KEYWORDS: Optical imaging, Arteries, Imaging systems, Image segmentation, In vivo imaging, Green fluorescent protein, Control systems, Luminescence, Ultrasonography, Tissue optics
This study develops a percutaneous optical imaging system for tracking fluorescent reporter gene expression in vasculatures. We build a percutaneous optical imaging system that primarily comprised a 1.5-mm, semi-rigid, two-port optical probe. The performance of the optical probe is first tested in vitro with cell phantoms, and then the feasibility of the percutaneous optical imaging system is validated in vivo in eight femoral artery segments of two pigs. The green fluorescent protein (GFP) gene is locally delivered into four arterial segments, while saline is delivered to the four contralateral arterial segments as controls. The targeted arteries are localized using color Doppler, and thereafter the optical probe is positioned to the target arterial segments under ultrasound guidance. Optical imaging captures are obtained using different exposure times from 10 to 60 s. Subsequently, the GFP- and saline-targeted arteries are harvested for fluorescent microscopy confirmation. The percutaneous optical probe is successfully positioned at a distance approximately 2 mm from the targets in all eight arteries. The in-vivo imaging shows higher average signal intensity in GFP-treated arteries than in saline-treated arteries. This study demonstrates the potential using the percutaneous optical imaging system to monitor, in vivo, reporter gene expression from vasculatures.
We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.
Noninvasive tracking of vascular gene delivery and expression forms an important part of successfully implementing vascular gene therapy methods for the treatment of atherosclerosis and various cardiovascular disorders. While ultrasound and MR imaging have shown promise in the monitoring of gene delivery to the vasculatures, optical imaging has shown promise for tracking gene expression. Optical imaging using bioreporter genes like Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP) and Luciferase to track and localize the therapeutic gene have helped provide an in vivo detection method of the process. The usage of GFP and RFP entails the detection of the fluorescent signal emitted by them on excitation with light of appropriate wavelength. We have developed a novel percutaneous optical imaging system that may be used for in vivo tracking vascular fluorescent gene expression in deep-seated vessels. It is based on the detection of the fluorescent signal emitted from GFP tagged cells. This phantom study was carried out to investigate the performance of the optical imaging system and gain insights into its performance record and study improvisation possibilities.
KEYWORDS: Green fluorescent protein, Signal detection, Optical imaging, Imaging systems, Luminescence, Monte Carlo methods, Gaussian beams, Tissues, Optical tracking, Convolution
Vascular gene therapy is an exciting approach to the treatment of cardiovascular diseases. However, to date, there are no imaging modalities available for non-invasive detection of vascular gene expression. We have developed an optical imaging method to track vascular gene expression by detecting fluorescent signals emitted from arterial walls following gene transfer. To investigate the feasibility of this new technique, we performed experiments on a set of human tissue-like phantoms using a common biological marker in gene therapy, the green fluorescent protein (GFP). The phantoms were constructed to mimic the arterial geometry beneath a tissue layer. Human smooth muscle cells transfected with GFP were embedded in a capillary tube in the phantom. Monte Carlo modeling of the phantom experiment was performed to optimize the performance of the optical imaging system. We compared the fluence rates among three types of light beams, including ring beam, Gaussian beam, and flat beam. The results showed that our optical imaging system was able to detect fluorescent signals up to 5-mm depth in the phantom, and that flat beam geometry would produce the optimum fluorescence remittance. This study provides valuable insights for improvements to the optical imaging system and refinement of the new technique to non-invasively detect/track vascular gene expression.
Three dimensional shape, volume and depth of penetration of a skin lesion are significant factors for early diagnosis and prognosis of melanoma. An optical imaging instrument, Nevoscope is pursued in this work to image and reconstruct pigmented lesions, in three dimensions. The Nevoscope provides a set of planar projections of the pigmented inhomogeneity using transillumination. This paper presents a novel and simple algorithm to reconstruct the volume of the skin lesion from the optical projections acquired using the Nevoscope. The annular ring source of the Nevoscope injects light in the visible spectrum into the skin area surrounding the skin lesion. Light in the visible spectrum undergoes absorption and multiple scattering in the skin. Light photons, which are not extinct, are back scattered and re-emerge carrying information of the structure of the skin-lesion. The transilluminated photons are detected by a set of mirrors functioning as detectors to form 2D projections of the skin-lesion. The multiple-scattering phenomenon renders the inverse problem of solving for the volume of lesion non-linear. A diffusion- theory based approach along with the physics of light propagation in superficial layers of the skin results in a proposition of a hybrid model for solving the forward problem. An iterative non-linear inversion method is pursued to solve the inverse problem. Reconstruction of the lesion volume based on iterative algebraic reconstruction technique involves computation of 'weights' (contribution of a given voxel for a given photon path between a source and a detector) to calculate the forward and inverse solution for every iteration. A previously proposed model computes these weights as a product of two fluences. The first is the fluence calculated at a given voxel due to the annular ring source (forward fluence) and the second is the fluence calculated at the same voxel due to an imaginary point source at the detector (adjoint fluence). A diffusion theory based solution for calculation of the weights results in an under-estimation of the volume. This is because, diffusion theory is not accurate for calculation of fluence very near the source. Conventional X-ray CT like approach over-estimates the volume of reconstruction as it assumes an imaginary light source emanating photons in straight-line paths between source and detector from within the volume of the medium, and ignores scattering. The proposed hybrid method uses both the solutions from a diffusion theory based approach and the X-ray CT like approach to solve the forward and the inverse problems. Milk- gelatin phantoms and a skin mole were used to validate the algorithm. The problem is solved using three different approaches; a straight line X-ray CT like approach, a Diffusion Theory approach and finally the proposed hybrid approach. Quantitative results show the hybrid model reconstructs the phantoms with less error as opposed to the other two.
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