PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.
Recent advances in light sources, detectors and other optical imaging technologies coupled with the development of
novel optical contrast agents have enabled real-time, high resolution, in vivo monitoring of molecular targets. Noninvasive
monitoring of molecular targets can help optimize photodynamic therapy (PDT) by providing the capabilities to
monitor the efficacy of treatment. Our lab has developed optical imaging technologies to investigate a wide range of
molecular, physiological and morphological responses to photodynamic therapy (PDT). With the idea that drug delivery
to the different compartments in the tumor is an important determinant of the treatment effect, we studied drug delivery
in vitro and in vivo using optical imaging tools. A molecular specific contrast agent that targets the vascular endothelial
growth factor (VEGF) was developed to monitor the changes in the protein expression following PDT. We also studied
the PDT-induced physiological changes in vascular permeability and metastasis with in vivo imaging.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Electron paramagnetic resonance imaging (EPRI) is one of the recent functional imaging modalities that can provide
valuable in vivo physiological information on its own merit and aids as a complimentary imaging technique to MRI and
PET of tissues especially with respect to in vivo pO2 (oxygen partial pressure), redox status and pharmacology. EPR
imaging mainly deals with the measurement of distribution and in vivo dynamics and redox changes using special nontoxic
paramagnetic spin probes that can be infused into the object of investigation. These spin probes should be
characterized by simple EPR spectra, preferably with narrow EPR lines. The line width should be reversibly sensitive
to the concentration of in vivo pO2 with a linear dependence. Several non-toxic paramagnetic probes, some particulate
and insoluble and others water-soluble and infusible (by intravenous or intramuscular injection) have been developed
which can be effectively used to quantitatively assess tissue redox status, and tumor hypoxia. Quantitative assessment
of the redox status of tissue in vivo is important in investigating oxidative stress, and that of tissue pO2 is very important
in radiation oncology. Other areas in which EPR imaging and oxymetry may help are in the investigation of tumorangiogenesis,
wound healing, oxygenation of tumor tissue by the ingestion of oxygen-rich gases, etc. The correct choice
of the spin probe will depend on the modality of measurement (whether by CW or time-domain EPR imaging) and the
particular physiology interrogated. Examples of the available spin probes and some EPR imaging applications
employing them are presented.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Photodynamic therapy (PDT) involves the combined action of light, oxygen and a photosensitizer (PS). It offers
unique control in the PS's action because the key cytotoxic agent, singlet oxygen (1O2), is only produced in situ upon
irradiation. The 1O2 production can be controlled in three levels. The first level involves the judicious use of fiber optics
to selectively deliver light to disease tissues. The second level is to exert control over the PS's localization by selectively
delivering PS to cancer cells. The third level is to exert control of the PS's ability to generate 1O2 in responding to specific cancer biomarkers. Here, we present two PDT agents based on the latter two levels of 1O2 control. The first PDT agent "PPF" contains a PS (Pyro) and a tumor homing molecule (folate) and a peptide linker. PPF was found to be
selectively accumulated in cancer cells via folate receptor (FR) pathway. The second PDT agent "PPMMP7B" is a matrix
metalloproteinase-7 (MMP7)-triggered photodynamic molecular beacon (PMB) containing a PS (Pyro), a 1O2 quencher
(BHQ3) and a MMP7-cleavable peptide linker. Thus, the 1O2 production of PPMMP7B is highly sequence-specific and its photodynamic cytotoxicity is MMP7-dependent. Since these agents are designed to share functional modules (PS and peptide linker) and common cancer cell model (KB cells overexpress both FR and MMP7), it forms the basis for rational
design of receptor-targeted PMB for achieving a multi-level control of 1O2 production in cancer cells, which in term,
could provide a much higher level of PDT selectivity.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Development of Single and Multiphoton Molecular Probes
Near-Infrared (NIR) absorbing chromophores have been used in analytical and bioanalytical chemistry extensively, including for determination of properties of biomolecules, DNA sequencing, immunoassays, capillary electrophoresis (CE) separations, etc. The major analytical advantages of these dyes are low background interference and high molar absorptivities. NIR dyes have additional advantages due to their sensitivity to microenvironmental changes. Spectral changes induced by the microenvironment are not desirable if the labels are used as a simple reporting group, e.g., during a biorecognition reaction. For these applications upconverting phosphors seem to be a better choice. There are several difficulties in utilizing upconverting phosphors as reporting labels. These are: large physical size, no reactive groups and insolubility in aqueous systems. This presentation will discuss how these difficulties can be overcome for bioanalytical and forensic applications. During these studies we also have investigated how to reduce physical size of the phosphor by simple grinding without losing activity and how to attach reactive moiety to the phosphor to covalently bind to the biomolecule of interest. It has to be emphasized that the described approach is not suitable for medical applications and the results of this research are not applicable in medical applications. For bioanalytical and forensic applications upconverting phosphors used as reporting labels have several advantages. They are excited with lasers that are red shifted respective to phosphorescence, resulting in no light scatter issues during detection. Also some phosphors are excited using eye safe lasers. In addition energy transfer to NIR dyes is possible, allowing detection schemes using donor-acceptor pairs. Data is presented to illustrate the feasibility of this phenomenon. If microenvironmental sensitivity is required, then specially designed NIR dyes can be used as acceptor labels. Several novel dyes have been synthesized in our laboratories for that purpose.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Indocyanine Green (ICG) is a photosensitive dye frequently used in clinical settings for retinal imaging and measuring
cardiac output function. Its low toxicity, high absorption in the near infrared region, and vascular retention have
generated much interest in its utility for several therapeutic and diagnostic applications. Its potential, however, is limited
by factors such as rapid circulation kinetics and lack of target specificity. Additionally, the optical properties of ICG vary
significantly with a wide range of influences such as concentration, solvent, pH, and temperature. In an effort to
overcome these limitations and improve ICG's effectiveness as a chromophore for photothermal treatment, we have
developed a novel nanometer-sized capsule system as a delivery vehicle for ICG. In our capsules, the ICG molecules are
encapsulated within a polymer-salt aggregate core coated with dextran. In this study, we report how the optical
properties of ICG are affected by its entrapment within the nano-assembled complexes (NACs). The absorption profiles
of both freely dissolved ICG and encapsulated ICG were recorded under various conditions to determine the effects of
encapsulation on ICG's light sensitivity and stability at different temperatures. Dilute preparations of freely dissolved
ICG demonstrated more rapid degradation kinetics in response to intense broadband light (360 - 1100 nm, 56 mW/ cm2)
than NACs containing ICG. Encapsulated ICG also demonstrated greater stability at room and elevated temperatures
than freely dissolved ICG. The results suggest that the entrapment of ICG within the capsules influences its ability to
undergo physicochemical transformations, including oligomerization and degradation to leucoforms.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
For biomedical applications, various optical techniques have been explored, all of which are inherently subject to the
limited penetration depth of observation. Nanoparticles have thus been introduced as a potential agent to overcome the
limit and to reveal more information on biomedical samples. Optical properties of nanoparticles in a turbid medium,
however, have remained difficult to measure precisely. In this paper, we investigate the scattering and absorbing
properties of gold nanoparticles of varying diameter and different concentration in absorbing liquid phantoms of silica
colloid, based on diffuse photometry. Diffuse reflectance patterns have been collected for each phantom with
nanoparticles of different size and concentrations relative to base media. The results imply that particle concentration and
size perturbs the medium properties, and the effect is dependent on the scattering properties of the particle. For the case
where the particle size stays within a range, the scattering property is dominated by the concentration.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Metal/dendrimer nanocomposites (DNCs) uniquely combine the properties of metallic clusters and the biofriendly
polymer host in a nanosized hybrid particle. DNCs can biochemically target tissues and locally reduce femtosecond
optical breakdown thresholds, making highly precise and selective photodisruption possible. In this study, we have used
high-frequency acoustic monitoring of bubble production dynamics to investigate how DNC properties, solution
concentration, and optical parameters affect threshold reduction, actual waiting time, and mechanical characteristics of
breakdown. Breakdown is defined here as bubble production with an onset of less than 20 seconds after laser exposure.
DNC properties varied include metal content (silver, gold) and terminal group (amino-NH2, glycidol-OH, and carboxyl-
COOH) which determine pH values. Results indicate that DNC metal content markedly influences solution threshold
reduction, while DNC terminal group (and thus net surface charge) and solution concentration influence the details of
breakdown at these reduced threshold fluences. {Ag(0)} DNCs reduce breakdown threshold fluence 1-2 orders of
magnitude more than {Au(0)} DNCs. Furthermore, concentrated DNC solutions and DNCs carrying a net negative
charge (carboxyl terminal groups) increase bubble production up to four times and shorten waiting time for breakdown
from seconds to milliseconds. Increasing laser fluence for a given DNC solution concentration also shortens breakdown
waiting time. Lastly, utilizing the fluorescence properties of silver nanocomposites, we use confocal microscopy to
examine KB cell uptake of folate targeted silver DNCs. Cells incubated with folate targeted silver DNCs exhibit a
measurable increase of intracellular fluorescence compared to control cells (no DNC incubation). However, while we
observe a threshold reduction in KB cells incubated with 500nM folate-targeted DNC solution, there is no threshold
reduction in cells incubated with 50nM folate-targeted DNC solution. This suggests that a specific minimum DNC
concentration may be required for localized reduced-threshold breakdown to occur.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Luminescent nanoparticles are increasingly used as emitting labels in the design of fluorescence imaging probes, because
of their outstanding optical properties, such as in the case of quantum dots, or their role in nano-assembling different
functionalities, such as imaging, drug delivery, and therapy. In this work, the potentiality of up-converting nano-crystals
for non invasive fluorescence imaging of small animal is explored. These up-converting nano-crystals are lanthanide
doped oxide matrices, such as Y2O3:Er,Yb compositions. They are produced by a bottom-up process and a thermal
treatment. They are functionalized by the coating of a thin polysiloxane shell layer, which can be further derivatized in
order to graft biomolecules such as antibodies, peptides, or DNA. Contrary to classical luminescent particles for which
light emission occurs at a wavelength superior to that of excitation, these materials emit at 564 and 661 nm upon
excitation at 980 or 815 nm. These unique emission properties, due to a multi-photonic process, can allow imaging
without any auto-fluorescence from the tissues in the wavelength detection window. Experiments in phantoms
mimicking the optical absorption and diffusion of tissues show that these crystals can be detected 4 mm deep at a 10
mg/mL concentration. Luminescence measurements in mouse and the potentiality of these nano-crystals for in vivo
imaging are discussed.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
The physio-pathological roles of sulfide biomolecules in cellular environments involves redox processes and
radical reactions that alter or protect the functional properties of enzymatic systems, proteins and nucleic acids repair.
We focus on micromolar monitoring of sulfur-centered radical anions produced by direct electron attachment, using
sulfide molecules (a thioether and a disulfide biomolecule) and two complementary spectroscopic approaches: low
energy radiation femtochemistry (1-8 eV) and high energy radiation femtochemistry (2.5-15 MeV). The early step of a
disulfide bond making RS∴SR from thiol molecules involves a very-short lived odd-electron bonded intermediate for
which an excess electron is transiently localized by a preexisting two sulfide monomers complex. The reactive center of
oxidized glutathione (cystamine), a major cytoplasmic disulfide biomolecule, is also used as sensor for the real-time IR
investigation of effective reaction radius reff in homogenous aqueous environments and interfacial water of biomimetic
systems. Femtosecond high-energy electrons beams, typically in the 2.5 - 15 MeV range, may conjecture the picosecond
observation of primary radical events in nanometric radiation spurs. The real-time investigation of sulfide and disulfide
molecules opens exciting opportunities for sensitisation of confined environments (aqueous groove of DNA, protein
pockets, sub-cellular systems) to ionizing radiation. Low and high-energy femtoradical probing foreshadow the
development of new applications in radiobiology (low dose effect at the nanometric scale) and anticancer radiotherapy
(pro-drogue activation).
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize
fundamental processes related to cancer development and response at cellular and molecular levels. This
technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major
concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of
treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to
limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in
vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the
bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9Lluc cells).
Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive
CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at
administered pO2 levels below about 5%, falling by approximately 50% at 0.2% pO2. Additional experiments
showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic
period reducing the ATP concentration to limiting levels for BLI.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
This paper presents a novel fiber optic Fabry-Perot (FP) structure for direct detection of deoxyribonucleic acid (DNA)
sequences. The capture DNA is immobilized onto the surface of a silica optical fiber tip by means of the layer-by-layer
electrostatic self-assembly (L-b-L ESA) technique. With the FP cavity inside the fiber, the change in optical path length
difference (OPD) caused by the immobilization and hybridization can be demodulated. Experimental results demonstrate
short (~5 min) response time. In addition, the sensor fabrication is simple and low cost. The signal is stable with high
visibility.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Fluorescent techniques developed for probing molecular events in vitro have been translated to small animal in-vivo fluorescence imaging using narrow-band, high-power laser sources, fast photon detection electronics and sophisticated image reconstruction algorithms. Targeted near-infrared fluorescent probes are being developed to increase image contrast and functional information collected by the in-vivo optical images. Besides fluorescence intensity, fluorescence lifetime can also be utilized to probe tissue physiology. Fluorescence lifetime, the average time that a fluorophore remains in the excited state, is specific to the molecule and its environment, but not necessarily to its concentration. Therefore, fluorescence lifetime imaging can be used to probe microenvironments within tissues to differentiate, for example, cancer from surrounding tissue by physiological differences. In the current study we demonstrate the ability to distinguish between two molecular probes in mice using fluorescence lifetime while using single excitation and emission wavelengths. Fluorescence detection was accomplished by measuring diffuse emission at 3 mm distance from excitation and raster-scanning whole-body regions of interest using time-correlated single photon counting technique. The resulting temporal point-spread function data was collected for grid points 1.5 mm apart. Fluorescence decays were de-convolved to determine measured fluorescence lifetimes by least-squares fit with associated chi-square error values. Relative signal from each fluorophore was then determined for specific tissues including liver, kidney and tumor areas. The results demonstrate that relative biodistributions of individual fluorophores with similar photonic characteristics can be simultaneously monitored in vivo using fluorescence lifetime imaging.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
The framework of fluorescent targeting probes for optical imaging is similar to that of contrast agents for other
modalities. They generally include a biological ligand, specific of the biological process to image, and a label, which
confers the probe its optical properties. Moreover, more sophisticated labeling functions, termed "activatable" can be
designed. An "activatable probe" will be initially non fluorescent. Only a specific molecular process, such as an
enzymatic reaction or cell internalization, is able to activate the probe fluorescence. Such probes are particularly easy to
design for the optical imaging modality, because of the easily triggered and well known fluorescence inhibition
processes. The optical properties of different commercially available organic dyes and their fluorescence inhibition are
therefore examined. Three classes of activatable probes have been listed: (i) activatable probes which rely on the selfquenching
of the label; (ii) activatable probes which use RET (Resonance Energy Transfer) between the label acting as a
donor, and an organic non-emissive acceptor; (iii) activatable probes which use an inorganic nanostructure as the
inhibitor, such as a gold nano-particle. Whereas activatable probes of class (i) can lead to higher fluorescence levels after
activation, their initial fluorescence inhibition can be hindered by structural constraints. Probes of class (ii) can therefore
be more interesting according to the probe design. The efficiency of probes of class (iii) using nanometer gold particles is
reduced because of their plasmon band lying in the visible and not near-infrared domain.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Highly luminescent nanoparticles, such as, trivalent erbium-doped yttrium-oxide, Er3+:Y2O3, are expected to have a wide
range of applications, including imaging, range finding, flash lidar, and other remote-sensing possibilities as well as
medical applications. These particles are synthesized by the precipitation from a homogeneous solution of the metal ions
and urea at elevated temperatures. The morphology of the calcinated materials, revealed through SEM, shows uniformly
spherical aggregates 200 nm or less depending on the ratio of the metal ions in the initial solution. Room temperature
optical absorption and emission spectra show that the trivalent erbium ions in Er3+:Y2O3 nanocrystals possess sharp
absorption lines and strong emission in near infrared region that are characteristic of Er 3+:Y2O3 grown as large single
crystals. Low temperature (8 K) spectra obtained from these particles were analyzed in detail for the crystal-field
splitting of the 2S+1LJ multiplet manifolds of Er3+(4f11) including the ground-state manifold 4I15/2, and excited manifolds
4I9/2, 4F9/2, 4S3/2, 2H11/2, 4F7/2, 4F5/2, and 4F3/2. Fluorescence lifetimes and results from an analysis of the intensities of
manifold-to-manifold transitions are also reported. Similarity of the nanocrystalline and large single crystal Er3+:Y2O3,
we propose that the simple, inexpensive method described in this study will lead to further investigation of these
nanocrystals for their optical properties, especially in the near infrared region of the spectrum.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
For investigatoin of intracellular protein interactions, quantum dots are widely used for fluorescent live cell imaging such
as total internal reflection fluorescence (TIRF) microscopy and confocal microscopy. In this paper, we performed a
quantitative analysis based on fluorescent intensity. For the measurement, A431 cell lines are imaged live with quantum
dots using TIRF microscopy. The distribution of quantum dots is affected by a TATHA2 peptide sequence in live cells.
This paper also presents the location change of quantum dots due to a nuclear localization signal in A431 cell lines.
Confocal microscopy was used to confirm the relation with fluorescent intensity and quantum dot concentration in live
cells.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
The steady state absorption and fluorescence spectroscopy of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and (2,2'-bipyridine)-3,3'-diol (BP(OH)2) were studied here free in solution and in human serum albumin (HSA) in order to test
their applicability as new biological probes. HBO and BP(OH)2 are known to undergo intramolecular proton transfers in
the excited state. Their absorption and fluorescence spectra are sensitive to environmental change from hydrophilic to
hydrophobic, thus allowing the opportunity to use them as environment-sensitive probes. The effect of water on the
steady state spectra of the two molecules also shows unique features which may position them as water sensors in
biological systems. For HBO in buffer, fluorescence is only due to the syn-keto tautomer, whereas in HSA the
fluorescence is due to four species in equilibrium in the excited state (the syn-keto tautomer, the anti-enol tautomer, the
solvated syn-enol tautomer, and the anion species of HBO). Analysis of the fluorescence spectra of HBO in HSA
indicates that HBO is exposed to less water in the HBO:HSA complex. For the BP(OH)2 molecule, unique absorption
due to water was observed in the spectral region of 400-450 nm. This absorption decreases in the presence of HSA due
to less accessibility to water as a result of binding to HSA. Fluorescence of BP(OH)2 is due solely to the di-keto
tautomer after double proton transfer in the excited state. The fluorescence peak of BP(OH)2 shows a red-shift upon
HSA recognition which is attributed to the hydrophobic environment inside the binding site of HSA. We discuss also the
effect of probe-inclusion inside well-defined hydrophobic cavities of cyclodextrins.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
We address the problem of quantitative evaluation of the absorption S0-S1 peaks &lgr; max of biological chromophores in
vacuo by using the state-of-art computational methods of quantum theory. In particularly, we rely on the second order
multiconfigurational quasidegenerate perturbation theory (MCQDPT2) following the complete active space selfconsistent
field (CASSCF) calculations. The use of augmented effective Hamiltonian operators in the MCQDPT2
framework allows us to correct deficiencies of the standard multistate approaches and to obtain stable saturated
solutions for the target low-lying excited states. A high accuracy of the proposed methodology is illustrated for several
photoactive protein chromophores in the gas phase including all-trans retinal, green fluorescent protein type
chromophores and photoactive yellow protein chromophores. It is shown that our approach provides correct ordering of
states and predicts maxima of absorption bands for the S0-S1 transitions within only a few nanometers from
experimental data.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
We present the results of modeling properties of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-
imidazolone (AHBMI), from the newly discovered fluorescent protein asFP595 inside the protein environment by using
the combined quantum mechanical - molecular mechanical (QM/MM) method. In this approach, the chromophore unit
and the side chains of the nearest amino acid residues are assigned to the quantum subsystem. The starting coordinates
of heavy atoms were taken from the relevant crystal structures of the protein. Hydrogen atoms were added manually,
and the structure of the model protein system was optimized by using QM/MM energy minimization for the trans-form
of the chromophore. The Hartree-Fock/6-31G quantum chemical approximation and the AMBER force field parameters
were employed in geometry optimization. The points on potential energy surfaces of the ground and first and second
excited electronic states were computed with the complete active space self-consistent field approximation in the
quantum subsystem under different choices of the QM/MM partitioning. Possible pathways for the trans-cis photo
isomerization presumably responsible for the kindling properties of asFP595 as well as other mechanisms of photo
excitation are discussed.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Biochemistry and Engineering of the Color Proteins
GFP is extensively used in molecular imaging applications. Its fluorescence is due to a autocatalytically formed
chromophore located in the center of the protein. Yellow and blue fluorescent mutants of GFP have been created. The
protein matrix that surrounds the chromophore influences both the intensity and the wavelength of the fluorescence. We
have used conformational searching methods and molecular dynamics simulations to examine the &tgr; and &fgr; dihedral space
available to a freely rotating and pyramidalizing chromophore. The calculations have shown that there seems to be a
relationship between the quantum yield of the fluorescent protein and the dihedral &tgr; and &fgr; space available to the
chromophore. The bright YFP has less rotational space available to a freely rotating chromophore than does wild-type
GFP or BFP, which has the most rotational freedom.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells
have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire
of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold
based on a single &bgr;-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the
measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein
(CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant &bgr;-hairpin peptides to be
rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a
16mer tryptophan zipper &bgr;-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer &bgr;-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of
the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the
20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we
experimentally demonstrate that the in vitro determined &bgr;-hairpin stabilities are well correlated with in vivo &bgr;-hairpin
stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked
by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer &bgr;-hairpin peptides
through the use of fluorescence-based library screening.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Bioluminescent photoproteins, such as aequorin and obelin, are proteins that emit light upon
binding calcium. Aequorin and obelin contain four EF-hand domains arranged into a globular structure.
The loop region of these EF-hand domains binds calcium by coordinating it in a pentagonal bipyramidal
structure with oxygen atoms. The binding of calcium to these EF-hands causes a slight conformational
change in the protein, which leads to the oxidation of the internally sequestered chromophore,
coelenterazine, producing coelenteramide and CO2. The excited coelenteramide then relaxes radiatively,
emitting bioluminescence at 471 nm in aequorin or 491 nm in obelin. Although calcium is the traditional,
and generally the most powerful, triggering ligand in this bioluminescence reaction, alternative di- and
trivalent cations can also bind to the EF-hand loops and stimulate luminescence. Species capable of this
cross-reactivity include: Cd2+, Ba2+, Mn2+, Sr2+, Mg2+, and several lanthanides. Magnesium is also known
to modulate the bioluminescence of wild-type aequorin, increase its stability, and decrease its aggregation
tendency. Both wild-type aequorin and wild-type obelin contain several cysteine residues, aequorin has
three and obelin has five. It is believed that these cysteine residues play an important, but as of yet
unknown, role in the bioluminescence of these proteins, since mutating most of these residues causes significant loss in bioluminescent activity. In order to explore whether or not these cysteine residues contributed to the specificity of the EF-hand domains for cations we generated four aequorin and obelin mutants and observed their luminescent intensity and decay kinetics by stimulation with calcium, barium, and magnesium. It was found that the cysteine mutations do appear to alter the effects that alternative divalent cations have on the bioluminescence of both aequorin and obelin.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
An important goal for in vivo imaging is to be able to non-invasively image single cells. The Olympus IV100 Laser
Scanning Microscope, with ultra-thin microscope objectives ("stick objectives"), was used for three-color whole-body
imaging of individual two-color cancer cells interacting with the GFP-expressing stromal cells. Cellular dynamics were
non-invasively imaged including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor
vasculature, and tumor blood flow. This imageable model should lead to a new paradigm of in vivo cancer cell biology
and to new visible real-time targets for cancer drug discovery.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Here we describe our cumulative experience with the development and preclinical application of several highly
fluorescent, clinically-relevant, metastatic orthotopic mouse models of pancreatic cancer. These models utilize the
human pancreatic cancer cell lines which have been genetically engineered to selectively express high levels of the
bioluminescent green fluorescent (GFP) or red fluorescent protein (RFP). Fluorescent tumors are established
subcutaneously in nude mice, and tumor fragments are then surgically transplanted onto the pancreas. Locoregional
tumor growth and distant metastasis of these orthotopic implants occurs spontaneously and rapidly throughout the
abdomen in a manner consistent with clinical human disease. Highly specific, high-resolution, real-time visualization of
tumor growth and metastasis may be achieved in vivo without the need for contrast agents, invasive techniques, or
expensive imaging equipment. We have shown a high correlation between florescent optical imaging and magnetic
resonance imaging in these models. Alternatively, transplantation of RFP-expressing tumor fragments onto the pancreas
of GFP-expressing transgenic mice may be used to facilitate visualization of tumor-host interaction between the
pancreatic tumor fragments and host-derived stroma and vasculature. Such in vivo models have enabled us to serially
visualize and acquire images of the progression of pancreatic cancer in the live animal, and to demonstrate the real-time
antitumor and antimetastatic effects of several novel therapeutic strategies on pancreatic malignancy. These fluorescent
models are therefore powerful and reliable tools with which to investigate human pancreatic cancer and therapeutic
strategies directed against it.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
More malignant tumors contain more hypoxic regions. In hypoxic tumor cells, expression of a series of hypoxiaresponsive
genes related to malignant phenotype such as angiogenesis and metastasis are induced. Hypoxia-inducible
factor-1 (HIF-1) is a master transcriptional activator of such genes, and thus imaging of hypoxic tumor cells where HIF-1
is active, is important in cancer therapy. We have been developing PTD-ODD fusion proteins, which contain protein
transduction domain (PTD) and the VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD)
domain of HIF-1 alpha subunit (HIF-1&agr;). Thus PTD-ODD fusion proteins can be delivered to any tissue in vivo through
PTD function and specifically stabilized in hypoxic cells through ODD function. To investigate if PTD-ODD fusion
protein can be applied to construct hypoxia-specific imaging probes, we first constructed a fluorescent probe because
optical imaging enable us to evaluate a probe easily, quickly and economically in a small animal. We first construct a
model fusion porein PTD-ODD-EGFP-Cy5.5 named POEC, which is PTD-ODD protein fused with EGFP for in vitro
imaging and stabilization of fusion protein, and conjugated with a near-infrared dye Cy5.5. This probe is designed to be
degraded in normoxic cells through the function of ODD domain and followed by quick clearance of free fluorescent
dye. On the other hand, this prove is stabilized in hypoxic tumor cells and thus the dye is stayed in the cells. Between
normoxic and hypoxic conditions, the difference in the clearance rate of the dye will reveals suited contrast for tumor-hypoxia
imaging. The optical imaging probe has not been optimized yet but the results presented here exhibit a potential
of PTD-ODD fusion protein as a hypoxia-specific imaging probe.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
We have developed an in vivo imaging system consisting of GFP- and DsRed-tagged human colonic
cancer cell line, which has peritoneal and lymph node metastatic potential and show high sensitivity to
EGFR targeting drugs, and convenient detection devices for GFP and DsRed. The latter includes a small
handy fluorescence detection device for external monitoring of the therapeutic effect of the drug and a
convenient stereo fluorescent microscope for internal visualization of micrometastases. We applied this
imaging system to investigate anti-metastatic effects of EGFR targeting drugs such as gefitinib (Iressa).
This system allowed sensitive detection of the development of peritoneal and lymph node metastases
from the micrometastasis stage at the cellular level and also permited noninvasive, non-anesthetic
monitoring of anti-metastatic effect of the drug in an animal facility without any pretreatment. Significant
decreases in the intraabdominal metastatic tumor growth and prevention of inguinal lymph node
metastasis by gefitinib treatment could be clearly monitored. These results suggest that convenient,
low-cost, true real-time monitoring of therapeutic effect using such a fluorescence-mediated whole body
imaging system seems to enhance the speed of preclinical study for novel anti-cancer agents and will
allow us to understand the action mechanism of molecular targeting drugs.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Early detection of primary tumors is the key for effective therapeutic intervention and successful patient survival. Small
animal models emulating human diseases are powerful tools for our comprehensive understanding of the
pathophysiology of tumor formation and metastasis to distant sites. Our long-term goal is to develop a non-invasive,
multiphoton-fluorescence lifetime imaging (MP-FLIM) modality that can precisely quantify these steps in animal tumor
models at a very early stage. The specific hypothesis is that fluorescence lifetime can be employed as reliable contrast
parameter for providing higher detection sensitivity as compared with conventional intensity-based tumor imaging
approaches and therefore it is possible to detect smaller tumor volumes (early detection) than those achieved by other
prevailing methods. We base this hypothesis on our recent observations that (1) fluorescence lifetime is "intrinsic" to
the fluorophore and its measurement is not affected by concentration and/or spectral artifacts as in intensity-based
methods, (2) multiphoton excitation can enable increased tissue penetrability and reduced phototoxicity and (3) MP-FLIM
approach can discriminate background autofluorescence from the fluorescent proteins in thick tissues thereby
achieving a ten-fold increase in signal-to-background ratio over the intensity-based approaches. We present our
preliminary data to support this hypothesis in primary tumor detection in nu/nu athymic mouse models.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable
target-specific agents are promising for clinical applications because of their high sensitivity and specificity.
Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in
animal models, and involves both conventional histology and immunohistochemistry, which requires large
numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of
genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to
produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells.
Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly
developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the
gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either
express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical
contrast agents with either green or near infrared fluorophores were developed to concurrently target and
validate cancer cells and their positive and negative controls, such as &bgr;-D-galactose receptor, HER1 and
HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect
separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo
imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical
contrast agents, thus reducing the number of animals needed to conduct these experiments.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by
fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically.
Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to
conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney
HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave
optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special
tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the
internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An
animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser
with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the
subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the
presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug
responce.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
The main reason green fluorescent protein (GFP) is so useful in molecular imaging is the fact that its chromophore is
formed autocatalytically. We have been using molecular mechanics to examine chromophore formation since 1995 that
is well before the first crystal structures of GFP were solved. Our calculations have resulted in a number of predictions
that have been borne out by subsequent experiments and a number of them that haven't. Recently we have been
supplementing these calculations with calculations based on the crystal structures of immature GFP mutants (i.e. the
precyclized form). Preliminary results from these calculations have shown that immature GFP does form a tight-turn in
the chromophore forming region, and that chromophore cyclization is probably catalyzed in the manner proposed by
Getzoff et al (Biochemistry 44: 1960-1970, 2005).
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
At the present time the phenomenon of "fluorescence kindling," which is typical of asCP595 protein
and some of its mutants, is of great interest for the studies of intracellular traffic. The range of
changing of intracellular pH may be rather wide, however, the effect of pH on the fluorescent state
of this protein is poorly investigated. Our studies have revealed that the fluorescence intensity of
asCP595 increases significantly in alkaline conditions. In addition, the observed change in the
position of excitation, emission, and absorption maxima indicates the appearance of new spectral
forms of the protein. These conformers are characteristics of new fluorescent states. The changes in
the absorption spectrum are indicative of a new dark form of the protein at alkaline pH.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) enhances tumor invasion and metastasis. To monitor MMP activity, we constructed plasmid that encoded a fluorescent sensor DC, in which an MMP substrate site (MSS) is sandwiched between DsRed2 and ECFP. MMPs are secretory proteins, only acting on the outside of cells; hence, an expressing vector was used that displayed the fluorescent sensor on the cellular surface. The DC was expressed in cells with high secretory MMP, so MSS was cleaved by MMP. Also, GM6001, an MMP inhibitor, causes DsRed2 signals to increase in living cells and on the chick embryo chorioallantoic membrane (CAM). Thus, this fluorescent sensor was able to sensitively monitor MMP activation in vivo. Potential applications for this sensor include high-throughput screening for MMP inhibitors for anti-cancer research, and detailed analysis of the effects of MMP
inhibitors.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.