KEYWORDS: Tomography, In vivo imaging, Modulation, Signal detection, Scanners, Continuous wave operation, Photography, Absorption, Acoustics, Data acquisition
Frequency domain optoacoustics relates to stimulation of optoacoustic signals using intensity modulated continuous wave light instead of pulsed laser light employed in time domain optoacoustic imaging. We present a method to generate frequency domain tomographic images of optical absorbers and cross sectional in-vivo mouse images, showing the changes of optical absorption before and after injection of indocyanine green (ICG). OCIS codes: 170.6960, 170.3880, 170.5220
The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.
Multi-Spectral Optoacoustic Tomography (MSOT) offers real time imaging that simultaneously exploits high ultrasound
resolutions and strong optical contrast. It allows visualizing absorbers in tissue by using their distinct spectral absorption
profiles. This work presents a non-invasive in vivo study of kinetics involved in the clearance of carboxylated dye in
mouse kidneys. The distinctio
Multispectral optoacoustic (photoacoustic) tomography (MSOT) exploits high resolutions given by ultrasound detection
technology combined with deeply penetrating laser illumination in the near infrared. Traces of molecules with different
spectral absorption profiles, such as blood (oxy- and de-oxygenated) and biomarkers can be recovered using multiple
wavelengths excitation and a set of methods described in this work. Three unmixing methods are examined for their
performance in decomposing images into components in order to locate fluorescent contrast agents in deep tissue in
mice. Following earlier works we find Independent Component Analysis (ICA), which relies on the strong criterion of
statistical independence of components, as the most promising approach, being able to clearly identify concentrations
that other approaches fail to see. The results are verified by cryosectioning and fluorescence imaging.
Multispectral optoacoustic (photoacoustic) tomography (MSOT) exploits the high resolutions provided by ultrasound
imaging technology in combination with the more biologically relevant optical absorption contrast. Traces of molecules
with different spectral absorption profiles, such as blood (oxy- and de-oxygenated) and biomarkers can be recovered
using multiple wavelengths excitation and a set of methods described in this work. Three unmixing methods are
examined for their performance in decomposing images into components in order to locate fluorescent contrast agents in
deep tissue in mice. Following earlier works we find Independent Component Analysis (ICA), which relies on the strong
criterion of statistical independence of components, as the most promising approach, being able to clearly identify
concentrations that other approaches fail to see. The results are verified by cryosectioning and fluorescence imaging.
Early detection of high-risk coronary atherosclerosis remains an unmet clinical challenge. We have
previously demonstrated a near-infrared fluorescence catheter system for two-dimensional intravascular
detection of fluorescence molecular probes [1]. In this work we improve the system performance by
introducing a novel high resolution sensor. The main challenge of the intravascular sensor is to provide a
highly focused spot at an application relevant distance on one hand and a highly efficient collection of
emitted light on the other.
We suggest employing a double cladding optical fiber (DCF) in combination with focusing optics to
provide a sensor with both highly focused excitation light and highly efficient fluorescent light collection.
The excitation laser is coupled into the single mode core of DCF and guided through a focusing element
and a right angle prism. The resulting side-fired beam exhibits a small spot diameter (50 μm) throughout a
distance of up to 2 mm from the sensor. This is the distance of interest for intravascular coronary imaging
application, determined by an average human coronary artery diameter. At the blood vessel wall, an
activatable fluorescence molecular probe is excited in the diseased lesions. Next light of slightly shifted
wavelength emits only in the places of the inflammations, associated with dangerous plaques [2]. The
emitted light is collected by the cladding of the DCF, with a large collection angle (NA=0.4). The doublecladding
acts as multimodal fiber and guides the collected light to the photo detection elements. The
sensor automatically rotates and pulled-back, while each scanned point is mapped according to the
amount of detected fluorescent emission. The resulting map of fluorescence activity helps to associate the
atherosclerotic plaques with the inflammation process. The presented detection system is a valuable tool
in the intravascular plaque detection and can help to differentiate the atherosclerotic plaques based on
their biological activity, identify the ones that prone to rupture and therefore require more medical
attention.
Over the last decade fluorescent reporter technologies (both fluorescent probes and proteins) have become a
very powerful imaging tool in everyday biomedical research. Multispectral optoacoustic tomography (MSOT)
is an emerging imaging technology that can resolve fluorophore concentration in small animals situated in deep
tissue by multispectral acquisition and processing of optoacoustic signals. In this work, we study the optimum
operating conditions of MSOT in imaging fluorescence activity in small animals. The performance of various
fluorochromes / fluorescent proteins is examined and it is shown that the new infrared fluorescent protein is an
order of magnitude brighter than the red ones. Finally, wavelength reduction after principle component analysis
shows, that accurate unmixing and 3D reconstruction of the distribution of fluorochromes is possible only with
2 or 3 wavelengths.
We interrogate the ability of free-space fluorescence tomography to image small animals in vivo using charge-coupled device (CCD) camera measurements over 360-deg noncontact projections. We demonstrate the performance of normalized dual-wavelength measurements that are essential for in-vivo use, as they account for the heterogeneous distribution of photons in tissue. In-vivo imaging is then showcased on mouse lung and brain tumors cross-validated by x-ray microcomputed tomography and histology.
KEYWORDS: Luminescence, Green fluorescent protein, Tissue optics, Tissues, In vivo imaging, Fluorescent proteins, Proteins, Absorption, Signal attenuation, Tumors
The discovery of new fluorescent proteins (FPs) that emit in the far-red part of the spectrum, where light absorption from tissue is significantly lower than in the visible, offers the possibility for noninvasive biological interrogation at the entire organ or small animal level in vivo. The performance of FPs in deep-tissue imaging depends not only on their optical characteristics, but also on the wavelength-dependent tissue absorption and the depth of the fluorescence activity. To determine the optimal choice of FP and illumination wavelength, we compared the performance of five of the most promising FPs: tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding of the performance of different illumination scenarios, especially as they pertain to tomographic imaging. It was found that the appropriate combination of red-shifted proteins and illumination wavelengths can improve detection sensitivity in small animals by at least two orders of magnitude compared with green FP. It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600-nm range may significantly affect the detection sensitivity and, therefore, necessitates the careful selection of illumination wavelengths for optimal imaging performance.
KEYWORDS: Luminescence, Natural surfaces, Fluorescence tomography, Tomography, 3D acquisition, 3D image processing, Tissue optics, In vivo imaging, Reconstruction algorithms, Free space
Complete projection (360°) free-space fluorescence tomography of opaque media is poised to enable highly performing
three-dimensional imaging through entire small animals in-vivo. This approach can lead to a new generation of
Fluorescence Molecular Tomography (FMT) systems since it allows high spatial sampling of photon fields propagating
through tissue at any projection, employing non-constricted animal surfaces.
Key features of this development is the implementation of non-contact illumination, for example by using beam
scanning techniques for light delivery on the tissue surface and direct non-contact imaging with CCD cameras.
Similarly, the development of free-space geometries, i.e. implementations that do not utilize immersion of the animal in
matching fluids are essential for obtaining appropriate experimental simplicity and avoid unnecessary diffusion through
scattering matching media.
To facilitate these developments it is important to retrieve the three-dimensional surface and a common coordinate
system for the illumination system, the detection system and the animal. Herein, we employ a volume carving method to
capture three-dimensional surfaces of diffusive objects from its silhouettes and register the captured surface in the
geometry of an FMT 360°-projection acquisition system to obtain three-dimensional fluorescence image reconstructions.
Using experimental measurements we evaluate the accuracy of the surface capture procedure by reconstructing the
surfaces of phantoms of known dimensions and demonstrate how this surface extraction method can be utilized in an
FMT inversion scheme. We then employ this methodology to characterize the animal movement of anaesthetized
animals and study the effects of animal movement on the FMT reconstructed image quality.
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