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Detection of magnetic particles in live DBA/2J mouse eyes
Objective: To demonstrate in vivo molecular imaging of the eye using spectral-domain magnetomotive optical coherence tomography (MMOCT).
Methods: A custom built, high speed and high resolution MMOCT was developed for imaging magnetic particle-coupled molecules in living mouse eyes by applying an external dynamic magnetic field gradient during OCT scanning. The magnetomotive signals were tested in vitro by scanning magnetic beads embedded within agarose gel (1.5%) and in vivo in the anterior segment of a mouse eye.
Results: Cross-sectional OCT images of the gel and anterior segment of eye were acquired by regular OCT structural scanning. MMOCT signals were successfully captured in the agarose gel with embedded magnetic beads. The signals were captured in the anterior segment of the mouse eyes after injecting the beads. The signal was overlaid successfully onto the structural OCT image.
Conclusions: We have demonstrated the ability to detect particles injected into the anterior chamber of the mouse eye using MMOCT. This demonstration suggests that MMOCT is effective for future live detection of molecular (protein) targets in various ocular diseases in mouse models.
Biological imaging at the molecular and cellular level is often critical for early detection and diagnosis of diseases. Signals from specific molecules and specific probes can be detected and localized when they exceed a certain threshold above the background.1-4 Light microscopy and histology, which utilize antibodies and stains, have been the standard method for detection of specific molecular protein targets. With the advancement of optical imaging methods such as optical coherence tomography (OCT), detection and localization of specific probes in live animals is now possible.2 Such detection ability could provide a great potential for obtaining morphological and functional information. Such information will be useful for early diagnosis of the diseases in the living tissue. Specific molecular probes targeted to intrinsic pathologic conditions or protein/biomacromolecular markers could be detected and may serve as diagnostic markers. The biodistribution of these probes can be imaged in vivo in relation to an underlying biological process.1,5,6 In contrast, other morphologic/anatomically based imaging modalities, such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, and structural OCT, are limited by their inability to detect diseases until tissue structural changes are present since they only detect the distortion of the structure caused by the disease. Fluorescent markers have been widely used to couple specific cell receptors and bioluminescent probes have been used for optical molecular imaging in small animal models.1,5,6 These probes are capable of detecting proteins that play key roles in diseases. They thereby enable the tracking of pathological development.1,5,6 Other non-fluorescent probes can provide equal benefit by enhancing image contrast. These methods include white light microscopy, reflectance confocal microscopy, and low-coherence, high depth resolution imaging like OCT with contrast enhancement.1,7
The magnetomotive OCT (MMOCT) is an advanced OCT technique that uses synchronized reorientation of nano magnetic beads to enhance the OCT imaging contrast. MMOCT has expanded molecular imaging in the eye, a technique that has not been previously explored. Magnetic beads coupled with antibodies will enable detection of proteins that are uniquely expressed or significantly up-regulated in pathologic conditions. The purpose of this study was to prove the feasibility of the MMOCT technique for molecular imaging of ocular proteins.
Instrumentation and method
Instrumentation: Ultra-high speed spectral domain MMOCT with ultra-high resolution
The prototype of the MMOCT (Figure 1) was based on our previously built high speed and ultra-high resolution spectral domain OCT (SD-OCT) instrument, described in prior studies.8,9 Briefly, a three-module superluminescent diode (SLD) light source (Broadlighter, T840-HP, Superlumdiodes Ltd, Moscow Russia) with a center wavelength of 840 nm and a FWHM (full width at half maximum) bandwidth of 100 nm was used. After passing through a fiber pigtailed isolator, the low coherence light was coupled into a 2-2 3dB fiber coupler (beam splitter/combiner) which splits the light into the reference arm and the sample arm. The sample light was transported to a telecentric optical delivery system. The delivery system consisted of an X-Y galvanometer scanner and the optics. The optics allows the transport of the sample light into the anterior segment of the eye. It also collected the back-reflected sample light. The power of the sample light was lowered to 750 µW by adjusting the source power with a fiber-based pigtail style attenuator to ensure that the light intensity delivered to the eye was safe. In the detection arm, a spectrometer with a line scan CCD camera (Aviiva-M2-CL-2014) was used to detect the combined reference and sample light. The scan depth was measured as 3.1 mm in air and axial resolution was about 3µm in the tissue, assuming a refractive index of ~1.33.
The system was modified with a magnetic coil powered by a regulated DC 24V power supply and with the attachment of a 6-axial stage, where a mouse was held for imaging. To obtain the magnetomotive signal, two images with the magnetic field ON and OFF were acquired. Pixel subtraction (the difference of scattering) was processed for each OCT scan and 2D image display. The magnetic field of 40 gauss within the coil was measured with a gauss meter.
Magnetic field and data acquisition
A flowing current in a coil generates a magnetic field on the coil axis when its length is much larger than its radius. We determined the current generated magnetic field strength on the central axis of the magnetic coil. This enabled us to determine the length of the magnetic coil necessary for the tissue sample or mouse eyes. In this experiment, we have demonstrated a magnetic flux density of about 40 gauss using a 24 V power supply and 50 ohm limiting resistor to the coil. The effective current generated is low at about 0.48 Amp. For the present experiment, separate MMOCT scan measurements have been performed during the current OFF, current ON with positive polarity (+), and current ON with negative polarity (-). The OCT scan control is the same as SD-OCT, except for the synchronization control of the current ON/OFF with the OCT scan. A total of two OCT images were obtained during current OFF and ON. The OCT images obtained during each of the current states have been processed and displayed.
MMOCT verification in vitro and in vivo
To verify the magnetic responses from the magnetic particles, the magnetomotive signals were acquired and compared between water and magnetic beads (Streptavidin Magnetic Beads S1420S, size 1µm; 4mg/mL; New England BioLabs, Ipswich, MA). One drop of each test component was placed on a plastic card and imaged with the MMOCT with the magnetic field OFF and ON (Figure 2A). In addition, the beads were injected into the agarose gel (1.5% Agarose; A9539-250G; Sigma Chemical Co. St. Louis, MO prepared in 400 mM Tris Acetate, 10 mM EDTA) with air for the simulation of the biodistribution of the beads. A solidified Agarose gel was used. The gel was kept on a transparent plastic holder that is standard for casting Agarose gels. MMOCT imaging was conducted. To test low concentration of the magnetic beads for MMOCT detection, the beads were diluted with normal saline (1:4), injected into the agarose gel, and imaged using MMOCT. To verify the magnetic responses in vivo from the magnetic particles, we injected 0.1 µL of the magnetic beads into the anterior segment of a DBA/2J mouse eye,10 followed by MMOCT imaging. Without turning ON the magnetic field, we imaged twice to demonstrate the repeatability of the imaging. MMOCT signals that were acquired when the magnetic field was turned ON.
These procedures were performed following an institutional animal care and use committee (IACUC) approved protocol. For these purposes, mice were anaesthetized using intraperitoneal injection (0.1µL) of ketamine (100mg/kg) and xylazine (9mg/kg). A topical anesthetic (0.5% tetracaine hydrochloride 0.5%) was applied to the selected eye. 0.5-1µL of the beads was injected using a 36G beveled needle (NF36BV-2, World precision instrument, Sarasota, FL) mounted on a 10 µL microsyringe (NanoFil 300329, World precision instrument). An UltraMicroPumpII (UMP2; World precision instrument) was used to control the injection volume. The needle was entered at the limbus and care was taken not to puncture the iris or the lens. As the needle was being withdrawn after the injection, a cotton tip applicator was applied for about 30 seconds to prevent the fluid from leaking out.
The in vitro determination showed the magnetomotive signals on the plastic card (Figure 2), as well as within the agarose gel with enhanced contrast (Figure 3). The signals were detectable with the 1:4 diluted beads in the agarose gel (Figure 4). The distribution of the beads in the agarose gel matched the location shown in the structural OCT image (Figures 2-4).
The entire anterior segment (including the ciliary body) of the mouse eye was also imaged successfully with the system (Figure 5). In the test on the mouse eye, there was no magnetic motion (Figure 6) detected by the MMOCT when the magnetic field was OFF. After turning ON the magnetic field, the signals pointed to the anterior segment angle by showing the responding scattering in addition to other boundaries (Figure 7). The front surface of the cornea showed some signals of the magnetic motion of the beads, possibly due to leakage of the beads during the injection. The anterior chamber was shallow due to leakage of aqueous humor during the injection.
Since OCT detects backscattering from the tissue, any changes in scattering will result in the detection of a difference, which will be used to localize the change in the structure. Biological tissues exhibit no ferromagnetism and only weak magnetic susceptibility. Imaging particles with high magnetic susceptibility and/or ferromagnetic properties has been successfully demonstrated.1,6,11 Iron oxide, such as magnetite, is a good candidate due to its known biocompatibility after polymer coating.12 When a high magnetic field gradient is present, particles with high magnetic susceptibility in tissue are exposed to a gradient force. This causes the ferromagnetic particles to change orientation, aligning their internal magnetization along the field. The rotation and movement of the particles result in the magnetomotion of the particle and perturbation of the surrounding cells and organelles that, in turn, induces the change in the localized scattering. When the magnetic field is removed, the particles in the elastic medium (tissue) return to their original position and orientation. These changes in position and orientation include displacement and rotation.11 When the magnetic field is repetitively switched ON and OFF, the induced magnetomotion causes a change of light scattering that can be captured by OCT. Thus, the differences between the two states can be imaged. The ON and OFF states can be rapidly switched during each A-scan data acquisition, resulting in the detection of the location where the magnetic nano-particles are embedded. The magnetic nano-particles can be coupled to an antibody or cell for targeting.1,6,11 Since OCT also acquires structural information, the magnetomotive signal can be projected (overlaid) onto the structural image.11
MMOCT is a combination of OCT with a synchronized magnetic field control system (control electronics and magnetic coil). MMOCT is the performance of OCT on a tissue that contains magnetic nano-particles that are changing orientation under different externally applied magnetic fields. The changes in orientations of magnetic nano-particles greatly affect the local tissue structure and thus enhance the local light scattering. Effective use of MMOCT can thus enhance 3D tomography image contrast. Rapid reorientation of magnetic nano-particles is achieved with a magnetic coil composed of a spool wound with a thin electrical wire. When the coil is connected to a power supply, the current flow in the coil generates the magnetic field. The greater the coil wiring loop density (number of wires per unit length) and the greater the current, the higher the generated central magnetic field. The central magnetic field direction can be changed by changing the current flow direction. For imaging, the tissue sample or mouse containing the magneto nano-particles needs to be placed near the center of the coil. The nano magnetic particles range in size from 50 - 500 nm and have minimal scattering to the MMOCT measurement light beam. Light scattering occurs when the particle sizes are close to and/or larger than the measurement light wavelength. With no applied electric field, the MMOCT measurement of the tissue sample with magnetic nano-particles is almost the same as the conventional SD-OCT measurement with no contrast enhancement. When a DC current is applied to the vertical magnetic coil, it generates a strong magnetic field pointing in a certain direction. Magnetic nano-particles react to the externally applied magnetic field by aligning in the same magnetic field direction. During the nano-particle reorientation and near-by nano-particle interactions, local larger scattering centers are formed through the change of local material (tissue) densities. This effectively scatters the measurement light beam and increases the MMOCT image contrast. When the current direction is reversed, the magnetic field direction also reverses and causes a reorientation of the magnetic nano-particles. By quickly switching the magnetic field direction and comparing the tomographic images obtained, we can further enhance the image contrast through image subtraction and processing.
This novel approach using MMOCT imaging in the mouse eye will tremendously widen the research applications of the OCT technique. By coupling magnetic beads to antibodies in future studies it could significantly advance our capability to image specific molecules in the eye and study diseases at the molecular level. These diseases include glaucoma, dry eye, multiple sclerosis (MS), and other diseases with ocular manifestation. Currently, mouse models are available for several of these diseases.13-16 Development of this novel technology will have a high impact on these studies. With the implementation of the MMOCT, the proteins or other biological macromolecules can be detected in live eyes, and their rate of generation and degradation may possibly be determined. Such detection in live tissues/eyes will provide insight into the mechanisms at the molecular level involving biomacromolecules. For instance, it will enable the detection of correlations among specific proteins and stages of disease and also therapeutic outcome for a given protein target at a given dose of inhibitor or activator. Fundamentally, the MMOCT will lead to the development of new approaches to the diagnosis and treatment of ocular abnormalities.
It appeared that the MMOCT signal was weaker in the agarose gel that on the plastic card. This may be due to the attention of the OCT light since the agarose appeared not totally transparent, as shown in Figures 3 and 4. We also noted that the anterior chamber after injection was shallow and this may be avoided by using a viscoleastic substance mixed with the beads to maintain a deep anterior chamber. This may make the MMOCT imaging easier as we may acquire the same quality image as shown in the pre-injection figure. This demonstration did not look at the specific targeting. The injected beads were not found specifically distributed in the anterior segment. In future studies, specific targeting may be used to locate proteins in the anterior segment and other parts of the eye. Further work in this field will be focused on the improvement of scanning speed and sensitivity. Using phase-resolved detection of magnetic nanoparticle displacements, Oldenburg et al. reported that the sensitivity was improved by a factor of 15 and speed was more than 10 times faster.3
In the present study, we have proven that it is feasible to use MMOCT to detect magnetic particles injected into the DBA/2J mouse eye. This demonstration suggests that MMOCT is effective for future live detection of molecular (protein) targets in various ocular diseases in mouse models.
Figure 1: Schematic of the MMOCT system. A broad superluminescent diode was connected to a fiber coupler, which was connected to sample and reference arms. The spectrometer was used to detect reflected light from both arms. Data acquisition was done on a computer with custom developed software. During the scanning, the X-Y cross aiming will be used for proper alignment with the aid of a video camera and the magnetic signal in 2D is displayed (shown in the inset). An electric magnetic coil is powered by a DC power supply to generate magnetic field, which is switched ON and OFF during imaging the mouse. SLD - superluminescent diode, OI - optical isolator, FC: 50:50 fiber coupler, PC - polarization controller, NDF - neutral density filter, DC - dispersion compensator, L1 to L6 - lenses, M - silver mirror, HDG - holographic volume diffraction grating.
Figure 2: OCT imaging water and magnetic particles. One drop of water (about 50 µl, marked in green bracket) was instilled onto a plastic card surface along with another drop (about 50 µl, marked in red bracket) of the magnetic particles. The card was placed on a magnetic coil. OCT linear scan (green line on Image A) was performed. OCT imaging was repeated during the magnetic modulation OFF (Image B) and ON (Image C). Magnetomotive signal in M-model (D) showed only the magnetic particles with great reduction of the background.
Figure 3: Magnetomotive OCT image of magnetic beads in the agar gel. The beads were injected into the gel. Two OCT images were acquired before and after switching on the magnetic modulation. The magnetomotive OCT image showed the signals of the magnetic beads, which matched the distribution of the beads in the structural OCT images.
Figure 4: Magnetomotive OCT image of magnetic beads at 1:4 dilutions. The beads (marked in green bracket) were diluted 1:4 with normal saline and injected into an agarose gel (1.5% agarose in 400 mM Tris Acetate, 10 mM EDTA). Two OCT images were acquired before and after switching on the magnetic modulation. The magnetomotive OCT image showed the boundaries of the magnetic beads.
Figure 5: The cross-sectional view of the anterior segment of the DBA/2J mouse eye. The image (A) was obtained with the ultra-high resolution spectral domain OCT with scan width of 3.5mm and scan depth 2mm. The entire cornea, anterior chamber, iris and the lens were clearly visualized. The image (B) was obtained when the scanning probe was turned to the limbus area of the mouse eye. The image showed the anterior chamber angle, iris and ciliary body clearly.
Figure 6: Magnetomotive OCT image of the DBA/2J mouse eye after injection of the magnetic beads. The beads were injected into the anterior chamber of the mouse eye and the anterior chamber was shallow after the injection due to the leak of aqueous humor. Two images (A and B) were captured with magnetic field OFF. The magnetic beads were imaged in the structural OCT images because of their scattering nature in the anterior chamber. Nothing was shown in the magnetomotive OCT image (C).
Figure 7: Magnetomotive OCT image of the DBA/2J mouse eye after injection of the magnetic beads with magnetic field. The first structural OCT image (A) of the mouse anterior segment was captured without magnetic field, and the second image (B) was acquired with the magnetic field. The magnetic beads appeared in the structural OCT images because of their scattering nature in the anterior chamber (A and B). In the magnetomotive OCT image (C), the magnetic beads appeared in the anterior chamber, anterior chamber angle, the back surface of the cornea and front surface of the crystal lens. There were almost none in the posterior chamber (marked as asterisk *) underneath the iris.