Magnetomotive optical coherence microscopy for cell dynamics and biomechanics
Magnetomotive microscopy techniques are introduced to investigate cell dynamics and biomechanics. These techniques are based on magnetomotive transducers present in cells and optical coherence imaging techniques. In this study, magnetomotive transducers include magnetic nanoparticles (MNPs) and fluorescently labeled magnetic microspheres, while the optical coherence imaging techniques include integrated optical coherence (OCM)and multiphoton (MPM) microscopy,and diffraction phase microscopy (DPM). Samples used in this study are murine macrophage cells in culture that were incubated with magnetomotive transducers. MPMis used to visualize multifunctional microspheres based on their fluorescence, while magnetomotive OCM detects sinusoidal displacements of the sample induced by a magnetic field. DPM is used to image single cells at a lower frequency magnetic excitation, and with its Fourier transform light scattering (FTLS) analysis, oscillation amplitude is obtained, indicating the relative biomechanical properties of macrophage cells. These magnetomotive microscopy method shave potential to be used to image and measure cell dynamics and biomechanical properties. The ability to measure and understand biomechanical properties of cells and their microenvironments, especially for tumor cells, is of great importance and may provide insight for diagnostic and subsequently therapeutic interventions.
Targeted multifunctional multimodal protein-shell microspheres as cancer imaging contrast agents
PURPOSE: In this study, protein-shell microspheres filled with a suspension of iron oxide nanoparticles in oil are demonstrated as multimodal contrast agents in magnetic resonance imaging (MRI), magnetomotive optical coherence tomography (MM-OCT), and ultrasound imaging. The development, characterization, and use of multifunctional multimodal microspheres are described for targeted contrast and therapeutic applications.PROCEDURES: A preclinical rat model was used to demonstrate the feasibility of the multimodal multifunctional microspheres as contrast agents in ultrasound, MM-OCT and MRI. Microspheres were functionalized with the RGD peptide ligand, which is targeted to α(v)β₃ integrin receptors that are over-expressed in tumors and atherosclerotic lesions.RESULTS: These microspheres, which contain iron oxide nanoparticles in their cores, can be modulated externally using a magnetic field to create dynamic contrast in MM-OCT. With the presence of iron oxide nanoparticles, these agents also show significant negative T2 contrast in MRI. Using ultrasound B-mode imaging at a frequency of 30 MHz, a marked enhancement of scatter intensity from in vivo rat mammary tumor tissue was observed for these targeted protein microspheres.CONCLUSIONS: Preliminary results demonstrate multimodal contrast-enhanced imaging of these functionalized microsphere agents with MRI, MM-OCT, ultrasound imaging, and fluorescence microscopy, including in vivo tracking of the dynamics of these microspheres in real-time using a high-frequency ultrasound imaging system. These targeted oil-filled protein microspheres with the capacity for high drug-delivery loads offer the potential for local delivery of lipophilic drugs under image guidance.
RGD coated protein microspheres as a dual fluorescent and magnetomotive contrast agent for targeted cancer imaging with magnetomotive optical coherence tomography
Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications ranging from ophthalmology to oncology. OCT is analogous to ultrasound technology but with micron by using light waves instead of sound waves providing detailed morphological or structural information at the cellular level about the tissue specimen. Magneto-motive OCT (MM-OCT) is a recently developed modality of OCT in which a magnetic field is modulated on and off during imaging. With the development of this modality, exogeneous contrast agents are becoming more important to target markers that are expressed prior to morphological changes that structural OCT can only detect. Modified protein microspheres consisting of an oil core and a hydrophilic BSA protein shell are being presented as a multi-modal contrast agent vehicle. The protein microspheres are encapsulated with iron oxide in the oil core to provide the magnetic signal contrast and a near infrared dye to provide a fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Under MM-OCT, these agents have been detected above various levels of background tissue scattering demonstrating that these agents can provide added contrast to OCT through the magnetic signal. These agents were incubated with various cell lines with differing levels of alpha(v)beta(3) integrin receptor expression that were quantified using western blotting and fluorescent antibody immunohistochemical staining. The normal control cell line used was the CRL-4010. The breast cancer cell lines studied included CRL-2314, SK-BR-3, MCF-7, and 13762 MAT B III cells. These studies address the binding specificity and sensitivity of the RGD functionalized protein microspheres to the alpha(v)beta(3) integrin receptors. In addition, a quantitative analysis is being performed to correlate the relative levels of bound microspheres to the cells, measured through MM-OCT measurements and through their fluorescence signals of the microspheres, and the cell’s alpha(v)beta(3) integrin receptor expression derived from the western blot experiments. Preliminary results indicate that these agents have a higher affinity to the cancer cells over the normal epithelial cells and are also internalized by the cells and could have to potential to become localized targeted drug delivery vehicles. In an NMU carcinogen induced rat animal model, the targeted protein microspheres were injected in-vivo. These preliminary results, using a multi-spectral dark box imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor. These microspheres are being presented as a novel contrast agent to a novel high resolution imaging modality targeted at cancer.
Multimodal biomedical imaging with asymmetric single-walled carbon nanotube/iron oxide nanoparticle complexes
Magnetic iron oxide nanoparticles and near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWNT) form heterostructured complexes that can be utilized as multimodal bioimaging agents. Fe catalyst-grown SWNT were individually dispersed in aqueous solution via encapsulation by oligonucleotides with the sequence d(GT)15, and enriched using a 0.5 T magnetic array. The resulting nanotube complexes show distinct NIR fluorescence, Raman scattering, and visible/NIR absorbance features, corresponding to the various nanotube species. AFM and cryo-TEM images show DNA-encapsulated complexes composed of a approximately 3 nm particle attached to a carbon nanotube on one end. X-ray diffraction (XRD) and superconducting quantum interference device (SQUID) measurements reveal that the nanoparticles are primarily Fe2O3 and superparamagnetic. The Fe2O3 particle-enriched nanotube solution has a magnetic particle content of approximately 35 wt %, a magnetization saturation of approximately 56 emu/g, and a magnetic relaxation time scale ratio (T1/T2) of approximately 12. These complexes have a longer spin-spin relaxation time (T2 approximately 164 ms) than typical ferromagnetic particles due to the smaller size of their magnetic component while still retaining SWNT optical signatures. Macrophage cells that engulf the DNA-wrapped complexes were imaged using magnetic resonance imaging (MRI) and NIR mapping, demonstrating that these multifunctional nanostructures could potentially be useful in multimodal biomedical imaging.
PNAS: Color-blind fluorescence detection for four-color DNA sequencing
We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.
Instrumentation for multi-modal spectroscopic diagnosis of epithelial dysplasia
Reflectance and fluorescence spectroscopies have shown great promise for early detection of epithelial dysplasia. We have developed a clinical reflectance spectrofluorimeter for multimodal spectroscopic diagnosis of epithelial dysplasia. This clinical instrument, the FastEEM, collects white light reflectance and fluorescence excitation-emission matrices (EEM’s) within a fraction of a second. In this paper we describe the FastEEM instrumentation, designed for collection of multi-modal spectroscopic data. We illustrate its performance using tissue phantoms with well defined optical properties and biochemicals of known fluorescence properties. In addition, we discuss our plans to develop a system that combines a multi-spectral imaging device for wide area surveillance with this contact probe device.