 1, the measurement is 100% vector averaging. For PIV 4. RESULTS AND DISCUSSION The effect of increasing Npairs on the measurement accuracy of correlation averaging and image overlapping was investigated using synthetic images. As shown in Figure 2 when Npairs = 1, the averaging) (the images are processed using 100% vector averaging the measured velocity is more than 10% greater than the true velocity of ∗ = 1. With increasing Npairs the measurement using the image overlapping technique ( ) converges rapidly to the true result and the standard deviation of the measurement also becomes very small. A similar trend occurs with correlation averaging (∆) except that the correlation averaging converges towards a value that overestimates the velocity by at least 5%. Results were also obtained for a measurement plane in the centre of the channel (not shown). Similar trends were found in the centre of the channel except without image overlapping e the finite depth of the measurement volume causes the measurements to underestimate the flow velocity. Figure 2. Variation of normalised near wall velocity (z = 0.9R) with the number of image pairs using correlation averaging ( ) or image overlapping ( ). Error bars e represent 1/10 of the velocity standard deviation. mage Velocity measured using image overlapping converges rapidly towards the true value (1) while correlation averaging overestimates the velocity near the wall. Unlike the synthetic images, the experimental images contain significant noise from various. The particles are of non non- uniform diameter, and therefore their brightness is non non- uniform. Also in these experiments, particles occasionally adhere to each other forming large and bright particle clumps skewing the velocity measurement towards the velocity of the clumps. As the images with the bright particle clumps will be preferentially collected in the overlapped image the effect of g these clumps is greatest on the overlapping technique. The velocity profile measured in the experimental channel ed Figure 3. Measured velocity profiles of channel flow using correlation averaging is shown in Figure 3a. The obtained with different image processing techniques, measured velocity is compared with the theoretical Poiseuille error bars represent the standard deviation and solid flow showing, as expected, that the measurements curves theoretical Poiseuille velocity profile. underestimate the velocity in the channel centre and decreases from 8.25px with a) correlation averaging overestimate it near wall. The standard deviation of the ith only, to 6.06px with b) correlation averaging & image measurements, represented by the error bars is significant band-pass filtering, to 3.53px with c) image overlapping across the flow. The value of is a relative measure of pass & image band-pass filtering. A reduction of 57% in the performance compared to the theoretical values averaged is achieved using image overlapping with image pre pre- over the channel depth. processing with the greatest improvement in velocity measurements occurring near the wall. The performance of correlation averaging can be improved by measurement accuracy. Image overlapping with band-pass image pre-processing as shown in Figure 3b. When an filtering reduces by 57% the measurement errors compared to optimized band-pass filter is applied prior to correlation that of plain correlation averaging, and by 42% the averaging, drops from 8.25px to 6.06px. The measurement errors compared to that of correlation averaging improvements are most significant in the flow centre and near with band-pass filtering the wall. The use of image thresholding as a pre-processing In summary, image overlapping produces significantly technique in combination with correlation averaging was also improved measurement accuracy with significantly less investigated. When the threshold levels were optimized their computational effort than correlation averaging. This simple effect on the accuracy of correlation averaging was similar to algorithm reduces the depth of measurement, producing 2 that of optimized band-pass filtering with = 6.79px. significant improvements in accuracy in regions where measurements would otherwise be biased by the velocities of The most accurate measurements were obtained using image out-of-focus particles. Image overlapping presents an overlapping with an image band-pass filter. This method opportunity to easily improve measurement accuracy in the produces excellent results as shown in Figure 3c. The critical wall region and thus is particularly relevant to value of 3.53 is almost half that of correlation averaging with biological flows. optimum image processing. In particular accuracy is significantly improved in the near wall region where the REFERENCES velocity gradients are greatest. [1] Gui L, Wereley ST, Lee SY (2002) Digital filters for The application of image band-pass filter as a pre-processing reducing background noise in micro PIV measurement. technique was found to successfully remove particle clumps 11th inter. symp. on the application of laser techniques to from the overlapped images. Image pre-processing was only fluid mechanics, Lisbon. necessary in images with particle clumps. Image thresholding prior to overlapping does not remove the particle clumps and [2] Meinhart CD, Wereley ST, Santiago JG (1999) PIV was found to produce little improvement in accuracy. This measurements of a microchannel flow. Experiments in result shows that even in images with particle clumps with Fluids 27(5): 414-419 appropriate pre-processing, image overlapping can produce significantly better measurement accuracy than correlation [3] Meinhart CD, Wereley ST, Santiago JG (2000) A PIV averaging. Algorithm for Estimating Time-Averaged Velocity Fields. Journal of Fluids Engineering 122: 285-289 5. CONCLUSIONS The velocities measured using micro PIV are biased by the [4] Olsen MG, Adrian RJ (2000) Out-of-focus effects on velocities of out-of-focus particles. The application of image particle image visibility and correlation in microscopic overlapping to reduce the depth of measurement can particle image velocimetry. Experiments in Fluids, 29(7): significantly reduce these errors. This was illustrated using S166-S174 channel flow where compared to standard correlation averaging, image overlapping produced a 57% reduction in the [5] Santiago J, Wereley S, Meinhart C, Beebe D, Adrian R value of . The improvements in measurement accuracy (1998) A particle image velocimetry system for were greatest in the near wall regions. microfluidics. Experiments in Fluids 25(4): 316-319 [6] Wereley ST, Gui L, Meinhart CD (2002) Advanced Image overlapping is performed by taking maximal intensities algorithms for microscale particle image velocimetry. from an image set to produce a single overlapped image. PIV AIAA Journal 40: 1047-1055 correlation is applied only once on a pair of overlapped images to produce an average velocity measurement. As the [7] Wereley ST, Meinhart CD, Gray MHB (1999) Depth in-focus particles are brightest, they are accumulated in the effects in volume illuminated particle image velocimetry. overlapped image. As a result, the measurement using Third International Workshop on Particle Image overlapped image pair provides the velocity of particles close Velocimetry,Santa Barbara, CA, USA to the focal plane. This effectively reduces the depth of measurement and the associated errors. Experimental images containing clumps of particles may require image pre- processing to remove these clumps before image overlapping can be effective. Correlation averaging has for the past decade been the gold standard in micro PIV processing. However, using both synthetic and experimental images image overlapping was shown to out-perform correlation averaging particularly in the near wall region. For synthetic images without particle clumps, with sufficient image pairs the velocity measured in the near wall region using overlapping converged to the true velocity, whereas correlation averaging significantly overestimated the velocity. For experimental images containing particle clumps, the measurements using image overlapping without pre-processing suffer bias errors from the clumps. With the application of band-pass filtering to remove the clumps, image overlapping continues to produce the best
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