Publications by authors named "Mikhail Y Berezin"

58 Publications

Antibody Conjugate Assembly on Ultrasound-Confined Microcarrier Particles.

ACS Biomater Sci Eng 2020 Nov 9;6(11):6108-6116. Epub 2020 Oct 9.

Washington University in St. Louis, 1 Brookings Drive, Jubel Hall, Room 203K, St. Louis, Missouri 63130, United States.

Bioconjugates are important next-generation drugs and imaging agents. Assembly of these increasingly complex constructs requires precise control over processing conditions, which is a challenge for conventional manual synthesis. This inadequacy has motivated the pursuit of new approaches for efficient, controlled modification of high-molecular-weight biologics such as proteins, carbohydrates, and nucleic acids. We report a novel, hands-free, semiautomated platform for synthetic manipulation of biomolecules using acoustically responsive microparticles as three-dimensional reaction substrates. The microfluidic reactor incorporates a longitudinal acoustic trap that controls the chemical reactions within a localized acoustic field. Forces generated by this field immobilize the microscale substrates against the continuous flow of participating chemical reagents. Thus, the motion of substrates and reactants is decoupled, enabling exquisite control over multistep reaction conditions and providing high-yield, high-purity products with minimal user input. We demonstrate these capabilities by conjugating clinically relevant antibodies with a small molecule. The on-bead synthesis comprises capture of the antibody, coupling of a fluorescent tag, product purification, and product release. Successful capture and modification of a fluorescently labeled antibody are confirmed via fold increases of 49 and 11 in the green (antibody)- and red (small-molecule dye)-channel median intensities determined using flow cytometry. Antibody conjugates assembled on acoustically responsive, ultrasound-confined microparticles exhibit similar quality and quantity to those prepared manually by a skilled technician.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01162DOI Listing
November 2020

Hyaluronan-Conjugated Carbon Quantum Dots for Bioimaging Use.

ACS Appl Mater Interfaces 2021 Jan 23;13(1):277-286. Epub 2020 Dec 23.

Department of Ophthalmology and Visual Sciences, Washington University in St. Louis, St. Louis, Missouri 63110, United States.

This work demonstrates the application of hyaluronan-conjugated nitrogen-doped carbon quantum dots (HA-nCQDs) for bioimaging of tumor cells and illustrates their potential use as carriers in targeted drug delivery. Quantum dots are challenging to deliver with specificity, which hinders their application. To facilitate targeted internalization by cancer cells, hyaluronic acid, a natural ligand of CD44 receptors, was covalently grafted on nCQDs. The HA-nCQD conjugate was synthesized by carbodiimide coupling of the amine moieties on nCQDs and the carboxylic acids on HA chains. Conjugated HA-nCQD retained sufficient fluorescence, although with 30% lower quantum efficiency than the original nCQDs. Confocal microscopy showed enhanced internalization of HA-nCQDs, facilitated by CD44 receptors. To demonstrate the specificity of HA-nCQDs toward human tumor cells, patient-derived breast cancer tissue with high-CD44 expression was implanted in adult mice. The tumors were allowed to grow up to 200-250 mm prior to the injection of HA-nCQDs. With either local or systemic injection, we achieved a high level of tumor specificity judged by a strong signal-to-noise ratio between the tumor and the surrounding tissue . Overall, the results show that HA-nCQDs can be used for imaging of CD44-specific tumors in preclinical models of human cancer and potentially used as carriers for targeted drug delivery into CD44-rich cells.
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http://dx.doi.org/10.1021/acsami.0c20088DOI Listing
January 2021

Hyperspectral imaging and characterization of allergic contact dermatitis in the short-wave infrared.

J Biophotonics 2020 Sep 18;13(9):e202000040. Epub 2020 Jun 18.

Department of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA.

Short-wave infrared hyperspectral imaging is applied to diagnose and monitor a case of allergic contact dermatitis (ACD) due to poison ivy exposure in one subject. This approach directly demonstrates increased tissue fluid content in ACD lesional skin with a spectral signature that matches the spectral signature of intradermally injected normal saline. The best contrast between the affected and unaffected skin is achieved through a selection of specific wavelengths at 1070, 1340 and 1605 nm and combining them in a pseudo-red-green-blue color space. An image derived from these wavelengths normalized to unaffected skin defines a "tissue fluid index" that may aid in the quantitative diagnosis and monitoring of ACD. Further clinical testing of this promising approach towards disease detection and monitoring with tissue fluid content quantification is warranted.
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http://dx.doi.org/10.1002/jbio.202000040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549435PMC
September 2020

New in vitro highly cytotoxic platinum and palladium cyanoximates with minimal side effects in vivo.

J Inorg Biochem 2020 07 6;208:111082. Epub 2020 May 6.

Department of Chemistry, Temple Hall 431, Missouri State University, 901 S. National, Springfield, MO 65897, USA. Electronic address:

Several biologically active bivalent Pd and Pt complexes with two structurally similar cyanoxime ligands abbreviated as H(DECO): 2-oximino-2-cyano-N,N'-diethylacetamide, and H(PyrCO): 2-oximino-2-cyan-N-pyrrolidine acetamide were synthesized and characterized using spectroscopic methods, thermal analysis and X-ray crystallography. Structures revealed planar cis-geometry of studied complexes. Freshly obtained Pt(DECO), Pd(DECO), Pt(PyrCO) and Pd(PyrCO) complexes were used in for in vitro cytotoxicity assays using two different etiology human cancer cell lines HeLa and WiDr cells. Investigated compounds showed cytotoxicity levels at or above cisplatin. Pt(DECO) was also tested in vivo in healthy C57BL/6 mice. The complex was administered at three different dosage (0, 7.5, 15 mg/kg, i.p. once/week), over a total period of 8 weeks. No changes were observed in the animal weight in the treated mice compared to the control dextrose-treated group. The levels of erythrocytes, leukocytes, and hemoglobin were within the normal level suggesting low myelotoxicity. Negligible cardiotoxicity was observed from the histological evaluation of the hearts from the treated animals. Results from the tail nerve conduction velocity (NCV) and nerve histomorphometry suggested no impact of Pt(DECO) on peripheral nerves. The complex, however, induced certain hepatotoxicity and lead to the elevation of IL-6, a pro-inflammatory cytokine. Overall, Pt(DECO)2 showed minimal in vivo toxicity, thus presenting a promising candidate for future testing in animal models of cancer.
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http://dx.doi.org/10.1016/j.jinorgbio.2020.111082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518941PMC
July 2020

Using Xenopus oocytes in neurological disease drug discovery.

Expert Opin Drug Discov 2020 01 1;15(1):39-52. Epub 2019 Nov 1.

Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA.

: Neurological diseases present a difficult challenge in drug discovery. Many of the current treatments have limited efficiency or result in a variety of debilitating side effects. The search of new therapies is of a paramount importance, since the number of patients that require a better treatment is growing rapidly. As an model, oocytes provide the drug developer with many distinct advantages, including size, durability, and efficiency in exogenous protein expression. However, there is an increasing need to refine the recent breakthroughs.: This review covers the usage and recent advancements of oocytes for drug discovery in neurological diseases from expression and functional measurement techniques to current applications in Alzheimer's disease, painful neuropathies, and amyotrophic lateral sclerosis (ALS). The existing limitations of oocytes in drug discovery are also discussed.: With the rise of aging population and neurological disorders, oocytes, will continue to play an important role in understanding the mechanism of the disease, identification and validation of novel molecular targets, and drug screening, providing high-quality data despite the technical limitations. With further advances in oocytes-related techniques toward an accurate modeling of the disease, the diagnostics and treatment of neuropathologies will be becoming increasing personalized.
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http://dx.doi.org/10.1080/17460441.2020.1682993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893134PMC
January 2020

Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes.

Methods Appl Fluoresc 2019 Nov 13;8(1):015001. Epub 2019 Nov 13.

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, United States of America.

The oocytes from Xenopus laevis are well known for their polarity, presenting a distinct animal and vegetal pole. Other heterogeneities are less known. To study the heterogeneity of the Xenopus oocyte, we expressed eGFP and analyzed the protein distribution with fluorescence lifetime microscopy. The vegetal pole exhibited higher levels of fluorescence, than the animal pole. However, the fluorescence lifetimes between the two areas were indistinguishable, suggesting similar environments. In contrast, we observed a substantial and gradual decrease in the fluorescence lifetime from 2.9 ns to 2.6 ns as slices approached the periphery. This has an important implication for future oocyte studies as it demonstrates the environment inside the oocyte is not uniform and might affect the fluorescence intensity. As a result, it cannot be assumed that the observed fluorescence intensity reflects the expression of the proteins but might reflect the environment within the oocyte.
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http://dx.doi.org/10.1088/2050-6120/ab51f8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584348PMC
November 2019

Imaging in the repair of peripheral nerve injury.

Nanomedicine (Lond) 2019 10 15;14(20):2659-2677. Epub 2019 Oct 15.

Department of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA.

Surgical intervention followed by physical therapy remains the major way to repair damaged nerves and restore function. Imaging constitutes promising, yet underutilized, approaches to improve surgical and postoperative techniques. Dedicated methods for imaging nerve regeneration will potentially provide surgical guidance, enable recovery monitoring and postrepair intervention, elucidate failure mechanisms and optimize preclinical procedures. Herein, we present an outline of promising innovations in imaging-based tracking of peripheral nerve regeneration. We emphasize optical imaging because of its cost, versatility, relatively low toxicity and sensitivity. We discuss the use of targeted probes and contrast agents (small molecules and nanoparticles) to facilitate nerve regeneration imaging and the engineering of grafts that could be used to track nerve repair. We also discuss how new imaging methods might overcome the most significant challenges in nerve injury treatment.
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http://dx.doi.org/10.2217/nnm-2019-0115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886568PMC
October 2019

Perfusion-based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near-infrared molecular probes.

J Biophotonics 2018 04 3;11(4):e201700232. Epub 2018 Jan 3.

Optical Radiology Laboratory, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri.

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near-infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor-selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor-to-background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real-time intraoperative procedures.
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http://dx.doi.org/10.1002/jbio.201700232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903995PMC
April 2018

Fluorescence lifetime imaging ophthalmoscopy.

Prog Retin Eye Res 2017 09 30;60:120-143. Epub 2017 Jun 30.

Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, University of Bern, Switzerland. Electronic address:

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review.
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http://dx.doi.org/10.1016/j.preteyeres.2017.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396320PMC
September 2017

Cell-free measurements of brightness of fluorescently labeled antibodies.

Sci Rep 2017 02 2;7:41819. Epub 2017 Feb 2.

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures.
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http://dx.doi.org/10.1038/srep41819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288790PMC
February 2017

Penetration depth of photons in biological tissues from hyperspectral imaging in shortwave infrared in transmission and reflection geometries.

J Biomed Opt 2016 12;21(12):126006

Washington University School of Medicine, Department of Radiology, St. Louis, Missouri, United States.

Measurement of photon penetration in biological tissues is a central theme in optical imaging. A great number of endogenous tissue factors such as absorption, scattering, and anisotropy affect the path of photons in tissue, making it difficult to predict the penetration depth at different wavelengths. Traditional studies evaluating photon penetration at different wavelengths are focused on tissue spectroscopy that does not take into account the heterogeneity within the sample. This is especially critical in shortwave infrared where the individual vibration-based absorption properties of the tissue molecules are affected by nearby tissue components. We have explored the depth penetration in biological tissues from 900 to 1650 nm using Monte–Carlo simulation and a hyperspectral imaging system with Michelson spatial contrast as a metric of light penetration. Chromatic aberration-free hyperspectral images in transmission and reflection geometries were collected with a spectral resolution of 5.27 nm and a total acquisition time of 3 min. Relatively short recording time minimized artifacts from sample drying. Results from both transmission and reflection geometries consistently revealed that the highest spatial contrast in the wavelength range for deep tissue lies within 1300 to 1375 nm; however, in heavily pigmented tissue such as the liver, the range 1550 to 1600 nm is also prominent.
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http://dx.doi.org/10.1117/1.JBO.21.12.126006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147011PMC
December 2016

Imaging of radicals following injury or acute stress in peripheral nerves with activatable fluorescent probes.

Free Radic Biol Med 2016 12 28;101:85-92. Epub 2016 Sep 28.

Division of Radiation Sciences, Department of Radiology, Washington University School of Medicine, 4515 McKinley Avenue, St. Louis, MO 63110, USA. Electronic address:

Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154790PMC
December 2016

Temperature-dependent shape-responsive fluorescent nanospheres for image-guided drug delivery.

J Mater Chem C Mater 2016 Apr 19;4(14):3028-3035. Epub 2016 Feb 19.

Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA.

Temperature-responsive nanoparticles used in conjunction with hyperthermia promise to provide synergistic effects for increasing drug efficacy. We propose a near-infared (NIR) fluorescent system based on a upper critical solution temperature (UCST) polymer, ISP2, integrated with a NIR fluorescent dye HITC for in vivo tracking. The system forms a nanoparticle that increases its volume as temperature increases, similar to the expansion of a Hoberman sphere. The nanospheres nearly doubled in size, from 80 nm to 140 nm, during a temperature increase from 40°C to 60°C.
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http://dx.doi.org/10.1039/C6TC00122JDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464001PMC
April 2016

Highly sensitive image-derived indices of water-stressed plants using hyperspectral imaging in SWIR and histogram analysis.

Sci Rep 2015 Nov 4;5:15919. Epub 2015 Nov 4.

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110.

The optical signature of leaves is an important monitoring and predictive parameter for a variety of biotic and abiotic stresses, including drought. Such signatures derived from spectroscopic measurements provide vegetation indices - a quantitative method for assessing plant health. However, the commonly used metrics suffer from low sensitivity. Relatively small changes in water content in moderately stressed plants demand high-contrast imaging to distinguish affected plants. We present a new approach in deriving sensitive indices using hyperspectral imaging in a short-wave infrared range from 800 nm to 1600 nm. Our method, based on high spectral resolution (1.56 nm) instrumentation and image processing algorithms (quantitative histogram analysis), enables us to distinguish a moderate water stress equivalent of 20% relative water content (RWC). The identified image-derived indices 15XX nm/14XX nm (i.e. 1529 nm/1416 nm) were superior to common vegetation indices, such as WBI, MSI, and NDWI, with significantly better sensitivity, enabling early diagnostics of plant health.
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http://dx.doi.org/10.1038/srep15919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632122PMC
November 2015

Nanothermometry: From Microscopy to Thermal Treatments.

Chemphyschem 2016 Jan 3;17(1):27-36. Epub 2015 Nov 3.

Department of Radiology, Washington University School of Medicine, 510 S. Kingshighway, St. Louis, MO, 63110, USA.

Measuring temperature in cells and tissues remotely, with sufficient sensitivity, and in real time presents a new paradigm in engineering, chemistry and biology. Traditional sensors, such as contact thermometers, thermocouples, and electrodes, are too large to measure the temperature with subcellular resolution and are too invasive to measure the temperature in deep tissue. The new challenge requires novel approaches in designing biocompatible temperature sensors-nanothermometers-and innovative techniques for their measurements. In the last two decades, a variety of nanothermometers whose response reflected the thermal environment within a physiological temperature range have been identified as potential sensors. This review covers the principles and aspects of nanothermometer design driven by two emerging areas: single-cell thermogenesis and image guided thermal treatments. The review highlights the current trends in nanothermometry illustrated with recent representative examples.
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http://dx.doi.org/10.1002/cphc.201500753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396319PMC
January 2016

Fluorescence anisotropy (polarization): from drug screening to precision medicine.

Expert Opin Drug Discov 2015 3;10(11):1145-61. Epub 2015 Aug 3.

a 1 Washington University School of Medicine, Department of Radiology , St. Louis 63110, USA.

Introduction: Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format.

Areas Covered: This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery.

Expert Opinion: Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine.
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http://dx.doi.org/10.1517/17460441.2015.1075001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871695PMC
May 2016

Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

Cytometry A 2015 May 23;87(5):419-27. Epub 2015 Mar 23.

Department of Radiology, Washington University School of Medicine, St. Louis, Missouri.

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.
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http://dx.doi.org/10.1002/cyto.a.22658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860906PMC
May 2015

Fluorescence lifetime for studying ophthalmic diseases in animal models.

Invest Ophthalmol Vis Sci 2014 Nov 11;55(11):7216. Epub 2014 Nov 11.

Department of Radiology, Washington University School of Medicine, St. Louis, Missouri, United States;

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http://dx.doi.org/10.1167/iovs.14-15769DOI Listing
November 2014

Multispectral measurement of contrast in tissue-mimicking phantoms in near-infrared spectral range of 650 to 1600 nm.

J Biomed Opt 2014 Aug;19(8):086008

In order to identify the optimal imaging conditions for the highest spatial contrast in biological tissue, we explored the properties of a tissue-mimicking phantom as a function of the wavelengths in a broad range of near-infrared spectra (650 to 1600 nm). Our customized multispectral hardware, which featured a scanning transmission microscope and imaging spectrographs equipped with silicon and InGaAs charge-coupled diode array detectors, allowed for direct comparison of the Michelson contrast obtained from a phantom composed of a honeycomb grid, Intralipid, and India ink. The measured contrast depended on the size of the grid, luminance, and the wavelength of measurements. We demonstrated that at low thickness of the phantom, a reasonable contrast of the objects can be achieved at any wavelength between 700 and 1400 nm and between 1500 and 1600 nm. At larger thicknesses, such contrast can be achieved mostly between 1200 and 1350 nm. These results suggest that distinguishing biological features in deep tissue and developing contrast agents for in vivo may benefit from imaging in this spectral range.
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http://dx.doi.org/10.1117/1.JBO.19.8.086008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407673PMC
August 2014

Design of fluorescent nanocapsules as ratiometric nanothermometers.

Chemistry 2014 Aug 13;20(33):10292-7. Epub 2014 Jul 13.

Department of Radiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (USA), Fax: (+1) 314-747-5191.

We have developed a novel design of optical nanothermometers that can measure the surrounding temperature in the range of 20-85 °C. The nanothermometers comprise two organic fluorophores encapsulated in a crosslinked polymethacrylate nanoshell. The role of the nanocapsule shell around the fluorophores is to form a well-defined and stable microenvironment to prevent other factors besides temperature from affecting the dyes' fluorescence. The two fluorophores feature different temperature-dependent emission profiles; a fluorophore with relatively insensitive fluorescence (rhodamine 640) serves as a reference whereas a sensitive fluorophore (indocyanine green) serves as a sensor. The sensitivity of the nanothermometers depends on the type of nanocapsule-forming lipid and is affected by the phase transition temperature. Both the fluorescence intensity and the fluorescence lifetime can be utilized to measure the temperature.
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http://dx.doi.org/10.1002/chem.201402828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477229PMC
August 2014

Minimization of self-quenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores.

Contrast Media Mol Imaging 2014 Sep-Oct;9(5):355-62. Epub 2014 Apr 25.

Department of Radiology, Washington University School of Medicine in St Louis, St Louis, MO, 63110, USA.

Self-aggregation of dyes even at low concentrations poses a considerable challenge in preparing sufficiently bright molecular probes for in vivo imaging, particularly in the conjugation of near infrared cyanine dyes to polypeptides with multiple labeling sites. Such self-aggregation leads to a significant energy transfer between the dyes, resulting in severe quenching and low brightness of the targeted probe. To address this problem, we designed a novel type of dye with an asymmetrical distribution of charge. Asymmetrical distribution prevents the chromophores from π-stacking thus minimizing the energy transfer and fluorescence quenching. The conjugation of the dye to polypeptides showed only a small presence of an H-aggregate band in the absorption spectra and, hence, a relatively high quantum efficiency.
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http://dx.doi.org/10.1002/cmmi.1585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198576PMC
July 2015

Central memory CD8+ T lymphocytes mediate lung allograft acceptance.

J Clin Invest 2014 Mar 24;124(3):1130-43. Epub 2014 Feb 24.

Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade-mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory CD8+ T cells (CD44(hi)CD62L(hi)CCR7+). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on CD8+ T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for CD8+ central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung.
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http://dx.doi.org/10.1172/JCI71359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938255PMC
March 2014

Synthesis of nitric oxide probes with fluorescence lifetime sensitivity.

Org Biomol Chem 2013 Dec 28;11(47):8228-34. Epub 2013 Oct 28.

Department of Radiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA.

We present the rationale, synthesis and evaluation of the first activatable fluorescent probe that utilizes fluorescence lifetime change for detection of nitric oxide. The new probe DAP-LT1 features a near-infrared polymethine skeleton with a diaminobenzene functionality incorporated into the meso-position. The probe is partially quenched, and upon reaction with nitric oxide shows an increase in the fluorescence lifetime from 1.08 ns to 1.24 ns.
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http://dx.doi.org/10.1039/c3ob41498aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3872128PMC
December 2013

Multispectral imaging in the extended near-infrared window based on endogenous chromophores.

J Biomed Opt 2013 Oct;18(10):101318

Washington University School of Medicine, Department of Radiology, St. Louis, Missouri 63110, USA.

To minimize the problem with scattering in deep tissues while increasing the penetration depth, we explored the feasibility of imaging in the relatively unexplored extended near infrared (exNIR) spectral region at 900 to 1400 nm with endogenous chromophores. This region, also known as the second NIR window, is weakly dominated by absorption from water and lipids and is free from other endogenous chromophores with virtually no autofluorescence. To demonstrate the applicability of the exNIR for bioimaging, we analyzed the optical properties of individual components and biological tissues using an InGaAs spectrophotometer and a multispectral InGaAs scanning imager featuring transmission geometry. Based on the differences in spectral properties of tissues, we utilized ratiometric approaches to extract spectral characteristics from the acquired three-dimensional "datacube". The obtained images of an exNIR transmission through a mouse head revealed sufficient details consistent with anatomical structures.
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http://dx.doi.org/10.1117/1.JBO.18.10.101318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739874PMC
October 2013

Application of time-resolved fluorescence for direct and continuous probing of release from polymeric delivery vehicles.

J Control Release 2013 Nov 20;171(3):308-14. Epub 2013 Jun 20.

School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, 92093, USA.

Though accurately evaluating the kinetics of release is critical for validating newly designed therapeutic carriers for in vivo applications, few methods yet exist for release measurement in real time and without the need for any sample preparation. Many of the current approaches (e.g. chromatographic methods, absorption spectroscopy, or NMR spectroscopy) rely on isolation of the released material from the loaded vehicles, which require additional sample purification and can lead to loss of accuracy when probing fast kinetics of release. In this study we describe the use of time-resolved fluorescence for in situ monitoring of small molecule release kinetics from biodegradable polymeric drug delivery systems. This method relies on the observation that fluorescent reporters being released from polymeric drug delivery systems possess distinct excited-state lifetime components, reflecting their different environments in the particle suspensions, i.e., confined in the polymer matrices or free in the aqueous environment. These distinct lifetimes enable real-time quantitative mapping of the relative concentrations of dye in each population to obtain precise and accurate temporal information on the release profile of particular carrier/payload combinations. We found that fluorescence lifetime better distinguishes subtle differences in release profiles (e.g. differences associated with dye loading) than conventional steady-state fluorescence measurements, which represent the averaged dye behavior over the entire scan. Given the method's applicability to both hydrophobic and hydrophilic cargo, it could be employed to model the release of any drug-carrier combination.
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http://dx.doi.org/10.1016/j.jconrel.2013.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795917PMC
November 2013

Sensitivity of activatable reactive oxygen species probes by fluorescence spectroelectrochemistry.

Analyst 2013 Aug 4;138(15):4363-9. Epub 2013 Jun 4.

Washington University School of Medicine, Department of Radiology, 510 S. Kingshighway, St. Louis, MO 63110, USA.

We have developed a new analytical method of evaluating activatable fluorescent probes for ROS detection using integrated fluorescence spectroelectrochemistry. The Tafel formalism was applied to describe the process of the probes' oxidation under electrochemical conditions and identify a novel parameter defined as the threshold oxidation potential. This potential can serve as an approximation to the equilibrium potential and can be utilized for determining the sensitivity of a probe to oxidation. Based upon the measured values of threshold potentials, the order of sensitivity towards oxidation among several commonly used probes was determined to be the following (from highest to lowest): 2,7-dihydrodichlorofluorescein > dihydroethidium > dihydrorhodamine 123 > dihydrorhodamine 6G. The presented approach opens up a new direction in synthesizing and screening novel ROS probes with a well-defined sensitivity for in vitro and in vivo applications.
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http://dx.doi.org/10.1039/c3an00459gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722072PMC
August 2013

BLOOD TRIGGERED RAPID RELEASE POROUS NANOCAPSULES.

RSC Adv 2013 Jan;3(16):5547-5555

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110.

Rapid-release drug delivery systems present a new paradigm in emergency care treatments. Such systems combine a long shelf life with the ability to provide a significant dose of the drug to the bloodstream in the shortest period of time. Until now, development of delivery formulations has concentrated on slow release systems to ensure a steady concentration of the drug. To address the need for quick release system, we created hollow polyacrylate nanocapsules with nanometer-thin porous walls. Burst release occurs upon interaction with blood components that leads to escape of the cargo. The likely mechanism of release involves a conformational change of the polymer shell caused by binding albumin. To demonstrate this concept, a near-infrared fluorescent dye indocyanine green (ICG) was incorporated inside the nanocapsules. ICG-loaded nanocapsules demonstrated remarkable shelf life in aqueous buffers with no release of ICG for twelve months. Rapid release of the dye was demonstrated first in vitro using albumin solution and serum. SEM and light scattering analysis demonstrated the retention of the nanocapsule architecture after the release of the dye upon contact with albumin. In vivo studies using fluorescence lifetime imaging confirmed quick discharge of ICG from the nanocapsules following intravenous injection.
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http://dx.doi.org/10.1039/C3RA22693JDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627417PMC
January 2013

Evaluation of inflammatory response to acute ischemia using near-infrared fluorescent reactive oxygen sensors.

Mol Imaging Biol 2013 Aug;15(4):423-30

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Purpose: Ischemia-related processes associated with the generation of inflammatory molecules such as reactive oxygen species (ROS) are difficult to detect at the acute stage before the physiologic and anatomic evidence of tissue damage is present. Evaluation of the inflammatory and healing response early after an ischemic event in vivo will aid in treatment selection and patient outcomes. We introduce a novel near-infrared hydrocyanine molecular probe for the detection of ROS as a marker of tissue response to ischemia and a precursor to angiogenesis and remodeling. The synthesized molecular probe, initially a non-fluorescent hydrocyanine conjugated to polyethylene glycol, converts to a highly fluorescent cyanine reporter upon oxidation.

Procedures: The probe was applied in a preclinical mouse model for myocardial infarction, where ligation and removal of a portion of the femoral artery in the hindlimb resulted in temporary ischemia followed by angiogenesis and healing.

Results: The observed increase in fluorescence intensity was approximately sixfold over 24 h in the ischemic tissue relative to the uninjured control limb and was attributed to the higher concentration of ROS in the ischemic tissue.

Conclusions: These results demonstrate the potential for non-invasive sensing for interrogating the inflammatory and healing response in ischemic tissue.
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http://dx.doi.org/10.1007/s11307-013-0614-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940059PMC
August 2013

Design of irreversible optical nanothermometers for thermal ablations.

Chem Commun (Camb) 2013 Jan;49(7):680-2

Department of Radiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.

Nanothermometers composed from a gold nanorod core, temperature sensitive linker and fluorescent dye are reported. The nanothermometers have low fluorescence due to a self-quenching mechanism at temperatures below 50 °C and become highly fluorescence above 70 °C.
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http://dx.doi.org/10.1039/c2cc37271aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556505PMC
January 2013

Pyrazole-substituted near-infrared cyanine dyes exhibit pH-dependent fluorescence lifetime properties.

Photochem Photobiol 2013 Mar-Apr;89(2):326-31. Epub 2012 Dec 10.

Department of Radiology, Washington University, St. Louis, MO, USA.

Near-infrared heptamethine cyanine dye is functionalized with pyrazole derivatives at the meso-position to induce pH-dependent photophysical properties. The presence of pyrazole unsubstituted at (1) N-position is essential to induce pH-dependent fluorescence intensity and lifetime changes in these dyes. Replacement of meso-chloro group of cyanine dye IR820 with (1) N-unsubstituted pyrazole resulted in the pH-dependent fluorescence lifetime changes from 0.93 ns in neutral media to 1.27 ns in acidic media in DMSO. Time-resolved emission spectra (TRES) revealed that at lower pH, the pyrazole consists of fluorophores with two distinct lifetimes, which cor-responds to pH-sensitive and non-pH-sensitive species. In contrast, (1) N-substituted pyrazoles do not exhibit pH response, suggesting excited state electron transfer as the mechanism of pH-dependent fluorescence lifetime sensitivity for this class of compounds.
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http://dx.doi.org/10.1111/php.12009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578979PMC
November 2013