[Show abstract][Hide abstract] ABSTRACT: Photocontrol of reversibly switchable fluorescent proteins (RSFPs) was used
to program optoacoustic signal time courses that were temporally unmixed to
increase the proteins contrast-to-noise-ratios (CNRs) in optoacoustic imaging.
In this way, two variants of the RSFP Dronpa with very similar optoacoustic
spectra could be readily discriminated in the presence of highly absorbing
blood. Addition of temporal unmixing to multispectral optoacoustic tomography
(tuMSOT) in conjunction with synthetic or genetically controlled photochromic
contrast agents and customized photoswitching schedules can increase the
performance of multiplexed and high contrast molecular optoacoustic imaging.
[Show abstract][Hide abstract] ABSTRACT: Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.
[Show abstract][Hide abstract] ABSTRACT: Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation.We conducted ex-vivo and Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) in-vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers.Ex-vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions at all ages studied. The panel of immunofluorescence experiments demonstrated that pulmonary lesions, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be non-invasively detected in-vivo on the fluorescence channel of FMT-XCT.Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring. This article is protected by copyright. All rights reserved.
International Journal of Cancer 12/2014; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the major challenges in dynamic multispectral optoacoustic imaging is its relatively low signal-to-noise ratio which often requires repetitive signal acquisition and averaging, thus limiting imaging rate. The development of denoising methods which prevent the need for signal averaging in time presents an important goal for advancing the dynamic capabilities of the technology.
[Show abstract][Hide abstract] ABSTRACT: We have imaged for the first time to our knowledge human skin in vivo with a raster-scan optoacoustic mesoscopy system based on a spherically focused transducer with a central frequency of 102.8 MHz and large bandwidth (relative bandwidth 105%). Using tissue phantoms we have studied the ability of the system to image vessels of sizes within the anatomically significant range from the key anatomical vasculature sites. The reconstructed images from experiments in vivo show several structures from the capillary loops at the dermal papillae, the horizontal plexus, and the difference between the dermis and the epidermis layers. Histopathological analysis of biopsied skin is an invasive, slow, and expensive medical process in dermatology, but necessary for accurately diagnosing skin diseases . State-of-the-art skin optical-imaging techniques like der-moscopy or linear and nonlinear microscopy methods are proposed as an alternative to reduce the number of biopsies [2–6], but they are intrinsically limited by light scattering, which limits the imaging depth to a few hundred microns. Optical coherence tomography (OCT) may penetrate deeper than dermoscopy or confocal im-aging to depths of ∼1 mm. However, the skin depth varies from 1.5 to 4 mm  and imaging at such depths is necessary in different pathologies. Optoacoustic mesoscopy, based on high-frequency acoustic resolution, may be an interesting alternative for skin imaging. Optoacoustic mesoscopy refers to opto-acoustic imaging that goes beyond the depth of optical microscopy, reaching several millimeters deep in tissue . Compared to confocal imaging or OCT it also offers alternative contrast mechanisms by resolving the absorp-tion of light by tissue [9,10]. Cross-sectional optoacoustic imaging of the skin has been already demonstrated using a linear array of trans-ducers operating at 24 MHz . The depth of skin lesions and burns was resolved with 97 μm lateral resolution and 22 μm axial resolution. Skin vasculature [12,13] has also been resolved using interferometry  or piezoelec-tric-focused detectors [15,16] operating at central frequencies of up to 50 MHz. The highest lateral resolution achieved so far was ∼40 μm, whereas the axial resolution reached ∼15 μm. At this resolution, only the larger skin vessels (25–100 μm) are visible, i.e., vessels situated rela-tively deep in the dermis . Smaller vessels (7–25 μm) situated close to the epidermal-dermal junction  blur the images and typically have to be removed for rendering purposes . Most mesoscopy implementations show vascular images as maximum intensity projections along the axial direction (coronal views) of the entire skin, a view that is not favorable to assess lesion depth. Skin im-aging with a high-frequency unfocused LiNbOb 3 detector has been also considered for skin imaging, yielding lateral resolution of 200 μm . An ultrawideband (20–200 MHz) system, implemented in transillumination, recently achieved ∼30 μm lateral and 7 μm an axial resolution . In this work we applied an epi-illumination version of this technology  to skin visualization and interrogated whether operation at a broader bandwidth could differentiate skin layers in axial and coronal skin views. In particular, we investigated if features not visible in the 25–50 MHz are better resolved when collecting broader frequency content. Imaging was based on a custom-made 102.8 MHz central frequency spherically focused transducer constructed out of LiNbOb 3 to yield ultrawideband measurements ranging from a few millihertz to ∼200 MHz. The active element of the transducer had a diameter of 1.5 mm and an f-number of ∼1 (numerical aperture ∼0.5). The frequency response of the transducer is shown in Fig. 1 Detected optoacoustic signals were preamplified by a low-noise amplifier (63 dB, AU-1291, Mited Inc., Haupppauge, New York, USA) and collected by a high-speed digitizer
[Show abstract][Hide abstract] ABSTRACT: Raster-scan optoacoustic mesoscopy (RSOM) comes with high potential for in vivo diagnostic imaging in dermatology, since it allows for high resolution imaging of the natural chromophores melanin, and hemoglobin at depths of several millimeters. We have applied ultra-wideband RSOM, in the 10 MHz to 160 MHz frequency band, to image healthy human skin at distinct locations. We analyzed the anatomical information contained at different frequency ranges of the optoacoustic (photoacoustic) signals in relation to resolving features of different skin layers in vivo. We further compared results obtained from glabrous and hairy skin and identify that frequencies above 60 MHz are necessary for revealing the epidermal thickness, a prerequisite for determining the invasion depth of melanoma in future studies. By imaging a benign nevus we show that the applied RSOM system provides strong contrast of melanin-rich structures. We further identify the spectral bands responsible for imaging the fine structures in the stratum corneum, assessing dermal papillae, and resolving microvascular structures in the horizontal plexus.
[Show abstract][Hide abstract] ABSTRACT: Although in vivo targeting of tumors using fluorescently-labeled probes has greatly gained in importance over the last few years, most of the clinically applied reagents lack tumor cell specificity. Our novel tumor cell-penetrating peptide-based probe (TPP) recognizes an epitope of Hsp70 that is exclusively present on the cell surface of a broad variety of human and mouse tumors and metastases, but not on normal tissues. Due to the rapid turn-over rate of membrane-Hsp70, fluorescently-labeled TPP is continuously internalized into syngeneic, spontaneous, chemically/genetically induced and xenograft tumors following intravenous administration, thereby enabling site-specific labeling of primary tumors and metastases. In contrast to the commercially available non-peptide small molecule alpha v beta3-integrin antagonist IntegriSense (trademark), TPP exhibits a significantly higher tumor-to-background contrast and stronger tumor-specific signal intensity in all tested tumor models. Moreover, in contrast to IntegriSense (trademark), TPP reliably differentiates between tumor cells and cells of the tumor microenvironment, such as tumor-associated macrophages and fibroblasts which were found to be membrane-Hsp70 negative. Therefore, TPP provides a useful tool for multimodal imaging of tumors and metastases that might help to improve our understanding of tumorigenesis and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70- based targeted therapies.
[Show abstract][Hide abstract] ABSTRACT: Molecular optoacoustic (photoacoustic) imaging typically relies on the spectral identification of absorption signatures from molecules of interest. To achieve this, two or more excitation wavelengths are employed to sequentially illuminate tissue. Due to depth-related spectral dependencies and detection related effects, the multispectral optoacoustic tomography (MSOT) spectral unmixing problem presents a complex non-linear inversion operation. So far, different studies have showcased the spectral capacity of optoacoustic imaging, without however relating the performance achieved to the number of wavelengths employed. Overall, the dependence of the sensitivity and accuracy of optoacoustic imaging as a function of the number of illumination wavelengths has not been so far comprehensively studied. In this paper we study the impact of the number of excitation wavelengths employed on the sensitivity and accuracy achieved by molecular optoacoustic tomography. We present a quantitative analysis, based on synthetic MSOT datasets and observe a trend of sensitivity increase for up to 20 wavelengths. Importantly we quantify this relation and demonstrate an up to an order of magnitude sensitivity increase of multi-wavelength illumination vs. single or dual wavelength optoacoustic imaging. Examples from experimental animal studies are finally utilized to support the findings. In vivo MSOT imaging of a mouse brain bearing a tumor that is expressing a near-infrared fluorescent protein. (a) Monochromatic optoacoustic imaging at the peak excitation wavelength of the fluorescent protein. (b) Overlay of the detected bio-distribution of the protein (red pseudocolor) on the monochromatic optoacoustic image. (c) Ex vivo validation by means of cryoslicing fluorescence imaging.
Journal of Biophotonics 10/2014; 9999. · 3.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Therapeutic applications of gene silencing using siRNA have seen increasing interest and research over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained.
[Show abstract][Hide abstract] ABSTRACT: We present a time-efficient backprojection image reconstruction approach applied to frequency-domain (FD) optoacoustic tomography based on tissue illumination at multiple, discrete frequencies. The presented method estimates the Fourier transform of a spatial, circular profile of the underlying image using the amplitude and phase data. These data are collected over multiple frequencies using an acoustic transducer positioned at several locations around the sample. Fourier-transform values for absent frequencies are estimated using interpolation based on low-pass filtering in the image domain. Reconstruction results are presented for synthetic measurements using numerical phantoms, and the results are compared with FD model-based reconstructions.
[Show abstract][Hide abstract] ABSTRACT: Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm−1 at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.
Journal of Pharmaceutical and Biomedical Analysis 09/2014; · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We introduce optoacoustic tomographic imaging using intensity modulated light sources and collecting amplitude and phase information in the frequency domain. Imaging is performed at multiple modulation frequencies. The forward modeling uses the Green's function solution to the pressure wave equation in frequency domain and the resulting inverse problem is solved using regularized least squares minimization. We study the effect of the number of frequencies and of the bandwidth employed on the image quality achieved. The possibility of employing an all-frequency domain optoacoustic imaging for experimental measurements is studied as a function of noise. We conclude that frequency domain optoacoustic tomography may evolve to a practical experimental method using light intensity modulated sources, with advantages over time-domain optoacoustics.
[Show abstract][Hide abstract] ABSTRACT: Chromophore quantification in optoacoustic tomography is challenging due to signal contributions from strongly absorbing background tissue chromophores and the depth-dependent light attenuation. Herein we present a procedure capable of correcting for wavelength-dependent light fluence variations using a logarithmic representation of the images taken at different wavelengths assisted with a blind unmixing approach. It is shown that the serial expansion of the logarithm of an optoacoustic image contains a term representing the ratio between absorption of the probe of interest and other background components. Under assumptions of tissue-like background absorption variations, this term can be readily isolated with an unmixing algorithm, attaining quantitative maps of photo-absorbing agent distribution.
Physics in Medicine and Biology 08/2014; 59(17):4785-4797. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We interrogated whether optoacoustic tomography could be employed to study blood functional parameters and biodistribution of injected fluorescent agents in humans. Using a multichannel scanner at a frame rate of 10 images per second, we obtained cross-sectional images of the human finger in real time, before and after the administration of indocyanine green. We demonstrated that multispectral optoacoustic tomography can sense fast flow kinetics and resolve spatiotemporal characteristics of a common fluorochrome in human vasculature at clinically relevant concentrations. We further register ICG images with oxygen saturation maps and anatomical views of the proximal interphalangeal joint of a healthy volunteer.