Classic histology still represents the gold standard in tumor tissue analytics. However, two-dimensional analysis of single tissue slides does not provide a representative overview of the inhomogeneous tumor physiology, and a detailed analysis of complex three-dimensional structures is not feasible with this technique. To overcome this problem, we applied multispectral fluorescence ultramicroscopy (UM) to the field of tumor analysis. Optical sectioning of cleared tumor specimen provides the possibility to three-dimensionally acquire relevant tumor parameters on a cellular resolution. To analyze the virtual UM tumor data sets, we created a novel set of algorithms enabling the fully automatic segmentation and quantification of multiple tumor parameters. This new postmortem imaging technique was applied to determine the therapeutic treatment effect of bevacizumab on the vessel architecture of orthotopic KPL-4 breast cancer xenografts at different time points. A significant reduction of the vessel volume, number of vessel segments, and branching points in the tumor periphery was already detectable 1 day after initiation of treatment. These parameters remained virtually unchanged in the center of the tumor. Furthermore, bevacizumab-induced vessel normalization and reduction in vascular permeability diminished the penetration behavior of trastuzumab-Alexa 750 into tumor tissue. Our results demonstrated that this newimaging method enables the three-dimensional visualization and fully automatic quantification of multiple tumor parameters and drug penetration on a cellular level. Therefore,UM is a valuable tool for cancer research and drug development. It bridges the gap between common macroscopic and microscopic imaging modalities and opens up new three-dimensional (3D) insights in tumor biology.
We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied.
gold nanorods; optoacoustic; dendritic polyglycerolsulfate; inflammation; polyanion; MSOT.
White-light surveillance colonoscopy is the standard of care for the detection and removal of premalignant lesions to prevent colorectal cancer, and the main screening recommendation following treatment for recurrence detection. However, it lacks sufficient diagnostic yield, exhibits unacceptable adenoma miss-rates and is not capable of revealing functional and morphological information of the detected lesions. Fluorescence molecular guidance in the near-infrared (NIR) is expected to have outstanding relevance regarding early lesion detection and heterogeneity characterization within and among lesions in these interventional procedures. Thereby, superficial and sub-surface tissue biomarkers can be optimally visualized due to a minimization of tissue attenuation and autofluorescence by comparison with the visible, which simultaneously enhance tissue penetration and assure minimal background. At present, this potential is challenged by the difficulty associated with the clinical propagation of disease-specific contrast agents and the absence of a commercially available endoscope that is capable of acquiring wide-field, NIR fluorescence at video-rates. We propose two alternative flexible endoscopic fluorescence imaging methods, each based on a CE certified commercial, clinical grade endoscope, and the employment of an approved monoclonal antibody labeled with a clinically applicable NIR fluorophore. Pre-clinical validation of these two strategies that aim at bridging NIR fluorescence molecular guidance to clinical translation is demonstrated in this study.
(170.2150) Endoscopic imaging; (170.2680) Gastrointestinal
Intravascular near-infrared fluorescence (iNIRF) imaging can enable the in vivo visualization of biomarkers of vascular pathology, including high-risk plaques. The technique resolves the bio-distribution of systemically administered fluorescent probes with molecular specificity in the vessel wall. However, the geometrical variations that may occur in the distance between fibre-tip and vessel wall can lead to signal intensity variations and challenge quantification. Herein we examined whether the use of anatomical information of the cross-section vessel morphology, obtained from co-registered intravascular ultrasound (IVUS), can lead to quantification improvements when fibre-tip and vessel wall distance variations are present. The algorithm developed employs a photon propagation model derived from phantom experiments that is used to calculate the relative attenuation of fluorescence signals as they are collected over 360 degrees along the vessel wall, and utilizes it to restore accurate fluorescence readings. The findings herein point to quantification improvements when employing hybrid iNIRF, with possible implications to the clinical detection of high-risk plaques or blood vessel theranostics.
The progression of atherosclerosis involves complex changes in the structure, composition and biology of the artery wall. Currently, only anatomical plaque burden is routinely characterized in living patients, whereas compositional and biological changes are mostly inaccessible. However, anatomical imaging alone has proven to be insufficient for accurate diagnostics of the disease. Multispectral optoacoustic tomography offers complementary data to anatomical methods and is capable of imaging both tissue composition and, via the use of molecular markers, the biological activity therein. In this paper we review recent progress in multispectral optoacoustic tomography imaging of atherosclerosis with specific emphasis on intravascular applications. The potential capabilities of multispectral optoacoustic tomography are compared with those of established intravascular imaging techniques and current challenges on the road towards a clinically viable imaging modality are discussed.
atherosclerosis; intravascular imaging; molecular imaging; multispectral imaging; optical imaging; optoacoustic imaging; photoacoustic imaging; ultrasound
Cardiomyocyte loss via apoptosis plays a crucial role in ventricular remodeling following myocardial infarction (MI). Cell-based therapy approaches using bone marrow derived c-kit+ pluripotent cells may attenuate apoptosis following ischemic injury. We therefore thought to examine the early course of apoptosis following myocardial infarction - in-vivo - and non-invasively determine the effect of c-kit+ bone marrow cells on post-MI remodeling. We studied apoptosis in wild-type Kit+/+, c-kit mutant KitW/KitW-v and KitW/KitW-v mice after cell therapy with bone-marrow derived c-kit+ cells after ischemia-reperfusion injury. Mice were followed by hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) at 6h, 24h and 7 days after ischemia-reperfusion injury using an Annexin V-based fluorescent nanosensor targeting phosphatidylserine. KitW/KitW-v mice showed increased and prolonged apoptosis compared to control Kit+/+ mice while c-kit cell therapy was able to attenuate the altered apoptosis rates. Increased apoptosis was accompanied by severe decline in heart function, determined by cardiac Magnetic Resonance Imaging, and cell therapy was able to rescue the animals from deleterious heart failure. Post-mortem cryoslicing and immunohistochemistry localized the fluorescence signal of the Annexin V sensor within the infarcted myocardium. Flow cytometry of digested infarct specimens identified apoptotic cardiomyocytes as the major source for the in-vivo Annexin V signal.
In-vivo molecular imaging using hybrid FMT-XCT reveals increased cardiomyocyte apoptosis in KitW/KitW-v mice and shows that c-kit+ cardioprotective cells are able to attenuate post-MI apoptosis and rescue mice from progressive heart failure.
apoptosis; heart failure; infarction; imaging; reperfusion.
The hybrid nature of optoacoustic imaging might impose limitations on concurrent placement of optical and ultrasonic detection components, especially in high resolution microscopic applications that require dense arrangements and miniaturization of components. This hinders optimal deployment of the optical excitation and ultrasonic detection paths, leading to reduction of imaging speed and spatial resolution performance. We suggest a compact coaxial design for optoacoustic microscopy that allows optimizing both the light illumination and ultrasonic detection parameters of the imaging system. System performance is showcased in phantoms and in vivo imaging of microvasculature, achieving real time operation in two dimensions and penetration of 6 mm into optically dense human tissues.
(170.5120) Photoacoustic imaging; (110.6880) Three-dimensional image acquisition; (170.3880) Medical and biological imaging; (120.3890) Medical optics instrumentation
To develop a two-dimensional intravascular near-infrared fluorescence (NIRF) imaging strategy for investigation of arterial inflammation in coronary-sized vessels.
Molecular imaging of arterial inflammation could provide new insights into the pathogenesis of acute myocardial infarction stemming from coronary atheromata and implanted stents. Presently few high-resolution inflammation approaches can image inflammation in coronary-sized arteries in vivo.
A new 2.9F rotational, automated pullback two-dimensional imaging catheter was engineered and optimized for 360-degree viewing intravascular NIRF imaging. In conjunction with the cysteine protease-activatable imaging reporter Prosense VM110, intra-arterial 2D NIRF imaging was performed in rabbit aortas with atherosclerosis (n=10) or implanted coronary bare metal stents (n=10, 3.5mm diameter, day 7 post-implantation). Intravascular ultrasound (IVUS) provided co-registered anatomical images of arteries. After sacrifice, specimens underwent ex vivo NIRF imaging, fluorescence microscopy, and histological and immunohistochemical analyses.
Imaging of coronary artery-scaled phantoms demonstrated 8-sector angular resolution and submillimeter axial resolution, nanomolar sensitivity to NIR fluorochromes, and modest NIRF light attenuation through blood. High-resolution NIRF images of vessel wall inflammation with signal-to-noise ratios > 10 were obtained in real-time through blood, without flushing or occlusion. In atherosclerosis, 2D NIRF, IVUS-NIRF fusion, microscopy, and immunoblotting studies provided insight into the spatial distribution of plaque protease activity. In stent-implanted vessels, real-time imaging illuminated an edge-based pattern of stent-induced arterial inflammation.
A new 2D intravascular NIRF imaging strategy provides high-resolution in vivo spatial mapping of arterial inflammation in coronary-sized arteries, and reveals increased inflammation-regulated cysteine protease activity in atheromata and stentinduced arterial injury.
intravascular imaging; inflammation; atherosclerosis; stent; molecular imaging
Advancing our understanding of human coronary artery disease requires new methods that can be used in patients for studying atherosclerotic plaque microstructure in relation to the molecular mechanisms that underlie its initiation, progression, and clinical complications, including myocardial infarction and sudden cardiac death. Here we report a dual-modality intra-arterial catheter for simultaneous microstructural and molecular imaging in vivo using a combination of optical frequency domain imaging (OFDI) and near-infrared fluorescence (NIRF) imaging. By providing simultaneous molecular information in the context of the surrounding tissue microstructure, this novel catheter could provide new opportunities for investigating coronary atherosclerosis and stent healing, and for identifying high-risk biological and structural coronary arterial plaques in vivo.
To date there is a lack of tools to map the spatio-temporal dynamics of diverse cells in experimental heart models. Conventional histology is labor intensive with limited coverage, whereas many imaging techniques do not have sufficiently high enough spatial resolution to map cell distributions. We have designed and built a high resolution, dual channel Born-normalized near-infrared fluorescence optical projection tomography system to quantitatively and spatially resolve molecular agents distribution within whole murine heart. We validated the use of the system in a mouse model of monocytes/macrophages recruitment during myocardial infarction. While acquired, data were processed and reconstructed in real time. Tomographic analysis and visualization of the key inflammatory components were obtained via a mathematical formalism based on left ventricular modeling. We observed extensive monocyte recruitment within and around the infarcted areas and discovered that monocytes were also extensively recruited into non-ischemic myocardium, beyond that of injured tissue, such as the septum.
Tumor targeting is of high clinical and biological relevance, and major efforts have been made to develop molecular imaging technologies for visualization of the disease markers in tissue. Of particular interest is apoptosis which has a profound role within tumor development and has significant effect on cancer malignancy.
Herein, we report on targeting of phosphatidylserine-exposing cells within live tumor allograft models using a synthetic near infrared zinc(II)-dipicolylamine probe. Visualization of the probe biodistribution is performed with whole body multispectral optoacoustic tomography (MSOT) system and subsequently compared to results attained by planar and tomographic fluorescence imaging systems.
Compared to whole body optical visualization methods, MSOT attains remarkably better imaging capacity by delivering high-resolution scans of both disease morphology and molecular function in real time. Enhanced resolution of MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that its tumor selectivity is gained by targeting the phosphatidylserine exposed on the surface of tumor vessels.
The current study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development.
Optoacoustic imaging; Tumor targeting; Molecular imaging; Phosphatidylserine targeting
The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research.
New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. While molecular imaging agents are available for lower-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries [AU: ok? YES]. Here, we have demonstrated that indocyanine green (ICG), an FDA-approved near-infrared fluorescence (NIRF) emitting compound, targets atheromas within 20 minutes of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aortae, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging studies showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG, compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.
Elevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology.
Methods and results
MMP-sensitive activatable fluorescent probe (MMPSense™ 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by in situ zymography, accompanied by increased macrophage influx.
We demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making.
Atherosclerosis; Optoacoustic imaging; Carotid arteries; Plaque; Contrast media; Inflammation; Medicine & Public Health; Imaging / Radiology
Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).
We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.
(170.6960) Tomography; (170.3880) Medical and biological imaging
The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.
Biomedical imaging has become an important tool in the study of “-omics” fields by allowing the noninvasive visualization of functional and molecular events using in vivo staining and reporter gene approaches. This capacity can go beyond the understanding of the genetic basis and phenotype of such respiratory conditions as acute bronchitis, adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and asthma and investigate the development of disease and of therapeutic events longitudinally and in unperturbed environments. Herein, we show how the application of novel quantitative optical imaging methods, using transillumination and fluorescence molecular tomography (FMT), can allow visualization of pulmonary inflammation in small animals in vivo. The results confirm prior observations using a protease-sensitive probe. We discuss how this approach enables in vivo insights at the system level as to the dynamic role of proteases in respiratory pathophysiology and their potential as therapeutic targets. Overall, the proposed imaging method can be used with a significantly wider range of possible targets and applications in lung imaging.
fluorescence; tomography; proteases; lung; inflammation, in vivo
Optical projection tomography is a new ex vivo imaging technique that allows imaging of whole organs in three dimensions at high spatial resolutions. In this Letter we demonstrate its capability to tomographically visualize molecular activity in whole organs of mice. In particular, eosinophil activity in asthmatic lungs is resolved using a Born-normalized fluorescence optical projection tomography and employing a near-IR molecular probe. The possibility to achieve molecularly sensitive imaging contrast in optical projection tomography by means of targeted and activatable imaging reporter agents adds a new range of capabilities for investigating molecular signatures of pathophysiological processes and a wide variety of diseases and their development.
Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimer's, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered.
We present a normalized Born approach for fluorescence optical projection tomography that takes into account tissue absorption properties. This approach can be particularly useful to study fluorochrome distribution within tissue. We use the algorithm to three-dimensionally reconstruct and characterize a fluorescein isothiocyanate containing absorptive phantom and an infarcted mouse heart previously injected with a fluorescent molecular probe.
We report on a systematic study of upconverting fluorescence signal generation within turbid phantoms and real tissues. An accurate three-point Green's function, describing the forward model of photon propagation, is established and experimentally validated. We further demonstrate, for the first time to our knowledge, autofluorescence-free transillumination imaging of mice that have received biocompatible upconverting nanoparticles. The method holds great promise for artifact-free whole-body visualization of optical molecular probes.
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 non-invasive 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. In order to determine the optimal choice of FP and illumination wavelength we compared the performance of five of the most promising FPs, i.e. tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding on the performance of different illumination scenarios, especially as it pertains 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 2 orders of magnitude compared to Green FP (GFP). It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600nm range may significantly affect the detection sensitivity and necessitates the careful selection of illumination wavelengths for optimal imaging performance.
Fluorescent proteins; optical tomography; whole body imaging
Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5–1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) . Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging.
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography , which appropriate for non-invasive in-vivo imaging at dimensions of 1mm–5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.