A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the “imaging window,” long-term shelf colloidal and optical stabilities with high brightness (>10 6 L mol −1 cm −1 ) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.
Prostate cancer diagnosis is based on PSA rate measurement and ultrasound guided biopsy. Recently criticized for its lack of specificity, new approaches are currently investigated: MRI, elastography, TEP, NIRS and Time Resolved (TR) fluorescence tomography. The advantage of TR fluorescence tomography relies on its good complementarity with the standard ultrasound protocol and on the possible localization of prostate tumors marked by specific probes. After a first TR system based on a bulky titanium-sapphire laser, we designed a new one taking advantage of a more compact white pulsed laser (supercontinuum). The improved compactness is now fully compatible with clinical environment. The light, filtered by two linear variable filters to select a 770±20 nm window, is driven to the transrectal probe which also collects the fluorescence light emitted by the marker. The signal is detected by photomultipliers connected to TCSPC boards. A reconstruction algorithm based on intensities and time of flight allows a fast localization of the fluorophore. We compared the performances of the new white laser system to the previous titanium-sapphire on prostate mimicking phantoms. The laser power delivered on the phantom by the new laser appeared to be suitable to fluorescence measurements, just below cutaneous maximum permitted exposure. The new system allowed us to localize fluorescent inclusions of a fluorescent nanoemulsion at fixed positions inside a prostate mimicking phantom.
This paper presents a tomograph for small animal fluorescence imaging. The compact and cost-effective system described in this article was designed to address the problem of tumor detection inside highly absorbent heterogeneous organs, such as lungs. To validate the tomograph's ability to detect cancerous nodules inside lungs, in vivo tumor growth was studied on seven cancerous mice bearing murine mammary tumors marked with Alexa Fluor 700. They were successively imaged 10, 12, and 14 days after the primary tumor implantation. The fluorescence maps were compared over this time period. As expected, the reconstructed fluorescence increases with the tumor growth stage.
We developed an endorectal time-resolved optical probe aiming at an early detection of prostate tumors targeted by
fluorescent markers. Optical fibers are embedded inside a clinical available ultrasound endorectal probe. Excitation light
is driven sequentially from a femtosecond laser (775 nm) into 6 source fibers. 4 detection fibers collect the medium
responses at the excitation and fluorescence wavelength (850 nm) by the mean of 4 photomultipliers associated with a 4
channel time-correlated single photon counting card.
We also developed the method to process the experimental data. This involves the numerical computation of the
forward model, the creation of robust features which are automatically correctly from numerous experimental possible
biases and the reconstruction of the inclusion by using the intensity and mean time of these features.
To evaluate our system performance, we acquired measurements of a 40 μL ICG inclusion (10 μmol.L-1) at
various lateral and depth locations in a phantom. Analysis of results showed we correctly reconstructed the
fluorophore for the lateral positions (16 mm range) and for a distance to the probe going up to 1.5 cm. Precision of
localization was found to be around 1 mm which complies well with precision specifications needed for the clinical
application.
The protocol for prostate cancer diagnosis, currently based on ultrasound guided biopsy, is limited by a lack of
relevance. To improve this protocol, a new approach was proposed combining optical and ultrasound measurements to
guide biopsy specifically to the tumors. Adding an optical measurement modality into an already existing ultrasound
probe is challenging as the overall size of the system should not exceed a given dimension so as to fit the operative
environment. Moreover, examination should not take more than 15 min to avoid any complication.
A combined ultrasound and optical endorectal probe was designed to comply with the constraints of the
sterilization protocols, the examination duration and required compactness. Therefore a totally innovative pulsed laser
source has been designed to meet compactness requirements while providing accurate time-resolved measurements. A
dedicated multi-channel photon counting system was optimized to decrease the examination duration. A fast
reconstruction method based on the analysis of the intensity and time of flight of the detected photons has been
associated to provide 3D localization of fluorescent dots almost immediately after acquisition.
The bi-modal probe was capable of withstanding the sterilization procedures. The performance of the compact
laser source has been shown at the same level as that of a standard laboratory Titane:Sapphire laser. The dedicated
photon counting solution was capable of acquiring optical data in less than one minute. To evaluate the overall
performance of the system in dealing with a realistic background signal, measurements and reconstructions were
conducted on prostate mimicking phantom and in vivo.
Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early
detection of prostate cancer. A trans-rectal probe adapted to fluorescence diffuse optical tomography measurements was
developed by our team. This probe is based on a pulsed NIR laser source, an optical fiber network and a time-resolved
detection system. A reconstruction algorithm was used to help locate and quantify fluorescent prostate tumors.
In this study, two different kinds of time-resolved detectors are compared: High Rate Imaging system (HRI) and a
photon counting system. The HRI is based on an intensified multichannel plate and a CCD Camera. The temporal
resolution is obtained through a gating of the HRI. Despite a low temporal resolution (300ps), this system allows a
simultaneous acquisition of the signal from a large number of detection fibers. In the photon counting setup, 4
photomultipliers are connected to a Time Correlated Single Photon Counting (TCSPC) board, providing a better
temporal resolution (0.1 ps) at the expense of a limited number of detection fibers (4).
At last, we show that the limited number of detection fibers of the photon counting setup is enough for a good
localization and dramatically improves the overall acquisition time. The photon counting approach is then validated
through the localization of fluorescent inclusions in a prostate-mimicking phantom.
To increase prostate cancer diagnosis sensibility, we propose to add an optical modality to an US biopsy tool to localize
fluorophore marked tumors. Optical signals are acquired on a time-resolved acquisition chain composed by a 770 nm
femtosecond laser source and a four channels TCSPC device. The fluorescence concentration is reconstructed by using
intensity and mean time of flight acquired from each time-resolved source-detector signal. Validation experiments are
performed on a phantom mimicking prostate both on its optical and ultrasound properties with 10 μmol/L ICG 1 cm deep double fluorescent inclusions to simulate marked tumors. An exhaustive search algorithm succeeded in reconstructing the two distinct fluorescence dots with correct locations.
We present two major advances in preclinical fluorescence-enhanced diffuse optical tomography (fDOT) system and assess its performance. It is now possible to perform experiments without adaptation liquid or a glass plate over the animal, and our system is equipped with a filter wheel in order to discriminate two injected fluorophores. Evaluation carried out on characterization phantoms and in vivo on mice demonstrates enriched use of the system for biological studies on small animals.
Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early detection of prostate cancer. The ultrasound provides morphological information about the prostate, while the optical system detects and locates fluorophore-marked tumors. A tissue-mimicking phantom, which is representative of prostate tissues both on its optical (absorption µa and diffusion µ) and its ultrasound properties, has been made by our team. A transrectal probe adapted to fluorescence diffuse optical tomography measurements was also developed. Measurements were taken on the prostate phantom with this probe based on a pulsed laser and a time-resolved detection system. A reconstruction algorithm was then used to help locate and quantify fluorescent inclusions of different concentrations at fixed depths.
A fluorescence diffuse optical tomography instrument including a dedicated reconstruction scheme which accounts for the
medium optical heterogeneities is presented. It allows non-contact measurements and does not require animal immersion in
an optical adaptation liquid.
Prostate cancer diagnosis is based on PSA dosage and digital rectal examination. In case of positive test, a biopsy is
conducted and guided by ultrasound imaging. Today, however, as ultrasound imaging is not able to precisely detect
tumors, some biopsies have to be performed in the prostate and the only way to improve detection is to increase the
number of those uncomfortable biopsies. In order to decrease this number and to improve the patient wellness, we are
studying a way to couple ultrasound and fluorescence optical imaging on an endorectal probe. The ultrasounds are used
to get morphological information on the prostate and the optical system to detect and to localize fluorophore marked
tumors. To support the development of such a system, we have carried out a new tissue-mimicking phantom which
represents the three different kind of tissue concerned during prostate endorectal examination: prostate, rectum,
surrounding tissues. It was imaged by ultrasound and by fluorescence diffuse optical imaging. We have proved that the
optical system is able to detect and to localize a fluorescing inclusion at different depth inside the phantom which has
then been superimposed to the morphological image provided by the ultrasounds.
Fluorescence Diffuse Optical Tomography is an optical non-invasive molecular technique for cancer imaging.
Depending on the accessibility of the organ two main geometries might be considered, reflection or transmission. We
will present first experimental and reconstruction comparison between these two geometries, on a laboratory time
resolved bench. Both acquisitions were made using a fluorophore inclusion positioned in a liquid phantom, with breast
comparable optical properties. We successfully reconstructed all fluorophore positions examined in both geometries.
Reflection geometry suffers of many drawbacks that we have to deal with. We will present all challenges it implies, and
also what are the advantages to use time resolved techniques in both geometries.
We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.
KEYWORDS: Luminescence, Tumors, Lung, 3D modeling, In vivo imaging, Geometrical optics, Reconstruction algorithms, Animal model studies, Liquids, Glasses
This paper presents in vivo experiments conducted on cancerous mice bearing mammary murine tumors. In order to
reconstruct the fluorescence yield even in highly attenuating and heterogeneous regions like lungs, we developed a fDOT
reconstruction method which at first corrects the light propagation model from optical heterogeneities by using the
transmitted excitation light measurements. The same approach is also designed to enable working without immersing the
mouse in adaptation liquid. The 3D fluorescence map is then reconstructed from the emitted signal of fluorescence and
from the corrected propagation model by an ART (Algebraic Reconstruction Technique) algorithm. The system ability to
reconstruct fluorescence distribution in presence of high attenuating objects has been validated on phantoms presenting a
fluorescent absorbent inclusion. A study was conducted on mice to follow up lungs at different stages of tumor
development. The mice were imaged after intravenous injection to the animal of a cancer specific fluorescent marker. A
control experiment was conducted in parallel on healthy mice to ensure that the multiple injections of fluorophore did not
induce parasite fluorescence distribution. These results validate our system performances for studying small animal lungs
tumor evolution. Detection and localization of the fluorophore fixations expresses the tumor development.
Fluorescence enhanced diffuse optical tomography (fDOT) is envisioned to be useful to collect functional information
from small animal models. For oncology applications, cancer-targeted fluorescent markers can be used as a surrogate of
the cancer activity.
We are developing a continuous wave fDOT bench intended to be integrated in systems dedicated to whole
body small animal fluorescence analyses. The focus is currently put on the reconstruction of non immersed small animals
imaged by a CCD camera. The reconstruction stage already corrects the tissue heterogeneity artifacts through the
computation of an optical heterogeneity map. We will show how this formalism coupled with the determination of the
animal boundaries performed by a laser scanner, can be used to manage non contact acquisitions. The time of
reconstruction for a 10 × 9 laser source positions, 45 × 40 detector elements and 14 × 11 × 14 mesh voxels is typically 10
minutes on a 3GHz PCs corresponding to the acquisition time allowing the two tasks to be performed in parallel.
The system is validated on an in vivo experiment performed on three healthy nude mice and a mouse bearing a
lung tumor at 10, 12 and 14 days after implantation allowing the follow up of the disease. The 3D fluorescence
reconstructions of this mouse are presented and the total fluorescence amounts are compared.
Optical imaging of fluorescent probes is an essential tool for investigation of molecular events in small animals for drug developments. In order to get localization and quantification information of fluorescent labels, CEA-LETI has developed efficient approaches in classical reflectance imaging as well as in diffuse optical tomographic imaging with continuous and temporal signals. This paper presents an overview of the different approaches investigated and their performances. High quality fluorescence reflectance imaging is obtained thanks to the development of an original "multiple wavelengths" system. The uniformity of the excitation light surface area is better than 15%. Combined with the use of adapted fluorescent probes, this system enables an accurate detection of pathological tissues, such as nodules, beneath the animal's observed area. Performances for the detection of ovarian nodules on a nude mouse are shown. In order to investigate deeper inside animals and get 3D localization, diffuse optical tomography systems are being developed for both slab and cylindrical geometries. For these two geometries, our reconstruction algorithms are based on analytical expression of light diffusion. Thanks to an accurate introduction of light/matter interaction process in the algorithms, high quality reconstructions of tumors in mice have been obtained. Reconstruction of lung tumors on mice are presented.
By the use of temporal diffuse optical imaging, localization and quantification performances can be improved at the price of a more sophisticated acquisition system and more elaborate information processing methods. Such a system based on a pulsed laser diode and a time correlated single photon counting system has been set up. Performances of this system for localization and quantification of fluorescent probes are presented.
In the framework of Fluorescence-enhanced Diffuse Optical Tomography, a numerical approach (usually the Finite Element Method) is often required because of the complexity of the geometry of the diffusing systems studied. This approach is appropriate for handling problems modelled by elliptic coupled partial differential equations but is known to be time and memory consuming. The resolution of the adjoint problem considerably speeds up the treatment and allows a full 3D resolution. Nevertheless, because of the ill-posedness of the problem, the reconstruction scheme is sensitive to a priori knowledge on the parameters to be reconstructed. In the present work, a multiple step, self-regularized, reconstruction algorithm for the spatial distribution of the fluorescent regions is presented. The prior knowledge of the regions of interest is introduced via a segmentation. This one is performed on the results obtained with a first rough reconstruction. The results are then refined along iterations of the segmentation/reconstruction scheme. The technique is tested on experiments performed with a home made tomographer. A phantom study is presented.
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