Please explain Near-Infrared Fluorescence Imaging of Apoptotic

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Near-Infrared Fluorescence Imaging of Apoptotic Neuronal
Cell Death in a Live Animal Model of Prion Disease
Victoria A. Lawson,,, Cathryn L. Haigh,,, Blaine Roberts,,#
Vijaya B. Kenche,,,§ Helen M. J. Klemm, Colin L. Masters,,#
Steven J. Collins,, Kevin J. Barnham,,§, and Simon C. Drew*,,§,,^


Department of Pathology, The University of Melbourne, Victoria 3010, Australia, Mental Health Research Institute, Parkville, Victoria
3052, Australia, #Centre for Neuroscience, The University of Melbourne, Victoria 3010, Australia, and §The Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia

Abstract

encephalopathies (prion diseases). Current medical imaging paradigms focus strongly on the detection of amyloid (1), but deposition of protein aggregates may arise
after significant neuronal dysfunction has occurred (2, 3).
Therefore, imaging agents capable of identifying molecular events independent of protein deposition and prior
to the onset of clinical symptoms are most desirable.
Fluorescence imaging technology can provide a noninvasive means for disease detection and evaluation of
therapeutic strategies in animal models by reporting on
the activity of a host of biological events (4-6). Intrinsic
and extrinsic fluorophores that are excited and detected in
the visible spectrum have been used extensively for in vitro
biological studies employing fluorescence reflectance imaging and microscopy (4, 7). However, their suitability for
in vivo imaging applications, including brain imaging, is
limited due to the strong attenuation of visible wavelengths by biological tissue (oxy- and deoxyhemoglobin,
fat, melanin, water, bone), leading to poor tissue penetration depths (5, 8). Although an optically transparent
window chamber can be surgically implanted to slightly
improve penetration depth, this requires an invasive
craniotomy to be performed. Diseased or dying tissues,
especially of the central nervous system, can also exhibit
higher levels of autofluorescence in the visible region as
compared with healthy tissues, due to the build up of
lipofuscin (9).
Near-infrared (NIR) wavelengths (650-900 nm) pass
more deeply through mammalian tissue and are therefore
more suitable for in vivo fluorescence imaging (6, 8). In
vivo optical imaging of amyloid plaques in a murine
Alzheimers model has already been demonstrated using
various NIR probes (10, 11). However, to monitor disease
onset, progression, and response to treatment, contrast
agents that are independent of amyloid deposits and
sensitive to early markers of disease must be sought. In
this study, we present an NIR imaging agent capable of
irreversibly binding to active caspases by conjugating a
hydrophobic NIR cyanine dye to the broad spectrum

Apoptotic cell death via activation of the caspase family of
cysteine proteases is a common feature of many neurodegenerative diseases including Creutzfeldt-Jakob disease.
Molecular imaging of cysteine protease activities at the
preclinical stage may provide valuable mechanistic information about pathophysiological pathways involved in
disease evolution and in response to therapy. In this study,
we report synthesis and characterization of a near-infrared
(NIR) fluorescent contrast agent capable of noninvasively
imaging neuronal apoptosis in vivo, by conjugating a
NIR cyanine dye to Val-Ala-Asp-fluoromethylketone
(VAD-fmk), a general inhibitor of active caspases. Following intravenous administration of the NIR-VAD-fmk
contrast agent, in vivo fluorescence reflectance imaging
identified significantly higher levels of active caspases in
the brain of mice with advanced but preclinical prion
disease, when compared with healthy controls. The contrast agent and related analogues will enable the longitudinal study of disease progression and therapy in animal
models of many neurodegenerative conditions.
Keywords: Near infrared, neurodegeneration, prion, mice,
apoptosis, caspase, fluorescence, optical imaging

T

he most common cause of dementia is neurodegeneration arising from the misfolding and aggregation of normal cellular proteins and their
deposition, often as amyloid. These neurodegenerative
conditions include Alzheimers disease, Parkinsons disease, Huntingtons disease, and transmissible spongiform
r 2010 American Chemical Society

Received Date: July 26, 2010
Accepted Date: September 17, 2010
Published on Web Date: September 30, 2010

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DOI: 10.1021/cn100068x |ACS Chem. Neurosci. (2010), 1, 720727

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side chain, indicating loss of the methyl ester during synthesis.
[M þ H]þ: m/z (calc) = 1015.3, m/z (exp) = 1015.0; [M þ Na]þ:
m/z (calc)=1038.3, m/z (exp) = 1039.0. The ester is not required
for cell permeability and is removed by intracellular esterases
prior to caspase inhibition. The excitation and emission maxima
of NIR-VAD-fmk were comparable to the unconjugated DY750 (λex 740 nm, λem 770 nm). The concentration of purified
NIR-VAD-fmk solutions was determined from the optical
density at 745 nm using an extinction coefficient of 270 000
M-1 cm-1.

Cell Culture
OBL-21 mouse neuronal cells (12, 13) were cultured in
Dulbeccos Modified Eagles Media (DMEM; Gibco - Invitrogen,
Victoria, Australia) supplemented with 10% fetal bovine serum
(Invitrogen), 50 U mL-1 penicillin, and 50 μg mL-1 streptomycin
solution (Sigma-Aldrich; New South Wales, Australia). All cell
lines were maintained at 37 °C with 5% CO2 in a humidified
incubator.

Figure 1. Structure of the contrast agent NIR-VAD-fmk. The
fluorophore is a near-infrared cyanine dye (DY-750; Dyomics,
GmbH). The VAD-fmk sequence irreversibly binds to the active site
of caspase enzymes and is a broad spectrum inhibitor of the caspase
family.

In Vitro Toxicity
Cells were plated to 20% confluency. Five microliters of
one-solution MTS reagent (Promega, Victoria, Australia)
per 100 μL media was added to the media of test and control
cultures and incubated under normal culture conditions for
90 min. Reaction product was quantified using absorbance at
462 nm in a Fluostar Optima (BMG Labtech). All results were
normalized to cell density.

caspase inhibitor VAD-fmk (Figure 1). To test the compound in vivo, we assessed its ability to detect neuronal
apoptosis in a live murine model of prion disease. Compared with healthy controls, a significant increase in active
caspases was measured in the brain of prion-infected mice
prior to onset of clinical signs such as ataxia and hind limb
paresis. The ability to detect neuronal cell death in living
mice prior to development of clinical signs of neurodegenerative disease makes it suitable candidate for
screening potential therapeutics targeting early pathogenic pathways.

In Vitro Active Caspase Detection
NIR-VAD-fmk (Figure 1) was solubilized in sterile phosphate buffered saline (PBS) (Gibco - Invitrogen) containing
10% v/v high quality (cell culture tested) sterile-filtered
DMSO (Sigma-Aldrich). Cells were incubated with 15 μM
NIR-VAD-fmk for 30 min, washed, and imaged under 20Â
magnification with a Nikon Eclipse TE2000-E epi-fluorescence microscope using an excitation wavelength of 620 (
30 nm and a 660 nm long-pass filter (Filter set 41008). To
correct for background, the average fluorescence intensity of
images taken without the addition of fluorescent marker was
subtracted in an identical manner from all images.

Methods
Synthesis of a Near-Infrared Fluorescent Inhibitor
of Active Caspases
Z-Val-Ala-Asp(OMe)-fmk (Z=benzyloxycarbonyl; fmk=
fluoromethylketone) was purchased from SM Biochemicals
LLC (Yorba Linda, USA). The peptide was dissolved in a
33% v/v solution of HBr in AcOH (Sigma-Aldrich, Castle Hill,
Australia) and stirred for 45 min to form the HBr salt of
NH2-VAD-fmk. The solution was evaporated to dryness and
residual HBr/AcOH was removed by washing with ethyl ether,
then resuspended in DMF at a concentration of 100 mM. The NIR
cyanine dye containing an amino-reactive N-hydroxysuccinimidyl-ester (DY-750 NHS-ester, Dyomics GmbH, Germany) was
dissolved in DMF and added to the peptide solution at a molar
ratio of 1.1:1 in the presence of 3 molar equiv of N,N-diisopropylethylamine. The reaction was allowed to proceed in the dark
overnight at room temperature (RT) with stirring, before the
solution was again evaporated to dryness. The labeled product
(denoted NIR-VAD-fmk) was purified by RP-HPLC using a
Shimadzu LCMS-2010EV LC/MS system with a 5 μm, 2.1 Â
150 mm cyano column (Waters Corp., Massachusetts, USA)
and a 40-45% MeCN/H2O gradient in the presence of 0.1%
formic acid. Purity was g98% as determined from the
RP-HPLC trace at 700 nm (Figure S1, Supporting Information).
MS analysis indicated the conjugate contained a free aspartyl

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Mice
All animal procedures conformed to National Health and
Medical Research Council of Australia guidelines and were
approved by the University of Melbourne Animal Experimentation Ethics Committee. The M1000 prion strain used in
this study was derived from the Fukuoka-1 strain of mouseadapted human prions (14). This strain was originally isolated
and maintained by passage in Balb/c mice (15). Five 6-week
old Tga 20 mice were inoculated under methoxyflurane
anesthetic in the left parietal region with 30 μL of 1% (w/v)
homogenate prepared in PBS from a pool of brains derived
from Balb/c mice with clinical prion disease induced by
M1000 infection. The Tga20 transgenic mouse line overexpresses murine PrPC and mice develop signs of clinical prion
disease (bradykinesis, kyphosis, ataxia, and hind limb paresis)
approximately 60 days after an intracerebral prion inoculation, with terminal disease occurring within days of symptom
onset (16). Three 5-week old Tga20 mice received an intracerebral prion inoculation with 30 μL of 1% w/v brain homogenate prepared from uninfected Balb/c mice and served as
age matched, sham inoculated controls.

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In Vivo Toxicity

methanol). Blots were blocked in 5% skim milk powder in PBS/
0.1% Tween-20 (PBST) for 1 h at room temperature (RT) and
probed for 1 h at RT with rabbit polyclonal antibody 03R19
raised to residues 89-103 of mouse PrP (19). The blot was
washed at RT 1 Â 10 min, and 3 Â 3 min, probed with
antirabbit-HRP for 1 h at RT, washed as described, and
developed using ECL-plus (GE Healthcare). The prepared
membrane was imaged using the LAS-3000 imaging system
and analysis was performed using Multi Gauge V3.0 software
(FujiFilm, Japan). Following normalization for total protein
(BCA; Pierce) homogenates prepared from U1-3 and I1-3
containing 50 μg of total protein were electrophoresed as
described above and probed overnight at 4 °C with an antibody
to cleaved caspase-3 (Asp175) (cat. no. 9661; Cell signaling
Technology), washed, incubated with antirabbit HRP for 1 h at
RT, washed and developed with ECL-advance (GE Healthcare)
before imaging and analysis as described above. Statistical
analyses were carried out using Minitab 15.1.30.0 statistical
software.

NIR-VAD-fmk (Figure 1) was prepared in sterile phosphate buffered saline (PBS) (Gibco - Invitrogen) containing
10% v/v high quality (cell culture tested) sterile-filtered
DMSO (Sigma-Aldrich). To screen for possible toxicity of
the new compound, two healthy 5-week-old Tga20 mice were
administered a 100 μL injection of a 0.5 mM NIR-VAD-fmk
solution via the lateral tail vein. They were monitored for 2 h
immediately following treatment, and thereafter daily for
1 week, whereupon a second dose was administered.

In Vivo Active Caspase Detection
Fifty-four days after inoculation, three prion-infected (I1,
I2, I3) and three sham-inoculated Tga20 mice (U1, U2, U3)
were administered 200 μL of a 0.25 mM NIR-VAD-fmk
solution via the lateral tail vein. To assess possible contributions
from nonspecific autofluorescence of diseased tissue, an additional two prion-infected Tga20 mice (I4, I5) received 200 μL of
inactive PBS/DMSO diluent only. PBS was used in preference to
a NIR labeled version of the well-known inactive substrate FAfmk. Although inactive against caspases, FA-fmk inhibits cathepsins (17), whose activities are up-regulated in prion-infected
mouse neuronal N2a cells (18). Moreover, it is not possible to
predict whether a fluorescent NIR-FA-fmk analogue will
exhibit blood-brain-barrier permeability comparable with
NIR-VAD-fmk. Prior to imaging, NIR-VAD-fmk was permitted
to circulate for 30 min following injection to allow circulation
and excretion of any unbound probe. Mice were subsequently
anaesthetised by the intraperitoneal administration of ketamine
(80 mg kg-1)/xylazine (10 mg kg-1) and their heads shaved in
preparation for fluorescence reflectance imaging.
Anesthetized mice were transferred to the dark box of a
LAS-3000 imaging system (FujiFilm, Japan). Fluorescence
reflectance images (16 bit, 36.7 Â 36.7 μm pixel size) were
obtained using a 710 nm LED epi-illuminator and a 785 nm
long-pass filter with an exposure time of 5 s and an aperture of
F0.85. These were superimposed upon monochrome images
obtained using a white epi-illuminator with an exposure time
of 16.7 ms and an aperture of F2.8. Image analysis was
performed using Multi Gauge V3.0 software (FujiFilm,
Japan). Statistical analyses were carried out using Minitab
15.1.30.0 statistical software.
At the conclusion of imaging anaesthetized mice were
euthanised by cardiac perfusion with PBS and brains sectioned through the sagittal plane. One half was snap frozen in
liquid nitrogen and stored at -80 °C and the other half fixed in
10% neutral buffered formalin (NBF).

Immunohistochemistry
Brain hemispheres fixed in 10% NBF were immersed in 99%
formic acid for 1 h prior to routine processing and immunostaining. Half-brains were paraffin embedded and 7 μm sections
cut and mounted on glass slides (Superfrost plus; Thermo).
Sections were deparaffinized and rehydrated through a graded
ethanol series to deionized water and stained with hematoxylin
and eosin or immunostained with ICSM18 anti-PrP monoclonal antibody (D-Gen Ltd., London, UK) (20). For immunostaining, rehydrated sections were autoclaved for 20 min at
132 °C and once cooled, washed in deionized water, exposed to
4 M guanidine thiocyanate for 2 h at 4 °C, washed and treated
with 96% formic acid for 5 min. After blocking with 20% fetal
bovine serum for 30 min, sections were incubated with ICSM18
overnight at 4 °C. Sections were then processed using a avidinbiotin immunohistochemical process (LSABþ; Dako) and
developed using diaminobenzadine (DABþ; DAKO) and
counterstained with hematoxylin.

Results and Discussion
The ability of labeled VAD-fmk compounds to detect
active caspases is well characterized (21), and their specificity for labeling apoptotic neuronal cells in live mice has
been clearly established (3). Nevertheless, prior to testing
the new NIR-VAD-fmk contrast agent in vivo, we first
examined its toxicity and cell permeability in vitro using a
mouse neuronal (OBL-21) cell line. No significant reduction in cell viability (measured by MTS reduction) was
seen in response to treatments ranging in concentration by
4 orders of magnitude, even after 3 days constant exposure to 75 μM. (This exceeds the nominal NIR-VADfmk blood concentration of 55.5 μM in mice, based upon
a typical blood volume of 800 μL and a 200 μL injection of
0.25 mM NIR-VAD-fmk.) NIR-VAD-fmk (Figure S2,
Supporting Information). To assess the cell permeability
and the specificity of the NIR-VAD-fmk binding in response to apoptotic stimulus, the compound was applied
to the OBL-21 mouse neuronal cells following 10 min

Western Blotting
Brain homogenates (20% w/v) were prepared in PBS using
a FastPrep Homogenizer (ThermoSavant) using a single cycle of
homogenization before being further diluted to 6% (w/v) in PBS
and final concentration of 0.1% SDS (to aid digestion of PrPC)
and treated with proteinase K (100 μg mL-1, 1 h, 37 °C). PKdigestion was stopped by the addition of PefaBloc SC (Roche;
4 mM) and 4 Â LDS loading dye (containing 12% v/v betamercaptoethanol) and heated to 100 °C for 10 min. Samples
were electrophoresed on a 12% (bis/tris) gel (NuPAGE Invitrogen) at 200 V for 50 min in MES electrophoresis buffer (Invitrogen) and transferred to a PVDF membrane at 85 V for 60 min in
Tris/glycine transfer buffer (25 mM Tris, 200 mM glycine, 20%

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Figure 2. NIR-VAD-fmk localization of caspase activation in OBL-21 mouse neuronal cells (a-c) 24 h post exposure to 10 min UV
irradiation. (d-f) untreated control. After 24 h, cells were incubated with 15 μM NIR-VAD-fmk for 30 min, washed and imaged by
fluorescence microscopy at 60Â magnification. Scale bar = 20 μm.

yielded a negligible autofluorescent signal (Figure S5,
Supporting Information), indicating that the increased
fluorescence was not attributable to accumulation of
lipofuscin in prion disease (9).
Following imaging, animals were euthanized and the
brain pathology was examined using traditional markers
of disease neuropathology. Consistent with the in vivo
observation of NIR-VAD-fmk fluorescence in prioninfected mice, Western immunoblot analysis showed a
significant increase in cleaved caspase-3 (a central effector
caspase) in prion-infected mice (Figure 5). Western immunoblot analysis confirmed the presence of PrPSc in
prion infected mice and absence in sham-inoculated controls (Figure 6a). Hematoxylin and eosin staining was
consistent with the preclinical state of the animals with
little to no vacuolation apparent in the hippocampus,
thalamus, pons or occipital cortex of infected mice I1, I2,
and I3 that received NIR-VAD-fmk and mild to moderate
vacuolation in infected mice I4 and I5 that by chance
received the vehicle (Figure 6b). PrPSc deposition was
similarly mild or undetectable in all prion infected mice
(Figure 6).
In hippocampal CA2 neurons, apoptosis has been
shown to occur prior to the accumulation of PrPSc and
the onset of clinical disease in a mouse-passaged 87 V
scrapie strain (2). However, confirmation of apoptosis
required culling the mice to carry out terminal deoxynucleotidyl transferase-mediated uridine triphosphate
nick end labeling (TUNEL) staining to show DNA
fragmentation within the brain. In this study, we have
noninvasively measured caspase activation associated
with apoptotic neuronal cell death in live prion-infected
mice 54 days postinoculation prior to development of
disease (16). NIR imaging of molecular processes such
as caspase and other protease activities at the preclinical
stage promises to provide more valuable mechanistic

ultraviolet (UV) irradiation. UV exposure initiates apoptosis via an intrinsic cell death pathway involving mitchondrial release of cytochrome c, activation of the initiator
caspase 9 and subsequent activation effector caspases
3 and 6 (22). In comparison with the nonirradiated control,
the UV irradiated cells displayed strong intracellular NIR
fluorescence (Figure 2), demonstrating the cell-permeability of NIR-VAD-fmk and its ability to bind to active
caspases. Binding of the probe could be observed within
hours of UV insult (Figure S3, Supporting Information),
and at 24 h a large number of cells could be demarcated by
NIR-VAD-fmk binding (Figure S4, Supporting Information). Untreated OBL-21 cells showed only a low
level of intracellular binding, consistent with basal levels of
active caspases associated with normal cellular processes,
such as cell division and differentiation (23).
Having established that NIR-VAD-fmk was nontoxic and able to permeate and bind to active caspases
in cultured cells, we examined its ability to label active
caspases in live animals using a murine model of prion
disease. In healthy controls, no signs of toxicity or
adverse reaction was observed following administration
of a single dose of NIR-VAD-fmk, nor after a second
dose one week later. NIR-VAD-fmk was then administered to Tga20 mice 54 days post inoculation with
M1000 prions or a sham inoculation (Figure 3). At this
time there was no evidence of weight loss or overt
signs of clinical prion disease, which occurs approximately 60 days post inoculation in this transgenic mouse
line. Nevertheless, quantification of NIR-VAD-fmk
binding by fluorescence reflectance imaging revealed
increased levels of active caspases in the brains of prioninfected mice compared with mice that received an
intracerebral inoculation with uninfected brain homogenate (Figure 4). Prion-infected mice that received only
the DMSO/PBS vehicle instead of NIR-VAD-fmk
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Figure 3. Overlay of in vivo fluorescence and white-light images of (a-c) prion-infected (I1-I3) and (d-f) sham inoculated (U1-U3) Tga20
mice, following administration of a NIR-VAD-fmk dose, 54 days postinoculation.

Figure 4. Quantification of the images shown in Figure 3. Prioninfected mice that received the NIR-VAD-fmk exhibited a significantly higher fluorescence emission compared with the sham-inoculated healthy (uninfected) controls (Mann-Whitney, onetailed, W = 15.0, p = 0.040, n = 3). Black squares represent the
average fluorescence intensity per unit area of each NIR image.
Horizontal bars denote the group means.

information about pathophysiological pathways involved in neurodegeneration compared with diagnostic
imaging modalities that focus on detection of late-stage
markers of disease such as amyloid formation.
A number of other NIR contrast agents for detecting
cell death have been developed in recent years. For example, a NIR (Cy5.5) fluorophore has been conjugated to
annexin-V to detect externalization of phosphatidylserine
at the cell membrane during tumor apoptosis (24). NIRVAD-fmk is demonstrably blood-brain-barrier permeable
making it suitable for brain imaging applications and can
be further refined to detect specific caspases by changing
the caspase recognition sequence. Analogous NIR compounds based upon caspase substrates rather than inhibitors
have also been developed. One example is the enzymeactivatable Cy5.5-DEVD substrate tethered to cell-permeable nanoparticles, which has been shown to detect active
caspases 3 and 7 in cell culture (25). Another involves intracellular delivery of DEVD conjugated to Alexa Fluor 647
r 2010 American Chemical Society

Figure 5. (a) Western immunoblot analysis of brain homogenates
from prion-infected (I1-I3) and sham-inoculated (uninfected)
(U1-U3) mice administered NIR-VAD-fmk. Molecular weight
markers are shown. Consistent with in vivo imaging, quantification
of immunoreactivity against cleaved caspase-3 (b) indicated increased
levels in prion-infected mice (Mann-Whitney, one-tailed, W =
15.0, p = 0.040, n = 3). Horizontal bars denote the group means.

and QSY 21 dyes via a cell-penetrating KKKRKV peptide
sequence, which was demonstrated to detect NMDAinduced apoptosis of retinal ganglion cells following intravitreal injection in live rats (26). However, the bloodbrain-barrier permeability of such compounds for brain
imaging applications remains unknown. Moreover, such
compounds do not bind irreversibly to active caspases and
therefore must rely on self-quenching until the substrate
is cleaved. Depending upon disease pathophysiology,
the kinetics of uptake of the uncleaved (nonfluorescent)
substrate and clearance of the cleaved (fluorescent) substrate may vary between healthy and diseased individuals,
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Figure 6. Pathology associated with prion-infected Tga20 mice. (a) PK-treated (100 μg mL-1, 1 h, 37 °C) homogenates prepared from sham
inoculated (U1-U3) and prion-infected (I1-I5) Tga20 mice were Western immunoblotted with 03R19. Homogenates prepared from a Tga20
mouse with clinical disease are shown before (-) and after (þ) PK treatment for comparison. Molecular weight markers (kDa) are shown.
(b) Immunostaining of PrPSc using ICSM18 and (c) hematoxylin and eosin staining of sham inoculated (U3) or prion infected mice with low
PrPSc (I3) and high PrPSc (I4) load. Images show the CA1 region of the hippocampus (HC), thalamus (Th), Pons and occipital cortex (OC).
Images were taken at Â20 and Â40 magnification. Scale bars shown are 50 μm.

target peptide sequence (21), while additional improvements in depth resolution and spatial localization may
be achieved using a fluorescence tomographic imaging
approach (6, 8, 27).

making it difficult to compare relative concentrations
measured at the time of imaging. The use of the NIRVAD-fmk caspase inhibitor, rather than a substrate, can...
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