Small Animal Optical Imaging Research Articles

Evaluating blood-brain barrier disruption and infarction volume concurrently in rats subjected to ischemic stroke using an optical imaging system

BackgroundBlood-brain barrier (BBB) disruption is pivotal in the pathophysiological process of ischemic stroke and is often measured in rodent stroke studies. Traditionally, rodent BBB permeability increase is determined by measuring cerebral leakage of certain dyes such as Evans Blue or sodium fluorescein (NaFL). However, due to the special processing of samples for BBB permeability measurement, they cannot be used afterward for determining other essential parameters such as cerebral infarction volume. Therefore, using different batches of animals for assessing BBB permeability and infarction volume is typical. However, this would limit the stroke study’s statistical power and scientific value while hindering the implementation of procedures for high standard animal welfare. New methodThe rats subjected to middle cerebral artery occlusion (MCAO) were intraperitoneally injected with NaFL during the reperfusion phase. The brains were sliced and measured for BBB permeability using the small animal optical imaging system (IVIS® Lumia series III). Afterward, the same brain samples were either sliced or homogenized for tests that assessed infarction volume or other molecular changes. ResultsThe sum fluorescence intensity of the ischemic brain slices under the IVIS® Lumia series Ⅲ showed a strong correlation with the infarction volume determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (r = 0.7440, P = 0.0087). The fluorescence intensity of the whole ischemic brain was correlated with the NaFL concentration of brain tissue homogenates (r = 0.8653, P = 0.0026) and cerebral infarction volume (r = 0.7282, P = 0.0072). Comparison with existing methodsThe new method enables concurrent measurement of BBB permeability and infarction volume on the same batch of brain tissue samples without affecting most downstream biochemical assays. ConclusionsBy applying the new method, we could use the same batch of ischemic rodent brain tissue for multiple assays, including BBB permeability and infarction volume. Through this, we would reduce the animal numbers in each study and help to maximize the scientific and statistical potential of future rodent ischemic studies.

Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells.

Despite prophylactic vaccination campaigns, high-risk human papillomavirus (HPV)-induced cervical cancer remains a significant health threat among women, especially in developing countries. The initial occurrence and consequent progression of this cancer type primarily rely on, E6 and E7, two key viral oncogenes expressed constitutively, inducing carcinogenesis. Thus, E6/E7 have been proposed as ideal targets for HPV-related cancer diagnosis and treatment. In this study, three novel HPV16 E6-binding affibody molecules (ZHPV16E61115, ZHPV16E61171, and ZHPV16E61235) were isolated from a randomized phage display library and cloned for bacterial production. These affibody molecules showed high binding affinity and specificity for recombinant and native HPV16 E6 as determined by surface plasmon resonance, indirect immunofluorescence, immunohistochemistry, and near-infrared small animal optical imaging in vitro and in vivo. Moreover, by binding to HPV16 E6 protein, ZHPV16E61235 blocked E6-mediated p53 degradation, which increased the expression of some key p53 target genes, including BAX, PUMA and p21, and thereby selectively reduced the viability and proliferation of HPV16-positive cells. Importantly, ZHPV16E61235 was applied in combination with HPV16 E7-binding affibody ZHPV16E7384 to simultaneously target the HPV16 E6/E7 oncoproteins, and this combination inhibited cell proliferation more potently than either modality alone. Mechanistic studies revealed that the synergistic antiproliferative activity depends primarily on the induction of cell apoptosis and senescence but not cell cycle arrest. Our findings provide strong evidence that three novel HPV16 E6-binding affibody molecules could form a novel basis for the development of rational strategies for molecular imaging and targeted therapy in HPV16-positive preneoplastic and neoplastic lesions.

Accessing lanthanide-based, in situ illuminated optical turn-on probes by modulation of the antenna triplet state energy.

Luminescent lanthanides possess ideal properties for biological imaging, including long luminescent lifetimes and emission within the optical window. Here, we report a novel approach to responsive luminescent Tb(iii) probes that involves direct modulation of the antenna excited triplet state energy. If the triplet energy lies too close to the 5D4 Tb(iii) excited state (20 500 cm−1), energy transfer to 5D4 competes with back energy transfer processes and limits lanthanide-based emission. To validate this approach, a series of pyridyl-functionalized, macrocyclic lanthanide complexes were designed, and the corresponding lowest energy triplet states were calculated using density functional theory (DFT). Subsequently, three novel constructs L3 (nitro-pyridyl), L4 (amino-pyridyl) and L5 (fluoro-pyridyl) were synthesized. Photophysical characterization of the corresponding Gd(iii) complexes revealed antenna triplet energies between 25 800 and 30 400 cm−1 and a 500-fold increase in quantum yield upon conversion of Tb(L3) to Tb(L4) using the biologically relevant analyte H2S. The corresponding turn-on reaction can be monitored using conventional, small-animal optical imaging equipment in presence of a Cherenkov radiation emitting isotope as an in situ excitation source, demonstrating that antenna triplet state energy modulation represents a viable approach to biocompatible, Tb-based optical turn-on probes.

Weak light emission of soft tissues induced by heating

The main goal of this work is to show that soft tissue interaction with high-intensity focused ultrasound (HIFU) or direct heating leads to a weak light emission detectable using a small animal optical imaging system. Our results show that the luminescence signal is detectable after 30min of heating, resembling the time scale of delayed luminescence. The imaging of a soft tissue after heating it using an HIFU field shows that the luminescence pattern closely matches the shape of the cone typical of the HIFU beam. We conclude that heating a soft tissue using two different sources leads to the emission of a weak luminescence signal from the heated region with a decay half-life of a few minutes (4 to 6min). The origin of such light emission needs to be further investigated.

Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo.

Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.

Radioluminescence from Tc-99m in glass predicts local dose

The widely-used gamma-emitter Tc-99m has been shown to lead to optical emissions in mice and glass. We investigated the possibility that these emissions are due to the Cerenkov effect and whether the light emitted is proportional to local dose. By using a Geant4 Monte Carlo model matched to an experimental measurement, we show that the light detected by a small animal optical imaging system provides a 2D map of the dose throughout a glass sample. We conclude that radioluminescence from Tc-99m can be used to quantitatively measure dose in transparent materials, which could have applications in dosimetry and quality assurance.

Optical imaging of irradiated cyclotron target window foils using Cerenkov and radioluminescence imaging

Radioisotopes production for PET radiopharmaceuticals is performed using cyclotrons resulting in radio activation of different cyclotron components. It is thus necessary to measure the level of radiation exposure and, if possible, to image the areas where most of the radiations are emitted in particular during maintenance or decommissioning procedures. In this work we present a novel optical imaging approach using Cerenkov luminescence imaging (CLI) and radioluminescence imaging (RLI). CLI was performed by placing a glass Cerenkov radiator on a target window (Havar foils) and RLI data were obtained by covering the Havar foils with an intensifying screen. CLI or RLI were acquired using a small animal optical imaging system used in bioluminescence mode without the use of any optical filters. The analysis of the normalized radiance line profiles of both CLI and RLI images showed a similar pattern, however the absolute radiance of the RLI signal is several order of magnitude higher with respect to CLI. We conclude that optical imaging with CLI and RLI can be considered a novel method to detect and image activation areas in irradiated samples from a medical cyclotron.

Lumbrokinase for degradation and reduction of amyloid fibrils associated with amyloidosis

Amyloidosis is a group of diseases caused by the accumulation of insoluble protein aggregates in different parts of the body. Repeated subcutaneous injection of insulin hormones in diabetic patients leads to localized amyloidosis that is found to be cytotoxic. Thus, agents that can dissociate these aggregates are critically needed. In the present study, insulin amyloid dissociation was demonstrated by the treatment of an enzyme lumbrokinase (LK) isolated from earthworm. Thioflavin T (ThT) fluorescence, solution turbidity, particle size analysis, FTIR, CD, atomic force microscopy and cell viability assay were employed to support the dissociation of insulin amyloid in vitro. The small animal optical imaging was used to explore the dissociation of amyloid fibrils in vivo using zebrafish model. The activity of LK towards amyloid dissociation was compared with the standard amyloid fibril degrading agent nattokinase (NK). Our results indicated that LK can be a probable fibril degrading agent for the dissociation of amyloids.

InP/ZnSe/ZnS quantum dots with strong dual emissions: visible excitonic emission and near-infrared surface defect emission and their application in in vitro and in vivo bioimaging.

Quantum dots (QDs) exhibit many unique optical properties, and show great promise as fluorescent markers in molecular, cellular and in vivo imaging. In this thematic issue, a major concern is their cytotoxicity. Among various Cd-free alternatives, InP-based QDs without highly toxic heavy metal elements have received the most attention. This article first focuses on the synthetic control of oil-soluble InP/ZnSe/ZnS QDs, exhibiting strong dual emissions, namely, visible excitonic emission and near-infrared (NIR) surface defect emission. Next, the organic-to-aqueous phase transfer of the dual emissive QDs was explored systematically. It was found that the dual emissions are relatively stable against the water transfer strategies used here; among them, aqueous dual emissive QDs obtained by wrapping oil-soluble QDs with a poly(acrylic acid)-octylamine (PAA-based) amphiphilic polymer (or modified with the cRGD peptide) exhibit enhanced NIR emission. Finally, using in vitro cell and in vivo small animal optical imaging techniques, the bioactivities of the cRGD-modified amphiphilic polymer-wrapped QDs were also investigated. The results confirm that single-wavelength excitation with strong dual emissions ranging from 550 to >1000 nm will endow the InP-based QDs with the capability for biomedical optical imaging across different spatial scales, as a promising alternative for Cd- and Pb-based QDs.

Persistent luminescence induced by near infra-red photostimulation in chromium-doped zinc gallate for in vivo optical imaging

The analysis of the optical spectroscopy of the Cr3+ doped spinel was initiated by Prof. Georges Boulon more than twenty years ago. More recently persistent luminescence nanoparticles of Cr doped zinc gallate have found interest for in vivo imaging of small animals. Here we evaluated near infra-red (NIR) excitation (or NIR photostimulation) via photo-transfer mechanism as an additional tool for in vivo optical imaging. Investigation of the persistent luminescence induced by NIR photostimulation is studied after either a primary UV (band-to-band excitation) or visible irradiation (direct Cr 3d-3d excitation). UV or visible pre-excited ZnGa2O4:Cr (ZGO:Cr) nanoparticles are kept active during several days thanks to deep traps (with depths 1 eV–1.2 eV) observed in these samples which can be probed through thermally stimulated luminescence (TSL) technique showing glow curve maximums at 470 K and 530 K upon visible light excitation. These deep traps are stable at room temperature but can be emptied by NIR light photostimulation. Experiments were carried out to study the photostimulation induced trapping-detrapping in the ZGO:Cr phosphor. Photostimulation was also tested in vivo for small animal optical imaging to offer new perspectives and modalities.

Overcoming Instability of Antibody‐Nanomaterial Conjugates: Next Generation Targeted Nanomedicines Using Bispecific Antibodies

Targeted nanomaterials promise improved therapeutic efficacy, however their application in nanomedicine is limited due to complexities associated with protein conjugations to synthetic nanocarriers. A facile method to generate actively targeted nanomaterials is developed and exemplified using polyethylene glycol (PEG)-functional nanostructures coupled to a bispecific antibody (BsAb) with dual specificity for methoxy PEG (mPEG) epitopes and cancer targets such as epidermal growth factor receptor (EGFR). The EGFR-mPEG BsAb binds with high affinity to recombinant EGFR (KD : 1 × 10(-9) m) and hyperbranched polymer (HBP) consisting of mPEG (KD : 10 × 10(-9) m) and demonstrates higher avidity for HBP compared to linear mPEG. The binding of BsAb-HBP bioconjugate to EGFR on MDA-MB-468 cancer cells is investigated in vitro using a fluorescently labeled polymer, and in in vivo xenograft models by small animal optical imaging. The antibody-targeted nanostructures show improved accumulation in tumor cells compared to non-targeted nanomaterials. This demonstrates a facile approach for tuning targeting ligand density on nanomaterials, by modulating surface functionality. Antibody fragments are tethered to the nanomaterial through simple mixing prior to administration to animals, overcoming the extensive procedures encountered for developing targeted nanomedicines.

Bremsstrahlung radiation detection for small animal imaging using a CCD detector

The use of optical methods for the detection of radionuclides is becoming an established tool for preclinical molecular imaging experiments. In this paper we present a set of proof of principle experiments showing that planar bremsstrahlung radiation images can be detected with an intensifying screen using a small animal optical imager based on charge coupled device detector.We develop a bremsstrahlung source using a 32P-ATP vial placed in a Plexiglas box, the source with an intensifying screen on top was placed inside a small animal optical imaging system. Bremsstrahlung radiation images were produced with the 32P-ATP source only and also with a pair of pliers placed between the source and the screen. We found that the pair of pliers absorption image matches the shape of the object.Spatial resolution measurements were not performed however, the bremsstrahlung image of the pliers show that the resolution is relatively poor due to a large penumbra effect.We conclude that it is possible to produce planar bremsstrahlung images using optical imaging devices.

Fluorescence and Cerenkov luminescence imaging

This review addresses small animal optical imaging (OI) applications in diverse fields of basic research. In the past, OI has proven to be cost- and time-effective, allows real-time imaging as well as high-throughput analysis and does not imply the usage of ionizing radiation (with the exception of Cerenkov imaging applications). Therefore, this technique is widely spread - not only geographically, but also among very different fields of basic research - and is represented by a large body of publications. Originally used in oncology research, OI is nowadays emerging in further areas like inflammation and infectious disease as well as neurology. Besides fluorescent probe-based contrast, the feasibility of Cerenkov luminescence imaging (CLI) has been recently shown in small animals and thus represents a new route for future applications. Thus, this review will focus on examples for OI applications in inflammation, infectious disease, cell tracking as well as neurology, and provides an overview over CLI.

Use of BRET to Study Protein-Protein Interactions In Vitro and In Vivo.

Application of bioluminescence resonance energy transfer (BRET) assay has been of special value in measuring dynamic events such as protein-protein interactions (PPIs) in vitro or in vivo. It was only in the late 1990s the BRET assay using RLuc-YFP was introduced for biological research showing its use in determining interaction of two proteins involved in circadian rhythm. Several inherent attributes such as rapid and fairly sensitive ratiometric measurements, assessment of PPI irrespective of protein location in cellular compartment, and cost-effectiveness consenting to high-throughput assay development make BRET a popular genetic reporter-based assay for PPI studies. In BRET-based screening, within a defined proximity range of 10-100 Å, excited state energy of the luminescence molecule can excite the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using the Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP, or TurboFP as acceptor, have been reported for use.In recent years, BRET-related research has become significantly versatile in the assay format and its applicability by adopting the assay on multiple detection devices such as small-animal optical imaging platform or bioluminescence microscope. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular optical imaging applications based on BRET assays have broadened its scope for screening of pharmacological compounds by unifying in vitro, live cell, and in vivo animal/plant measurement all on one platform. Taking examples from the literature, this chapter contributes to in-depth methodological details on how to perform in vitro and in vivo BRET experiments, and illustrates its advantages as a single-format assay.

Performance investigation of SP3 and diffusion approximation for three-dimensional whole-body optical imaging of small animals.

The third-order simplified harmonic spherical approximation (SP3) and diffusion approximation (DA) equations have been widely used in the three-dimensional (3D) whole-body optical imaging of small animals. With different types of tissues, which were classified by the ratio of µ s'/µ ɑ, the two equations have their own application scopes. However, the classification criterion was blurring and unreasonable, and the scope has not been systematically investigated until now. In this study, a new criterion for classifying tissues was established based on the absolute value of absorption and reduced scattering coefficients. Using the newly defined classification criterion, the performance and applicability of the SP3 and DA equations were evaluated with a series of investigation experiments. Extensive investigation results showed that the SP3 equation exhibited a better performance and wider applicability than the DA one in most of the observed cases, especially in tissues of low-scattering-low-absorption and low-scattering-high-absorption range. For the case of tissues with the high-scattering-low-absorption properties, a similar performance was observed for both the SP3 and the DA equations, in which case the DA was the preferred option for 3D whole-body optical imaging. Results of this study would provide significant reference for the study of hybrid light transport models.

A near-infrared phthalocyanine dye-labeled agent for integrin αvβ6-targeted theranostics of pancreatic cancer

Integrin αvβ6 is widely upregulated in variant malignant cancers but is undetectable in normal organs, making it a promising target for cancer diagnostic imaging and therapy. Using streptavidin-biotin chemistry, we synthesized an integrin αvβ6-targeted near-infrared phthalocyanine dye-labeled agent, termed Dye-SA-B-HK, and investigated whether it could be used for cancer imaging, optical imaging-guided surgery, and phototherapy in pancreatic cancer mouse models. Dye-SA-B-HK specifically bound to integrin αvβ6 in vitro and in vivo with high receptor binding affinity. Using small-animal optical imaging, we detected subcutaneous and orthotopic BxPC-3 human pancreatic cancer xenografts in vivo. Upon optical image-guidance, the orthotopically growing pancreatic cancer lesions could be successfully removed by surgery. Using light irradiation, Dye-SA-B-HK manifested remarkable antitumor effects both in vitro and in vivo. 18F-FDG positron emission tomography (PET) imaging and ex vivo fluorescence staining validated the observed decrease in proliferation of treated tumors by Dye-DA-B-HK phototherapy. Tissue microarray results revealed overexpression of integrin αvβ6 in over 95% cases of human pancreatic cancer, indicating that theranostic application of Dye-DA-B-HK has clear translational potential. Overall, the results of this study demonstrated that integrin αvβ6-specific Dye-SA-B-HK is a promising theranostic agent for the management of pancreatic cancer.

The importance of inversion disorder in the visible light induced persistent luminescence in Cr³⁺ doped AB₂O₄ (A = Zn or Mg and B = Ga or Al).

Cr(3+) doped spinel compounds AB2O4 with A = Zn, Mg and B = Ga, Al exhibit a long, near infrared persistent luminescence when excited with UV or X-rays. In addition, the persistent luminescence of ZnGa2O4, and to a lesser extent MgGa2O4, can also be induced by visible light excitation via (4)A2→(4)T2 transition of Cr(3+), which makes these compounds suitable as biomarkers for in vivo optical imaging of small animals. We correlate this peculiar optical property with the presence of antisite defects, which are present in ZnGa2O4 and MgGa2O4. By using X-ray absorption fine structure (XAFS) spectroscopy, associated with electron paramagnetic resonance (EPR) and optical emission spectroscopy, it is shown that an increase in antisite defects concentration results in a decrease in the Cr-O bond length and the octahedral crystal field energy. A part of the defects occurs in the close environment of Cr(3+) ions, as shown by the increasing strain broadening of EPR and XAFS peaks observed upon increasing antisite disorder. It appears that ZnAl2O4, which exhibits the largest crystal field splitting of Cr(3+) and the smallest antisite disorder, does not show considerable persistent luminescence upon visible light excitation as compared to ZnGa2O4 and MgGa2O4. These results highlight the importance of Cr(3+) ions with neighboring antisite defects in the mechanism of persistent luminescence exhibited by Cr(3+) doped AB2O4 spinel compounds.

Design of a multimodal fibers optic system for small animal optical imaging

Small animals optical imaging systems are widely used in pre-clinical research to image in vivo the bio-distribution of light emitting probes using fluorescence or bioluminescence modalities. In this work we presented a set of simulated results of a novel small animal optical imaging module based on a fibers optics matrix, coupled with a position sensitive detector, devoted to acquire bioluminescence and Cerenkov images. Simulations were performed using GEANT 4 code with the GAMOS architecture using the tissue optics plugin. Results showed that it is possible to image a 30 × 30 mm region of interest using a fiber optics array containing 100 optical fibers without compromising the quality of the reconstruction. The number of fibers necessary to cover an adequate portion of a small animal is thus quite modest. This design allows integrating the module with magnetic resonance (MR) in order to acquire optical and MR images at the same time. A detailed model of the mouse anatomy, obtained by segmentation of 3D MRI images, will improve the quality of optical 3D reconstruction.

Optical fluorescent imaging to monitor temporal effects of microbubble-mediated ultrasound therapy

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p <0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors.

Synthesis of Luminescent Near-Infrared AgInS2 Nanocrystals as Optical Probes for In Vivo Applications

Near infrared quantum dots have been receiving great attention as fluorescent optical probes for in vivo imaging applications. In this contribution, we report the synthesis and surface functionalization of cadmium free ternary AgInS2 nanocrystals emitting in the near infrared range for successful in vitro and in vivo bioimaging applications. The FDA approved triblock copolymer Pluronic F127 was used to encapsulate the nanocrystals and made them dispersible in aqueous solution. By employing a whole body small animal optical imaging setup, we were able to use the AgInS2 nanocrystals formulation for passive targeted delivery to the tumor site. The ultra-small crystal size, near-infrared emitting luminescence, and high quantum yield make the AgInS2 nanocrystals an attractive candidate as a biological contrast agent for cancer sensing and imaging.