Multiphoton microscopy (MPM) has the capacity to record second-harmonic generation (SHG) and endogenous two-photon excitation fluorescence (2PEF) signals emitted from biological tissues. The development of fiber-based miniaturized endomicroscopes delivering pulses in the femtosecond range will allow the transfer of MPM to clinical endoscopy. We present real-time SHG and 2PEF ex vivo images using an endomicroscope, which totally complies with clinical endoscopy regulations. This system is based on the proximal scanning of a commercial multicore image guide (IG). For understanding the inhomogeneities of the recorded images, we quantitatively characterize the IG at the single-core level during nonlinear excitation. The obtained results suggest that these inhomogeneities originate from the variable core geometries that, therefore, exhibit variable nonlinear and dispersive properties. Finally, we propose a method based on modulation of dispersion precompensation to address the image inhomogeneity issue and, as a proof of concept, we demonstrate its capability to improve the nonlinear image quality.
KEYWORDS: Radiotherapy, Magnetic resonance imaging, Polymers, Radio optics, Tomography, Signal attenuation, 3D image processing, Radon transform, Projection systems, Optical tomography
Recent advances in radiotherapy have created the need to develop novel methods for the accurate, three-dimensional
assessment of the applied radiation dose during specific radiotherapy plans. Here we present a
study based on the use of polymer gel dosimeters in combination with a novel Optical Projection Tomography
system, which allows the association of optical properties, namely the attenuation coefficient, to the irradiation
dose. Polymer gel dosimeters are polymerized after X-ray irradiation via free radical production during water
radiolysis resulting to increased optical opacity as well as change of the nuclear magnetic resonance relaxation
times, thus making it possible to study them with both optical and MRI techniques. The optical tomographic
system employs a sensitive CCD camera, a rotation stage allowing full 360 degrees rotation and a homogeneous
white light source transilluminating the samples. This setup allows the calculation of the optical attenuation
coefficient which can then be directly related to the applied radiotherapy dose, as well as the definition of the
surface of the sample in space. The experimental procedure involves the recording of transillumination images
of the polymer samples in steps of 1 degree to get the desired resolution. Data analysis is performed by back
propagating the photons using an inverse Radon transform resulting to the reconstruction of three dimensional
images of the attenuation coefficient or equivalently the dose distribution. The sensitivity and dynamic range
offered by the technique covers the range of radiotherapy doses in modern clinical practice and are compared
with the corresponding achieved with MRI.
KEYWORDS: Luminescence, Green fluorescent protein, Reconstruction algorithms, Tomography, Tissues, In vivo imaging, Optical filters, Control systems, Cameras, Skin
Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo
in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying
the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different
biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral
unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging
modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity
originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated
emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo.
The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of
organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is
highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental
parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to
measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral
unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for
specific applications.
KEYWORDS: Luminescence, Tomography, Fluorescence tomography, Green fluorescent protein, Skin, In vivo imaging, Tissues, Absorption, Signal detection, Reconstruction algorithms
Autofluorescence has been a significant disadvantage when dealing with tomographic imaging of biological samples or
tissue phantoms. Consequently, the accurate removal of autofluorescence signal has been a major concern in
fluorescence tomography. Here we present a study on three-dimensional mapping and removal of autofluorescence from
fluorescence molecular tomography (FMT) data, both for phantoms and small animal in vivo. The technique is based on
the recording of tomographic data in multiple spectral regions with different excitation light and on the application of a
linear unmixing algorithm for targeting multiple fluorescent probes. Two types of measurements are taken, one with the
excitation being in the region of the maximum absorption of the targeted fluorophore and one in a region away from the
maximum. The relative strengths of the different spectra are employed to calculate the signal to be removed from the
tomographic reconstruction. Autofluorescence spectra are recorded using identical reflection geometry as during the
FMT acquisitions allowing for the correct mapping of the autofluorescence signal. Results from phantoms exhibiting
different background autofluorescence strengths are presented and discussed. In this work we have also studied in vivo
fluorescent activity in mice, involving both subcutaneously implanted fluorescent phantoms and b10 transgenic mice.
Optical tomography has been proposed as a promising technique for probing deep in tissue with many medical applications. Recently, the adaptation of fluorescent probes by the radiologists, gave rise to a new imaging tool in the area of molecular imaging. Optical tomography can, provide three-dimensional images of fluorescent concentrations inside living systems of sizes in the order of many cm. Our optical tomographer was based on a technique which is called Fluorescence Molecular Tomography (FMT) and can quantify fluorescent signals in mice. The imaging procedure is performed in a non-contact geometry so that living subjects of arbitrary shapes can be imaged with no fibers attached to them. We have developed a way to reconstruct the 3D surface of the subject and we use theoretical models to account for the propagation of the emerging signal in the free space. The system consists of a rotating sample holder and a CCD camera in combination with a laser-scanning device. An Argon-ion laser is used as the source and different filters are used for the detection of various fluorophores or fluorescing proteins. So far, we have observed of the distribution of GFP expressing T-lymphocytes in-vivo for the study of the function of the immune system in a murine model. Then we investigated the performance of the FMT setup to quantify the different amounts of migrated cells in the different organs by comparing our results with the FACS measurements. Further experiments included the measurement of the variations of the T cell's concentration in-vivo, over time.
A way to determine the depth of an embedded fluorescent object, for example deep-lying tumors marked with a fluorescent probe, is to detect fluorescent light that has propagated through the medium at two different wavelength bands. A ratio can then be calculated between the corresponding intensities. The wavelength regions should be chosen such that there is a difference in the absorption in the medium. This spectral information could be used as a complement in other methods, for example in tomography, due to its straightforward implementation. In this study we have performed phantom measurements to determine the depth of a fluorescent object, filled with fluorophores. The transmission of yellow and red fluorescence was measured and a ratio of yellow to red fluorescence was calculated for several depths in tissue with a thickness of 2 cm. The ratio showed a clear dependence on the depth of the object.
Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. However, most imaging systems currently used in reasearch make use of fiber technology for delivery and detection, which restricts the size ofthe photon collecting arrays leading to insufficient spatial sampling and field of view. To enable large data sets and full 3600angular measurements, we developed a novel imaging system that enables 3D imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry in combination with a 3D surface reconstruction algorithm. The system is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled CCD camera in combination with a fiber coupled scanning device. The accuracy of the system in obtaining the surface reconstruction was measured to be in the order of 1μm.
KEYWORDS: Imaging systems, In vivo imaging, Tomography, 3D modeling, 3D image processing, Luminescence, Green fluorescent protein, Stereoscopy, CCD cameras, Tissues
Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. In this study we present a novel imaging system that enables three dimensional (3D) imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry, in combination with a 3D surface reconstruction algorithm, which is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled Charge Coupled Device (CCD) camera in combination with a fiber coupled laser scanning device. An Argon ion laser is used as the source and different filters are used for the detection of various fluorophores or fluorescing proteins. With this new setup a large measurements dataset can be achieved while the use of inversion models give a high capacity for quantitative 3D reconstruction of fluorochrome distributions as well as high spatial resolution. The system has already been tested in the observation of the distribution of Green Fluorescent Protein (GFP) expressing T-lymphocytes in order to study the function of the immune system in a murine model, which can then be related to the function of the human immune system.
KEYWORDS: Imaging systems, In vivo imaging, Tomography, 3D modeling, 3D metrology, Cameras, Green fluorescent protein, Optical tomography, Tissues, Luminescence
Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. However, most imaging systems currently used in research make use of fiber technology for delivery and detection, which restricts the size of the photon collecting arrays leading to insufficient spatial sampling and field of view. To enable large data sets and full 360o angular measurements, we developed a novel imaging system that enables 3D imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry in combination with a 3D surface reconstruction algorithm. The system consists of a rotating subject holder and a lens coupled Charge Coupled Device (CCD) camera in combination with a fiber coupled laser scanning device. An Argon ion laser is used as the source and different filters are used for the detection of various fluorophores or fluorescing proteins. With this new setup a large measurements dataset can be achieved while the use of inversion models give a high capacity for quantitative 3D reconstruction of fluorochrome distributions as well as high spatial resolution. The system is currently being tested in the observation of the distribution of Green Fluorescent Protein (GFP) expressing T-lymphocytes in order to study the function of the immune system in a murine model.
Optical characterization of biopsies can be used to discern between tissues when performing diffuse optical tomography of the female breast. The theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, focusing the study in the regime of geometries comparable to the scattering mean free path, the diffusion approximation must be rearranged. Here we present several theoretical assumptions in this direction. In order to investigate the validity of the improved theory, experiments were made involving the transmittance of laser light through turbid phantom models. After the validation of our theoretical model, we have managed to derive the optical properties of over 50 excised breast tissue samples.
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