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Patent Application Titled "Systems, Methods, and Apparatus for Imaging of Diffuse Media Featuring Cross-Modality Weighting of Fluorescent and...

May 8, 2014



Patent Application Titled "Systems, Methods, and Apparatus for Imaging of Diffuse Media Featuring Cross-Modality Weighting of Fluorescent and Bioluminescent Sources" Published Online

By a News Reporter-Staff News Editor at Politics & Government Week -- According to news reporting originating from Washington, D.C., by VerticalNews journalists, a patent application by the inventors Yared, Wael (Lexington, MA); Kempner, Joshua (Reading, MA); Ripoll Lorenzo, Jorge (Madrid, ES); Arranz, Alicia (Zurich, CH), filed on October 15, 2013, was made available online on April 24, 2014.

The assignee for this patent application is VisEn Medical, Inc.

Reporters obtained the following quote from the background information supplied by the inventors: "Imaging in diffusive media has become an attractive field of research mainly due to its applications in biology and medicine, showing great potential in, for example, cancer research, drug development, inflammation and molecular biology. With the development of highly specific activatable fluorescent probes, a new way of obtaining information at the molecular level in vivo has been made possible. Due to the inherent scattering present in tissues in the optical wavelengths, light is multiply scattered in tissue and its original direction of propagation is randomized after what is termed the scattering mean free path, lsc, a distance which represents the density and efficiency of scattering of the different constituents of tissue. Even though images of fluorescent or bioluminescent emission from tissues might be recorded, due to the scattering present, there is a non-linear relation of these images with the concentration, size and position of the fluorescent probes or bioluminescent reporters, resulting in what is termed an ill-posed problem. Recently developed methods and systems make use of tomographic approaches by introducing a spatial dependence of fluorescence on the excitation and appropriate modeling of light propagation in tissues through the diffusion approximation to alleviate this ill-posedness, enabling the recovery of the spatial distribution of the concentration of fluorescence with a resolution on the order of the scattering mean free path (lsc) or better. In an ideal situation, the sensitivity of these tomographic techniques would depend only on the detector efficiency and probe brightness together with the absorption present in tissue, assuming it is possible to completely block the excitation light when measuring fluorescence. In this ideal case, the sensitivity could be increased by augmenting the laser power since the emitted and excitation intensities are proportional to one another, at least for powers acceptable for small animal imaging, which ensure that no appreciable heating occurs in tissue.

"However, the reality of an in vivo measurement is quite different. Due to the excitation of surrounding auto-fluorescence always present in tissue, the sensitivity of a system is strongly dependent on the ability to distinguish the specific signal due to the fluorescent probe from the non-specific signal of the surrounding auto-fluorescence. This problem becomes more pronounced when exciting in reflection mode, since the greater auto-fluorescent contribution would be from the tissue sections closest to the camera. In practice, the sensitivity of the tomographic data, or in general any collection of fluorescent data, is determined by the level of auto-fluorescence when compared to the specific signal. This issue is overcome to some extent by using far-red or near infra-red (NIR) fluorescence signals, since tissue auto-fluorescence is slightly reduced in this part of the spectrum, with the added advantage that tissue presents lower absorption properties in this part of the spectrum. This is the case when using fluorescent probes that emit in the far-red or near infra-red part of the spectrum, which are activated when a specific molecular activity is present. An advantage of this kind of probes is that they provide a high signal-to-noise ratio. However, in most practical instances, the amount of signal that can be detected depends on how much specific signal surpasses the surrounding tissue auto-fluorescence.

"In order to separate the contribution of the specific from the non-specific signal, the most common approach is to employ multi-spectral measurements, assuming the emission spectrum is known. Even though this approach slightly increases the sensitivity, the problem still remains: at each emission wavelength measured there is an unknown contribution from tissue auto-fluorescence. The weaker the specific signal, the more dominant the effect of auto-fluorescence.

"Bioluminescent reporters offer the significant advantage of not requiring an external illumination to place them in an excited state such that they would emit light. Since the excited state is reached though a chemical reaction, the emitted light represents the background-free solution of the imaging problem, akin to the ideal case of fluorescence mentioned in the previous paragraphs. This inherent benefit of bioluminescence also has some drawbacks, specifically in relation to the ill-posed problem mentioned above. Since it is not currently possible to effectively introduce a spatial dependence on the intensity of this emission (whereas this is possible in the case of fluorescence), it is not possible to recover simultaneously the spatial distribution of bioluminescent probe concentration. Thus, because the main goal is to recover the spatial distribution of probe concentration, the ill-posedness of the problem cannot be substantially reduced in the case of bioluminescence as is possible in the case of fluorescence.

"There is a need for an optical imaging system in which the low-background and lack of auto-fluorescence of bioluminescent probes can be combined with the specificity, high quantum yield, and the external capability of emission intensity modulation exhibited by fluorescent probes."

In addition to obtaining background information on this patent application, VerticalNews editors also obtained the inventors' summary information for this patent application: "In certain embodiments, the invention relates to systems and methods for in vivo tomographic imaging of fluorescent probes and/or bioluminescent reporters, wherein a fluorescent probe and a bioluminescent reporter are spatially co-localized (e.g., located at distances equivalent to or smaller than the scattering mean free path of light) in a diffusive medium (e.g., biological tissue). Measurements obtained from bioluminescent and fluorescent modalities are combined per methods described herein.

"In one aspect, the invention provides a method for imaging a target region of a diffuse object, the method comprising: (a) administering a bioluminescent substrate (and/or chemiluminescent substrate) to the object; (b) detecting bioluminescent (and/or chemiluminescent) light emitted from the object by a bioluminescent reporter in the target region of the object (e.g., using an external detector); administering a probe comprising a fluorescent species (and/or a jointly fluorescent/bioluminescent species) to the object; (d) directing excitation light into the object at multiple locations and/or at multiple angles, thereby exciting the fluorescent species; (e) detecting fluorescent light (e.g., as a function of detector position, excitation light source position, or both), the detected fluorescent light having been emitted by the fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light; (f) detecting excitation light (e.g., as a function of detector position, excitation light source position, or both), the detected excitation light having been transmitted through the region of the diffuse object (transillumination) or having been reflected from the region of the diffuse object (epi-illumination); and (g) processing data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object.

"In certain embodiments, the bioluminescent reporter is endogenous. In certain embodiments, the bioluminescent reporter is expressed within the object by a bioluminescent cell line. In certain embodiments, the bioluminescent reporter is exogenously administered. In certain embodiments, the bioluminescent reporter is administered to the object as a component of a tandem bioluminescent-fluorescent probe.

"In certain embodiments, following step , at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object. In certain embodiments, following step , the bioluminescent reporter is also present in the object at one or more locations that are not substantially co-located with the (fluorescent) probe. In certain embodiments, the probe in step comprises both the fluorescent species and the bioluminescent reporter in tandem with the fluorescent species (e.g., the bioluminescent reporter and fluorescent probe comprises a single construct). In certain embodiments, it is not required that the bioluminescent reporter and the fluorescent species be conjugated or colocalized to within FRET-type distances. Co-located may mean located within a macro-level distance. For example, in certain embodiments, the method may be used even where the bioluminescent reporter and the fluorescent species are separated by a macro-level distance, for example, up to about 1 mm or up to about 2 mm, rather than up to about 5 to 10 nm as in FRET and BRET.

"In certain embodiments, the diffuse object comprises living biological tissue (in vivo imaging). In certain embodiments, the diffuse object is a mammal. In certain embodiments, the image of the target region is a tomographic image. In certain embodiments, the method further comprises placing the object within a holder prior to steps (b), (d), (e), and (f). For example, in certain embodiments, the image of the target region is a tomographic image and wherein a surface of the object is at least partially conformed by the holder such that the surface can be characterized by a continuous function (e.g., in Cartesian, polar, or cylindrical coordinates), thereby facilitating tomographic reconstruction.

"In certain embodiments, step (b) comprises detecting bioluminescent light using a detector located outside the object. In certain embodiments, step (b) comprises detecting bioluminescent light as a function of detector position. In certain embodiments, step (b) comprises detecting bioluminescent light using a detector in optical contact with the object (e.g., a component of the detector is in physical contact with either the object itself or a transparent surface in physical contact with the object, and/or optical guides, fiber guides, optical matching fluids, and or lenses are used such that optical contact is maintained during detection). In certain embodiments, step (b) comprises detecting bioluminescent light using a detector positioned such that there is a nondiffusive medium (e.g., a layer of air) between the detector and the object. In certain embodiments, step (b) comprises detecting bioluminescent light using an emission filter. In certain embodiments, step (b) comprises detecting bioluminescent light without using an emission filter.

"In certain embodiments, the probe in step is a visible or near-infrared fluorescent probe (e.g., a NIRF molecular probe, a red agent, etc.).

"In certain embodiments, step (d) comprises directing visible and/or near infrared light into the object. In certain embodiments, step (d) comprises directing a point source of excitation light into the object. In certain embodiments, step (d) comprises simultaneously directing multiple sources of excitation light into the object. In certain embodiments, step (d) comprises directing structured excitation light into the object. In certain embodiments, step (d) comprises directing the excitation light into the object at multiple discrete positions (e.g., an array of positions) and/or at multiple discrete angles (e.g., an array of angles), each instance of directing the excitation light into the object occurring at discrete times.

"In certain embodiments, the method comprises performing steps (e) and (f) after each instance of directing excitation light into the object at a discrete position and/or at a discrete angle. In certain embodiments, step (d) comprises directing the excitation light into the object at multiple discrete positions (e.g., an array of positions) and/or at multiple discrete angles (e.g., an array of angles) simultaneously. In certain embodiments, step (d) comprises scanning the excitation light over the object. In certain embodiments, the excitation light detected in step (f) comprises at least one of continuous wave (CW) light, time-resolved (TR) light, and intensity modulated (IM) light.

"In certain embodiments, step (e) comprises detecting fluorescent light emitted from the target region of the object using a detector located outside the object. In certain embodiments, step (e) comprises detecting fluorescent light on the same side of the object into which excitation light was directed in step (d) (e.g., epi-illumination). In certain embodiments, step (e) comprises detecting fluorescent light on a side of the object opposite the side into which excitation light was directed in step (d) (e.g., transillumination). In certain embodiments, step (e) comprises detecting fluorescent light emitted from the target region of the object using a detector in optical contact with the object (e.g., a component of the detector is in physical contact with either the object itself or a transparent surface in physical contact with the object, and/or optical guides, fiber guides, optical matching fluids, and or lenses are used such that optical contact is maintained during detection). In certain embodiments, step (e) comprises detecting fluorescent light emitted from the target region of the object using a detector positioned such that there is a nondiffusive medium (e.g., a layer of air) between the detector and the object.

"In certain embodiments, step (f) comprises detecting excitation light transmitted through the target region of the object or reflected from the target region of the object using a detector located outside the object. In certain embodiments, step (f) comprises detecting excitation light transmitted through the target region of the object or reflected from the target region of the object using a detector in optical contact with the object (e.g., a component of the detector is in physical contact with either the object itself or a transparent surface in physical contact with the object, and/or optical guides, fiber guides, optical matching fluids, and or lenses are used such that optical contact is maintained during detection). In certain embodiments, step (f) comprises detecting excitation light transmitted through the target region of the object or reflected from the target region of the object using a detector positioned such that there is a nondiffusive medium (e.g., a layer of air) between the detector and the object.

"In certain embodiments, step (e) comprises detecting the fluorescent light from multiple projections and/or views. In certain embodiments, step (f) comprises detecting the excitation light from multiple projections and/or views.

"In certain embodiments, the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the fluorescent species in the three-dimensional target region. In certain embodiments, the tomographic image indicates three-dimensional spatial distribution of the probe (e.g., wherein the probe comprises a fluorescent species and/or a jointly fluorescent/bioluminescent species) within the target region. In certain embodiments, the tomographic image indicates concentration of the probe as a function of position within the target region of the object. In certain embodiments, the tomographic image indicates concentration of the probe as a function of position in three dimensions.

"In certain embodiments, the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the bioluminescent reporter in the three-dimensional target region. In certain embodiments, the tomographic image indicates three-dimensional spatial distribution of the bioluminescent reporter within the target region. In certain embodiments, the tomographic image indicates concentration of the bioluminescent reporter as a function of position within the target region of the object.

"In certain embodiments, the bioluminescent reporter and the fluorescent species have emission spectra that differ.

"In certain embodiments, step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and normalizing data corresponding to the detected fluorescent light with data corresponding to the detected excitation light. In certain embodiments, step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and then normalizing the resulting weighted data with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. In certain embodiments, step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light, and weighting an associated weight matrix with data corresponding to the detected bioluminescent light, then inverting the weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. In certain embodiments, step (g) comprises weighting data corresponding to the detected bioluminescent light with data corresponding to the detected fluorescent light normalized with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the bioluminescent reporter in the target region of the object.

"In certain embodiments, the detecting in step (b) is performed at a different time than the detecting in steps (e) and (f) (e.g., because pharmacokinetics of the probes and the substrate may be different). In certain embodiments, one would time the administration (e.g., injection) of bioluminescent substrate and fluorescent probe such that the imaging of both modalities can be performed in close succession, in order to capture a single 'time point'. Imaging via the two modes need not occur in exact simultaneity, for example, in an instrument equipped to image in both modalities. It is possible to image using two single-modality instruments that are in proximity such that imaging the object (e.g., a living animal) in one instrument can be performed, and the animal may be shuttled to the second instrument, e.g., within a few minutes. An animal holder may be used, for example, such as that described in U.S. Patent Application Publication No. US 2011/0071388, incorporated herein by reference.

"In another aspect, the invention provides a method for imaging a target region of a diffuse object, the method comprising: (a) administering a bioluminescent substrate (and/or chemiluminescent substrate) to the object; (b) detecting bioluminescent (and/or chemiluminescent) light emitted from the object by a bioluminescent reporter in the target region of the object (e.g., using an external detector); administering a probe comprising a fluorescent species (and/or a jointly fluorescent/bioluminescent species) to the object; (d) detecting fluorescent light (e.g., as a function of detector position), the detected fluorescent light having been emitted by the fluorescent species in the target region of the diffuse object as a result of excitation by the bioluminescent light (e.g., bioluminescent stimulation of fluorescence by radiative transfer); and (e) processing data corresponding to the detected bioluminescent light and the detected fluorescent light to provide an image of the target region within the diffuse object.

"In certain embodiments, the method further includes directing light (e.g., at an excitation wavelength) into the object and detecting the light having been transmitted through the region of the diffuse object (transillumination) or having been reflected from the region of the diffuse object (epi-illumination) (e.g., to provide a proxy attenuation map), and using data corresponding to the detected light (e.g., using the proxy attenuation map) in step (g) along with the data corresponding to the detected bioluminescent light and the detected fluorescent light to provide the image of the target region within the diffuse object.

"In another aspect, the invention provides a system for imaging a target region within a diffuse object, the system comprising: an excitation light source; an optical imaging apparatus configured to direct light from the excitation light source into the diffuse object at multiple locations and/or at multiple angles; one or more detectors, the one or more detectors individually or collectively configured to detect and/or measure (e.g., as a function of detector position, excitation light source position, or both), (i) bioluminescent (and/or chemiluminescent) light emitted from the object, (ii) fluorescent light emitted by a fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light, and (iii) excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and a processor configured to process data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object.

"In another aspect, the invention provides a system for imaging a target region within a diffuse object, the system comprising: an excitation light source; an optical imaging apparatus configured to direct light from the excitation light source into the diffuse object at multiple locations and/or at multiple angles; one or more detectors, the one or more detectors individually or collectively configured to detect and/or measure (e.g., as a function of detector position, excitation light source position, or both), (i) bioluminescent (and/or chemiluminescent) light emitted from the object, (ii) fluorescent light emitted by a fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light, and (iii) excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and a processor configured to process data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object.

"In another aspect, the invention provides an apparatus for reconstructing a tomographic representation of a probe within a target region of a diffuse object, the apparatus comprising: a memory that stores code defining a set of instructions; and a processor that executes the instructions thereby to process data corresponding to: (a) establish a forward model of excitation light propagation from an excitation light source to the probe within the target region of the object and of emission light propagation from the probe to a detector using (i) data corresponding to detected fluorescent light from the probe, (ii) data corresponding to detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object, and (iii) data corresponding to detected bioluminescent light emitted from a bioluminescent reporter, wherein at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object, and wherein the forward model is established as a weight matrix; and (b) invert the weight matrix to obtain the tomographic representation of the probe within the target region of the diffuse object.

"In another aspect, the invention provides a non-transitory computer readable medium having instructions thereon that, when executed by a processor, cause the processor to: (a) establish a forward model of excitation light propagation from an excitation light source to the probe within the target region of the object and of emission light propagation from the probe to a detector using (i) data corresponding to detected fluorescent light from the probe, (ii) data corresponding to detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object, and (iii) data corresponding to detected bioluminescent light emitted from a bioluminescent reporter, wherein at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object, and wherein the forward model is established as a weight matrix; and (b) invert the weight matrix to obtain the tomographic representation of the probe within the target region of the diffuse object.

"In another aspect, the invention provides a method for imaging a target region of a diffuse object, the method comprising: (a) detecting bioluminescent (and/or chemiluminescent) light emitted from the object by a bioluminescent reporter in the target region of the object (e.g., using an external detector); (b) directing excitation light into the object at multiple locations and/or at multiple angles, thereby exciting a fluorescent species of a probe (e.g., a fluorescent probe or a fluorescent/bioluminescent tandem probe) in the target region of the diffuse object; detecting fluorescent light as a function of detector position, excitation light source position, or both, the detected fluorescent light having been emitted by the fluorescent species as a result of excitation by the excitation light; (d) detecting excitation light as a function of detector position, excitation light source position, or both, the detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and (e) processing data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object.

"Elements from embodiments of one aspect of the invention may be used in other aspects of the invention (e.g., elements of claims depending from one independent claim may be used to further specify embodiments of other independent claims). Other features and advantages of the invention will be apparent from the following figures, detailed description, and the claims.

"The objects and features of the invention can be better understood with reference to the drawings described below, and the claims. In the drawings, like numerals are used to indicate like parts throughout the various views.

BRIEF DESCRIPTION OF DRAWINGS

"FIG. 1 is a schematic drawing depicting a comparison between an image generated by a bioluminescent source and one generated by a fluorescence source, the intensity of which can be modulated externally by changing the intensity of the excitation source. The shape of both signals should be, in the absence of auto-fluorescence, identical, with the only difference residing in their absolute intensity values.

"FIG. 2 is a schematic drawing depicting how the information due solely to the fluorophore can be identified with the bioluminescence profile when the fluorescence signal is degraded through contribution of auto-fluorescence and background signal.

"FIG. 3 is a schematic drawing depicting how the contribution of auto-fluorescence changes with the source position, while the fluorescence emission changes in intensity but not in distribution.

"FIG. 4 is a block flow diagram depicting an example method of combining bioluminescence with normalized fluorescent measurements to obtain 3D images of fluorescent and/or bioluminescent sources, according to an illustrative embodiment of the invention.

"FIG. 5 shows the results of a simulation of the combination of bioluminescence with normalized fluorescent measurements to obtain accurate 3D images of fluorescent sources in the presence of non-specific background fluorescent signal, according to an illustrative embodiment of the invention.

"FIG. 6 shows a schematic of a diffuse optical tomography imaging system that may be used in various embodiments described herein."

For more information, see this patent application: Yared, Wael; Kempner, Joshua; Ripoll Lorenzo, Jorge; Arranz, Alicia. Systems, Methods, and Apparatus for Imaging of Diffuse Media Featuring Cross-Modality Weighting of Fluorescent and Bioluminescent Sources. Filed October 15, 2013 and posted April 24, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3454&p=70&f=G&l=50&d=PG01&S1=20140417.PD.&OS=PD/20140417&RS=PD/20140417

Keywords for this news article include: VisEn Medical Inc.

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