Mesoscopic epifluorescence tomography is a novel technique that discovers fluorescence bio-distribution in small animals by tomographic means in reflectance geometry. A collimated laser beam is scanned over the skin surface to excite fluorophores hidden within the tissue while a CCD camera acquires an image of the fluorescence emission for each source position. This configuration is highly efficient in the visible spectrum range where trans-illumination imaging of small animals is not feasible due to the high tissue absorption and scattering in biological organisms. The reconstruction algorithm is similar to the one used in fluorescence molecular tomography. However, diffusion theory cannot be employed since the source-detector separation for most image pixels is comparable to or below the scattering length of the tissue. Instead Monte Carlo simulations are utilized to predict the sensitivity functions. In a phantom study we show the effect of using enhanced source grid arrangements during the data acquisition and the reconstruction process to minimize boundary artefacts. Furthermore, we present ex vivo data that show high spatial resolution and quantitative accuracy in heterogeneous tissues using GFP-like fluorescence in B6-albino mice up to a depth of 1100 μm.
The recent development of hybrid imaging scanners that integrate fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) allows the utilization of x-ray information as image priors for improving optical tomography reconstruction. To fully capitalize on this capacity, we consider a framework for the automatic and fast detection of different anatomic structures in murine XCT images. To accurately differentiate between different structures such as bone, lung, and heart, a combination of image processing steps including thresholding, seed growing, and signal detection are found to offer optimal segmentation performance. The algorithm and its utilization in an inverse FMT scheme that uses priors is demonstrated on mouse images.
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 present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.
A novel hybrid imaging system for simultaneous X-ray and Fluorescence Tomography
is presented, capitalizing on 360°-projection free-space fluorescence tomography. The system is
implemented within a commercial micro-CT scanner allowing reconstructions with a resolution of
95μm. Acquired data sets are intrinsically coregistered in the same coordinate system and can be
used to correctly localize reconstructed fluorescence distributions with morphological features.
More importantly, the micro-CT data, automatically segmented into different organ and tissue
segments can be used to guide the fluorescence reconstruction algorithm and reduce the ill coditioning
of the inverse problem. We showcase the use of the system and the improvements in
image quality for lesions in brain and lung.
We describe an improved optoacoustic tomography method, that utilizes a diffusion-based photon propagation model in
order to obtain quantified reconstruction of targets embedded deep in heterogeneous scattering and absorbing tissue. For
the correction we utilize an iterative finite-element solution of the light diffusion equation to build a photon propagation
model. We demonstrate image improvements achieved by this method by using tissue-mimicking phantom
measurements. The particular strength of the method is its ability to achieve quantified reconstructions in non-uniform
illumination configurations resembling whole-body small animal imaging scenarios.
Recently, we have presented a thin optical detector assembly consisting of a microlens array (MLA) coupled to
a large area CMOS sensor through a septum mask. The sensor is placed in the physical focal plane of the MLA.
Each lens of the MLA forms a small image on the sensor surface, with individual images being separated from
each other by the septum mask. The resulting sensor image thus shows a multitude of small sub-images. A
low-resolution image can be attained by extracting only those pixels that are located on the optical axis of a
microlens, as reported previously. Herein we describe an improved post-processing method to extract images of
higher resolution (which can be focused to an arbitrary plane) from a single raw sensor image: Each lens of the
MLA results in a mapping from points in object space to corresponding sensor pixels. By tracing back the light
paths from sensor pixels through the lenses onto an arbitrary focal plane in object space this mapping can be
inverted. Intensities captured on individual sensor pixels can be attributed to virtual pixels on that focal plane
using the computed inverse mapping.
As a result, from a single acquisition by the detector, images focused to any plane in object space can be
calculated. In contrary to the approach of extracting focal point intensities, the spatial resolution is not limited
by microlens pitch. We present experimental examples of extracted images at various object plane distances and
studies determining the spatial resolution.
An optical detector suitable for inclusion in tomographic arrangements for non-contact in vivo bioluminescence
and fluorescence imaging applications is proposed. It consists of a microlens array (MLA) intended for field-of-view definition, a large-field complementary metal-oxide-semiconductor (CMOS) chip for light detection, a septum mask for cross-talk suppression, and an exchangeable filter to block excitation light. Prototype detector
units with sensitive areas of 2.5 cm x 5 cm each were assembled. The CMOS sensor constitutes a 512 x 1024 photodiode matrix at 48 μm pixel pitch. Refractive MLAs with plano-convex lenses of 480 μm in diameter and pitch were selected resulting in a 55 x 105 lens matrix. The CMOS sensor is aligned on the focal plane of
the MLA at 2.15mm distance. To separate individual microlens images an opaque multi-bore septum mask
of 2.1mm in thickness and bore diameters of 400 μm at 480 μm pitch, aligned with the lens pattern, is placed
between MLA and CMOS. Intrinsic spatial detector resolution and sensitivity was evaluated experimentally as a
function of detector-object distance. Due to its small overall dimensions such detectors can be favorably packed
for tomographic imaging (optical diffusion tomography, ODT) yielding complete 2 π field-of-view coverage. We
also present a design study of a device intended to simultaneously image positron labeled substrates (positron
emission tomography, PET) and optical molecular probes in small animals such as mice and rats. It consists of
a cylindrical allocation of optical detector units which form an inner detector ring while PET detector blocks
are mounted in radial extension, those gaining complementary information in a single, intrinsically coregistered
experimental data acquisition study. Finally, in a second design study we propose a method for integrated optical
and magnetic resonance imaging (MRI) which yields in vivo functional/molecular information that is intrinsically
registered with the anatomy of the image object.
Can time-resolved, high-resolution data as acquired by an intensified gated CCD camera (ICCD) aid in the tomographic
reconstruction of fluorescence concentration? Usually it is argued that fluorescence is a linear process and thus does not
require non-linear, time-dependent reconstructions algorithms, unless absorption and scattering coefficients need to be
determined as well. Furthermore, the acquisition of a number of time frames is usually prohibitive for fluorescence
measurements, at least in small animals, due to the increased total measurement time. On the other hand, it is obvious
that diffusion is less pronounced in images at early gates, due to selective imaging of photons of lower scatter order. This
will be the case also for photons emitted by fluorescent sources. Early-gated imaging might increase the contrast in
acquired images and could possibly improve fluorescence localization. Herein, we present early gated fluorescence
images obtained from phantoms and compare them to continuously acquired data. Increased contrast between
background and signal maximum can be observed in time-gated images as compared to continuous data. To make use of
the properties exhibited by early gated frames, it is necessary to use a modified reconstruction algorithm. We propose a
variant of the well-known Born approximation to the diffusion equation that allows to take into account single time
frames. The system matrix for the time-dependent Born approach is more complex to calculate, however the complexity
of the actual inverse problem (and the acquisition times) of single-frame reconstructions remains the same as compared
to continuous mode.
Non-contact detection schemes for optical or fluorescence tomography offer several advantages compared to classic approaches, most importantly the ability to obtain images with a CCD in the absence of a matching fluid or fiber optics. This allows the acquisition of high density datasets, as well as simplified experimental procedures. Herein we create a unified framework for contact and non-contact detection procedures and present experimental results that show the ability of the non-contact method to quantify the concentration of fluorochromes hidden in turbid media as well as the improvement in image quality between conventional and non-contact detection.
We present a simple and novel theoretical approach for modeling the intensity distribution from an arbitrarily shaped turbid volume in a non-contact geometry by considering diffuse light propagation in free-space. Optical tomography of turbid media has so far been limited by systems that require fixed geometries or fiber measurements. This novel theory is validated with experiments for a diffusive volume of known geometry in a non-contact situation, both with and without the presence of an embedded absorber. We also present a system that records non-contact optical measurements from diffuse media of arbitrary shapes and retrieves the three-dimensional surface information of the diffuse medium. This approach offers significant experimental simplicity and yields high-information content datasets. The performance of this novel tomographic approach is demonstrated with experimental reconstructions of phantoms. Finally, the implications of this new formulation in the context of optical tomography in turbid media are discussed.
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