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Nonlinear optical microscopy has enabled the possibility to explore inside the living organisms. It utilizes ultrashort laser pulse with long wavelength (greater than 800nm). Ultrashort pulse produces high peak power to induce nonlinear optical phenomenon such as two-photon excitation fluorescence (TPEF) and harmonic generations in the medium while maintaining relatively low average energy pre area. In plant developmental biology, confocal microscopy is widely used in plant cell imaging after the development of biological fluorescence labels in mid-1990s. However, fluorescence labeling itself affects the sample and the sample deviates from intact condition especially when labelling the entire cell. In this work, we report the dynamic images of Arabidopsis thaliana root cells. This demonstrates the multimodal nonlinear optical microscopy is an effective tool for long-term plant cell imaging.

Quantitative assessment of upper airway geometry using optical coherence tomography in burns patients could provide physicians with the information needed to make critical decisions. We have developed a high speed catheter based OCT system capable of real time imaging in airways up to 3cm in diameter. Preliminary scans of inhalation injured airways are presented to demonstrate the feasibility of aOCT as a diagnostic tool for assessing burns patients.

A compact fiber grating laser has been exploited as an ultrasound sensor to probe optically induced spherical elastic waves, taking advantage of its response in beat-frequency variation of the laser output. Optical-resolution photoacoustic microscopy (PAM) is further implemented with such a sensor by raster scanning the excitation light with a 2-axis galvo scanner. A PAM image of mouse ear with a field width of 2 mm is demonstrated. The wide field-of-view of the sensor allows the implementation of fast-scanning PAMs which is attractive for in vivo imaging applications.

Initial feasibility of a spatial frequency domain imaging system was studied consisting of a hand held miniaturized projector and a rigid endoscope. Three wavelengths and two spatial frequencies were used for imaging. The system was calibrated using tissue mimicking phantoms. In vivo imaging was performed on five live mouse tumor models, and the absorption, scattering, hemoglobin oxygen saturation was measured. The initial promising results indicate that the spatial frequency domain imaging can a very useful tool for quantitative wide field tissue evaluation during minimally invasive image guided surgery.

Recent years have seen rapid development of hybrid optical-acoustic imaging modalities with broad applications in research and clinical imaging, including photoacoustic tomography (PAT), photoacoustic microscopy, and ultrasound-modulated optical tomography. Tissue-mimicking phantoms are an important tool for objectively and quantitatively simulating in vivo imaging system performance. However, no standard tissue phantoms exist for such systems. One major challenge is the development of tissue-mimicking materials (TMMs) that are both highly stable and possess biologically realistic properties. To address this need, we have explored the use of various formulations of PVC plastisol (PVCP) based on varying mixtures of several liquid plasticizers. We developed a custom PVCP formulation with optical absorption and scattering coefficients, speed of sound, and acoustic attenuation that are tunable and tissue-relevant. This TMM can simulate different tissue compositions and offers greater mechanical strength than hydrogels. Optical properties of PVCP samples with varying composition were characterized using integrating sphere spectrophotometry and the inverse adding-doubling method. Acoustic properties were determined using a broadband pulse-transmission technique. To demonstrate the utility of this bimodal TMM, we constructed an image quality phantom designed to enable quantitative evaluation of PAT spatial resolution. The phantom was imaged using a custom combined PAT-ultrasound imaging system. Results indicated that this more biologically realistic TMM produced performance trends not captured in simpler liquid phantoms. In the future, this TMM may be broadly utilized for performance evaluation of optical, acoustic, and hybrid optical-acoustic imaging systems.

High resolution micro-optical coherence tomography (µOCT) technology has been demonstrated to be useful for imaging respiratory epithelial functional microanatomy relevant to the study of pulmonary diseases such as cystic fibrosis and COPD. We previously reported the use of a benchtop μOCT imaging technology to image several relevant respiratory epithelial functional microanatomy at 40 fps and at lateral and axial resolutions of 2 and 1.3μm, respectively. We now present the development of a portable μOCT imaging system with comparable optical and imaging performance, which enables the μOCT technology to be translated to the clinic for in vivo imaging of human airways. Published version

Currently, the majority of microscopic and endoscopic technologies utilize white light illumination. For a number of applications, hyper-spectral imaging can be shown to have significant improvements over standard white-light imaging techniques. This is true for both microscopy and in vivo imaging. However, hyperspectral imaging methods have suffered from slow application times. Often, minutes are required to gather a full imaging stack. Here we will describe the system and evaluate optimizations and applications of a novel excitation-scanning hyperspectral imaging system. We have developed and are optimizing a novel approach called excitation-scanning hyperspectral imaging that provides an order of magnitude increased signal strength. Optimization of the light path, optical components and illumination sources have allowed us to achieve high speed image acquisition. This high speed allows for potential live video acquisition. This excitation-scanning hyperspectral imaging technology has potential to impact a range of applications. The current system allows triggering of up to 16 wavelengths at less than 1 millisecond per image using digital strobing. Analog intensity control is also provided for a fully customizable excitation profile. A significant advantage of excitation-scanning hyperspectral imaging is can identify multiple targets simultaneously in real time. We are optimizing the system to compare sensitivity and specificity of excitation-scanning hyperspectral imaging with pathology techniques. Finally, we are exploring utilizing this technology to measure cAMP distribution in three dimensions within a cell.

We demonstrate a flexible stand-alone, minimally invasive video-endomicroscope with an outer diameter of 1.6 mm and a length of the rigid tip of 6.7 mm that enables surgeons and biologists to image hardly accessible regions in-vivo in epifluorescence mode. The 60 mg light device improves state-of-the-art objectives by a double deflection approach using a side-fire fiber in combination with spherical microlenses, GRIN-lenses with a specific adapted gradient index profile and an extremely miniaturized chip-on-the-tip camera to achieve an excellent imaging quality. A high NA of 0.7 enables the observation of subcellular features within the entire field of view with a diameter of 183 μm, assure a bright and high-contrast image and promise a good overview during the intervention. Ex-vivo measurements of biological samples confirmed the functionality of the probe.

We propose a rapid imaging method to monitor the spatial distribution of total hemoglobin concentration (CHbT), the tissue oxygen saturation, and the scattering power b in the expression of μs’=aλ-b as the scattering parameters in cerebral cortex using a digital red-green-blue camera. In the method, the RGB-values are converted into the tristimulus values in CIEXYZ color space which is compatible with the common RGB working spaces. Monte Carlo simulation (MCS) for light transport in tissue is used to specify a relation among the tristimulus XYZ-values and the concentration of oxygenated hemoglobin, that of deoxygenated hemoglobin, and the scattering power b. In the present study, we performed sequential recordings of RGB images of in vivo exposed rat brain during the cortical spreading depolarization evoked by the topical application of KCl. Changes in the total hemoglobin concentration and the tissue oxygen saturation imply the temporary change in cerebral blood flow during CSD. Decrease in the scattering power b was observed before the profound increase in the total hemoglobin concentration, which is indicative of the reversible morphological changes in brain tissue during CSD. The results in this study indicate potential of the method to evaluate the pathophysiological conditions in brain tissue with a digital red-green-blue camera.

In the case of infiltrative brain tumors the surgeon faces difficulties in determining their boundaries to achieve total resection. The aim of the investigation was to evaluate the performance of multimodal OCT (MM OCT) for differential diagnostics of normal brain tissue and glioma using an experimental model of glioblastoma. The spectral domain OCT device that was used for the study provides simultaneously two modes: cross-polarization and microangiographic OCT. The comparative analysis of the both OCT modalities images from tumorous and normal brain tissue areas concurrently with histologic correlation shows certain difference between when accordingly to morphological and microvascular tissue features.

"Surface-enhanced Raman spectroscopy" (SERS) nanoparticles have gained much attention in recent years for in silico, in vitro and in vivo sensing applications. Our group has developed novel generations of biocompatible "surfaceenhanced resonance Raman spectroscopy" (SERRS) nanoparticles as novel molecular imaging agents. Via rigorous optimization of the different variables contributing to the Raman enhancement, we were able to design SERRS nanoparticles with so far unprecedented sensitivity of detection under in vivo imaging conditions (femto-attomolar range). This has resulted in our ability to visualize, with a single nanoparticle, many different cancer types (after intravenous injection) in mouse models. The cancer types we have tested so far include brain, breast, esophagus, stomach, pancreas, colon, sarcoma, and prostate cancer. All mouse models used are state-of-the-art and closely mimic the tumor biology in their human counterparts. In these animals, we were able to visualize not only the bulk tumors, but importantly also microscopic extensions and locoregional satellite metastases, thus delineating for the first time the true extent of tumor spread. Moreover, the particles enable the detection of premalignant lesions. Given their inert composition they are expected to have a high chance for clinical translation, where we envision them to have an impact in various scenarios ranging from early detection, image-guidance in open or minimally invasive surgical procedures, to noninvasive imaging in conjunction with spatially offset (SESORS) Raman detection devices.

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

A hybrid system combining fluorescence molecular tomography (FMT) and magnetic resonance imaging (MRI) is attractive for preclinical imaging as it allows fusion of molecular information derived from FMT and anatomical reference data derived from MRI. We have previously developed such a system and demonstrated its performance in biological applications. For reconstruction slab geometry with homogeneous optical parameters was assumed, which led to undesirable artifacts. In order to exploit the power of the hybrid system, the use of MRI derived anatomical information, as a constraint for FMT reconstruction, appears logical. Heterogeneity of tissues and irregular surface derived from MRI can be accounted for by generating a mesh using the finite element method (FEM), and attributing optical parameters to individual mesh points. We have established a forward simulation tool based on TOAST ++ to mimic an FMT experiment. MRI images were recorded on a 9.4T MR scanner using a T1-weighted pulse sequence. The voxelized dataset was processed by iso2mesh to yield a 3D-mesh. Four steps of FMT simulation were included: 1) Assignment of optical properties, 2) Specification of boundary conditions and generation of 3) excitation and 4) emission maps. FEM-derived results were compared with those obtained using the analytical solution of Green's function and with experimental data with a single fluorescent inclusion in a silicon phantom. Once, the forward modeling method is properly validated it will be used as a central element of a reconstruction algorithm for analyzing data derived from a hybrid FMT/MRI setup.

Recently, photoacoustic tomography (PAT) has emerged as a remarkable non-invasive imaging modality that provides a strong optical absorption contrast, high ultrasonic resolution, and great penetration depth. Thus, PAT has been widely used as an in vivo preclinical imaging tool. Surface-enhanced Raman spectroscopy (SERS) is another attractive sensing technology in biological research because it offers highly sensitive chemical analyses and multiplexed detection. By performing dual-modal imaging of SERS and PAT, high-resolution structural PAT imaging and high-sensitivity SERS sensing can be achieved. At the same time, it is equally important to develop a dual modal contrast agent for this purpose. To perform both PAT and SERS, we synthesized PEGylated silver bumpy nanoshells (AgBSs). The AgBSs generate strong PA signals owing to their strong optical absorption properties as well as sensitive SERS signals because of the surface plasmon resonance effect. Then, multiplexed Raman chemicals were synthesized to enhance the sensitivity of Raman. We have photoacoustically imaged the sentinel lymph nodes of small animals after intradermal injection of multiplexed agents. Furthermore, the chemical composition of each agent has been distinguished through SERS.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique for microvasculature imaging at high spatial resolution and contrast. In this work, we present a practical visible laser-diode-based OR-PAM (LD-OR-PAM) prototype for vasculature imaging, which has the desirable properties of being portable, low-cost, and label-free. The prototype employs a 300 mW pulsed laser diode in a 3.8 mm diameter package, emitting 174 ns pulses at 405 ± 5 nm wavelength and a pulse energy of 52 nJ. An aspheric objective with a numerical aperture of 0.60 is used to achieve microscope optical illumination. The laser diode excitation has a compact size of 4.5 × 1.8 × 1.8 cm3 assembled with a cooling block. The lateral resolution was tested to be 0.95 μm on ~7 μm carbon fibers. The subcutaneous microvasculature on a mouse back was label-free imaged ex vivo, which demonstrates the potential of the LD-OR-PAM prototype for in vivo imaging skin chromosphores such as hemoglobin. Our ultimate aim is to provide a practical and affordable OR-PAM system as a routine instrument for standard clinical applications.

Ultrasound flow imaging is widely used for quantification of blood flow in vivo, but estimating low flow velocities remains challenging. Pulsed photoacoustic flowmetry could be an alternative – but has not been shown capable of deep in vivo imaging so far. A new photoacoustic system is proposed, that has more potential for deep in-vivo applications. In this work the system is tested in vitro. In contrast to earlier research, 1064 nm NIR laser irradiation is used, that would allow deeper in-vivo light penetration. For detection, a 15 MHz transducer with lower in-vivo tissue ultrasound attenuation is used. Both changes are not trivial, as they reduce the overall visibility of the photoacoustic signals from red blood cells. This work shows the flow estimation performance of this system in vitro, and aims to serve as a point of reference when moving to an in-vivo application. Using this 15 MHz transducer, 1D flow profiles of flowing blood were extracted. The jitter (standard error) in the velocity estimation over the profiles was 10% when estimating flow of particles and 20% with whole blood; the bias in flow estimation was roughly 30%.

Optoacoustic Tomography is a fast developing imaging modality, combining the high resolution and penetration depth of ultrasound detection with the high contrast available from optical absorption in tissue. The spectral profile of near infrared excitation light used in optoacoustic tomography instruments is modified by absorption and scattering as it propagates deep into biological tissue. The resulting images therefore provide only qualitative insight into the distribution of tissue chromophores. Knowledge of the spectral profile of excitation light across the mouse is needed for accurate determination of the absorption coefficient in vivo. Under the conditions of constant Grueneisen parameter and accurate knowledge of the light fluence, a linear relationship should exist between the initial optoacoustic pressure amplitude and the tissue absorption coefficient. Using data from a commercial optoacoustic tomography system, we implemented an iterative optimization based on the σ-Eddington approximation to the Radiative Transfer Equation to derive a light fluence map within a given object. We segmented the images based on the positions of phantom inclusions, or mouse organs, and used known scattering coefficients for initialization. Performing the fluence correction in simple phantoms allowed the expected linear relationship between recorded and independently measured absorption coefficients to be retrieved and spectral coloring to be compensated. For in vivo data, the correction resulted in an enhancement of signal intensities in deep tissues. This improved our ability to visualize organs at depth (> 5mm). Future work will aim to perform the optimization without data normalization and explore the need for methodology that enables routine implementation for in vivo imaging.

Optical imaging has long been hindered by the high absorption and scattering of light in biological tissue. This makes it difficult to probe beyond a few millimeters beneath the surface without sacrificing image resolution and quantitative accuracy. Strong scattering and the inherent nature of the inverse problem makes fluorescence diffuse optical tomography (FT) extremely challenging. To this end, multi-modality techniques that combine anatomical imaging with the functional optical information have been used to improve the resolution and accuracy of FT. Previously, we have reported on the feasibility of a new imaging method, "Thermal Outlining using Focused Ultrasound" (TOFU), which combines the sensitivity of FT with the resolution of focused ultrasound using temperature reversible fluorescent probes. In this method, the position of the temperature reversible fluorescent probes is localized by an increase in fluorescent signal when the hot spot of the focused ultrasound beam is scanned over the medium. This a priori information is then utilized to guide and constrain conventional reconstruction algorithm to recover the position and concentration of the probes more accurately. The small size of the focal spot (~1.4 mm) up to a depth of 6 cm, allows imaging the distribution of these temperature sensitive agents with not only high spatial resolution but also high quantitative accuracy in deep tissue. In this work, the performance of the system will be evaluated using simulation and phantoms to investigate the dependence that size of the fluorescent distribution has on the TOFU system performance.

International audience; Persistent luminescence nanoparticles have recently been proposed as innovative optical probes for small animal in vivo imaging. The main advantage of such probes is their ability to emit light for a long time after the end of their excitation, allowing in vivo imaging with low background. This work reports new information on the physico-chemical characterizations of Cr doped ZnGa2O4 nanoprobes in terms of synthetic procedure, luminescence properties as well as colloidal stabilities in different aqueous media and over the time.

We have developed an automated, wide-field optical coherence tomography (OCT)-based imaging device (OTIS TM Perimeter Medical Imaging) for peri-operative, ex-vivo tissue imaging. This device features automated image acquisition, enabling rapid capture of high-resolution (15 μm) OCT images from samples up to 10 cm in diameter. We report on the iterative progression of device development from phantom and pre-clinical (tumor xenograft) models through to initial clinical results. We discuss the challenges associated with proving a novel imaging technology against the clinical “gold standard” of conventional post-operative pathology.

Cone beam computed tomography (CBCT) is limited in guiding irradiation for soft tissue targets. As a complementary imaging modality, bioluminescence tomography (BLT) provides strong soft tissue contrast. We developed a dual-use BLT system which consists of an optical assembly, a mobile cart and an independent mouse bed. The system is motorized which can easily dock onto an independent mouse bed operating as a standalone system for longitudinal bioluminescence imaging (BLI)/BLT studies and also dock onto the SARRP for on-line radiation guidance. Our initial tests for the system demonstrate that (i) the imaging depth is 28 mm, (ii) the optical background is sufficiently low and uniform, (iii) the non-uniform response of the optical imaging can be corrected by the flat field correction, and (iv) the imaging acquisition speed was improved by an average of 3.7 times faster than our previous systems. We also presented a geometry calibration procedure to map the planar BLIs acquired at multi-projections onto the surface of the CBCT image. The CBCT is required to generate the mesh for BLT reconstruction and used for treatment planning and radiation delivery. Feasibility study of the geometry calibration was performed on a manual-docking prototype. The mean and maximum mapping accuracy is 0.3 and 0.6 mm. The performance of the proposed motorized dual-use system is expected to be superior to that of the manual-docking prototype because of the mechanism stability. We anticipate the dual-use system as a highly efficient and cost-effective platform to facilitate optical imaging for preclinical radiation research.

Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

In vivo imaging technologies such as optical coherence tomography (OCT) and rodent models enable longitudinal studies of different ophthalmic pathologies and their underlying physiological changes. In this work a custom-made high resolution polarization sensitive OCT system is used to image the posterior eye of small rodents like mice and rats. Enface projections at certain depths/levels in the three-dimensional (3D) reflectivity images enable to visualize different vascular structures in the retina as well as in the choroid. Further, phase resolved motion contrast based OCT angiography allows to create vascular maps with a high contrast to distinguish between static tissue and dynamic scatterers such as red blood cells. The levels of the vascular maps are determined by a semi-automatic approach in which layers of the retina are segmented in a first step. In a second step the projection level can be adjusted by the operator to restrict the volume in which the en-face projection is carried out. To demonstrate the functionality of this approach rats and mice of different strains were imaged. The anesthetized rodents were scanned with an angle of up to 28° × 28°. Respiration induced motion artefacts were compensated by post-processing. The vascular maps based on the OCT reflectivity information as well as on the phase variance showed that the resolution of the custom-made systems is suitable to resolve small retinal capillaries in the outer plexiform layer. Further, larger vessels within the nerve fiber layer and the inner nuclear layer as well as in the choroid were visualized by the proposed approach.

This paper presents a novel THz optical design that allows the acquisition of THz reflectivity maps of in vivo cornea without the need for a field flattening window and preliminary imaging results of in vivo rabbit cornea. The system’s intended use is to sense small changes in corneal tissue water content (CTWC) that can be precursors for a host of diseases and pathologies. Unique beam optics allows the scanning of a curved surface at normal incidence while keeping the source detector and target stationary. Basic system design principles are discussed and image sets of spherical calibration targets and corneal phantom models are presented. The presented design will enable, for the first time, non-contact THz imaging of animal and human cornea.

Fluorescence from endogenous fluorophores has been emerging as a promising biomarker for tissue discrimination resulting a noninvasive screening methodology to understand the biochemical and morphological variations in tissues associated with cancer development. We have developed a scan based fiber optic probe system to image increased flavin adenine dinucleotide (FAD) fluorescence from epithelial tissues under conditions mimicking dysplasia surrounded by normal tissues. Experiments were conducted on optical phantoms mimicking epithelial tissues excited by 450nm LED source. The spectral emission from the sample is collected via optical fibers and the imaging is performed by scanning the sample using a translation stage at desired resolution. Monte Carlo simulations were also performed by devising an optical model corresponding to epithelial tissue and the results were correlated with experimental fluorescence measurements. This whole field imaging approach could be useful for in vivo assessment of tissue pathologies based on auto fluorescence and can give a better quantitative approach for estimation of tissue properties by correlating the experimental and simulated data.

Photoacoustic tomography is expected to impact biology and medicine broadly by providing multiscale in vivo functional and molecular imaging of structures ranging from subcellular organelles to organs, enabling a noninvasive look at subcutaneous tissue at a deep level. Lihong Wang holds the Gene K. Beare Distinguished Professorship of Biomedical Engineering at Washington University in St. Louis, and is Editor-in-Chief of the Journal of Biomedical Optics. Wang was awarded the 2015 Britton Chance Biomedical Optics Award for his pioneering technical contributions and visionary leadership in the development and application of photoacoustic tomography, photoacoustic microscopy, and photon transport modeling.

Activatable photoacoustic probes hold great promise for in vivo imaging of enzyme activity as they exhibit high contrast and high selectivity at depths similar to that of ultrasound imaging. Here we report the synthesis and testing of a matrix metalloproteinase 2 (MMP-2) specific peptide probe capable of changing its transient photoacoustic lifetime from short to long after activation. The intact probe comprises an enzyme-specific cleavable sequence conjugated to a pair of methylene blue (MB) molecules that dimerize due to forced proximity, resulting in static quenching. Upon cleavage, the MB molecules dissociate and recover their intrinsic excited-state lifetime of more than 2 μs. We demonstrated using a pump-probe photoacoustic imaging approach that the cleaved probe exhibits a transient signal with lifetime comparable to that of MB monomers, whereas no lifetime was detected for the intact probe. This could allow for detection of the cleaved probe in the presence of high levels of intact probe as well as short transients due to endogenous tissue absorbers, thereby providing high contrast and low background noise. Finally, we have compared peptide sequences of varying length and structure using absorption spectrometry in order to select the probe best suited for our imaging needs. Our results suggest that both factors could potentially have an impact on dimerization efficiency and the cleavage rate of the peptide. However, all probes provided a high degree of dimerization and efficient separation after cleavage, indicating that a lifetime-based activatable peptide can be constructed for in vivo applications using a two-wavelength imaging approach.

Fluorescent tomography has been hindered by poor tissue penetration and weak signal which results in poor spatial resolution and quantification accuracy. Recently, it has been reported that activatable temperature responsive fluorescent probes which respond to focused ultrasound heating can improve the resolution and quantification of fluorescent tomography in deep tissue. This has lead to a new imaging modality, "Temperature-modulated fluorescent tomography." This technique relies on activatable thermo-sensitive fluorescent nanocapsules for whose fluorescence quantum efficiency is temperature dependent. Within a 4-5° C temperature range, the fluorescent signal increase more than 10-fold. In this molecular probe, Indocyanine Green (ICG) is encapsulated inside the core of a thermo-reversible pluronic micelle. Here we show the fluorescence response and temperature range of the nanocapsules which have been optimized for a higher temperature range to be used for in vivo animal imaging. We report on the feasibility of these temperature-sensitive reversible nanocapsules for in vivo applications by studying the pharmacokinetics in a subcutaneous mouse tumor model in vivo.

We demonstrate high-resolution imaging of the living human retina by computationally correcting highorder ocular aberrations. These corrections are performed post-acquisition and without the need for a deformable mirror or wavefront sensor that are commonly employed in hardware adaptive optics (HAO) systems. With the introduction of HAO to ophthalmic imaging, high-resolution near diffraction-limited imaging of the living human retina has become possible. The combination of a deformable mirror, wavefront sensor, and supporting hardware/software, though, can more than double the cost of the underlying imaging modality, in addition to significantly increasing the system complexity and sensitivity to misalignment. Optical coherence tomography (OCT) allows 3-D imaging in addition to naturally providing the complex optical field of backscattered light. This is unlike a scanning laser ophthalmoscope which measures only the intensity of the backscattered light. Previously, our group has demonstrated the utility of a technique called computational adaptive optics (CAO) which utilizes the complex field measured with OCT to computationally correct for optical aberrations in a manner similar to HAO. Until now, CAO has been applied to ex vivo imaging and in vivo skin imaging. Here, we demonstrate in vivo imaging of cone photoreceptors using CAO. Additional practical considerations such as imaging speed, and stability are discussed.

Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

We present an ultrahigh resolution dual modality optical resolution photoacoustic microsopy (OR-PAM) and spectral domain optical coherence microscopy (SD-OCM) system. The ultrahigh sub-micron lateral resolution is provided by the high numerical aperture of the objective lens used while the ultrahigh axial resolution is provided by the broadband OCT laser that covers 107 nm with a central wavelength of 840 nm. The synchronized simultaneous acquisition for the two modalities is achieved using a 40MHz FPGA. 2D-scanning is realized by two orthogonal translation stages (PI, 400 nm resolution). The transversal resolution of the system is 0.5 μm, the axial resolutions are 30 μm (PAM) and 4 μm (OCM), respectively. The values have been determined experimentally using nanospheres (diameter 10-200nm). For a demonstration of the imaging capability we present images from thin slices of different biological samples as well as in vivo imaging in the zebrafish embryo.

The oxygen partial pressure (pO2), which results from the balance between oxygen delivery and its consumption, is a key component of the physiological state of a tissue. Images of oxygen distribution can provide essential information for identifying hypoxic tissue and optimizing cancer treatment. Previously, we have reported a noninvasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor on small animals to identify hypoxia area. We also showed that PALI is able monitor changes of tissue oxygen, in an acute ischemia and breathing modulation model. Here we present our work on developing a treatment/imaging modality (PDT-PALI) that integrates PDT and a combined ultrasound/photoacoustic imaging system. The system provides real-time feedback of three essential parameters namely: tissue oxygen, light penetration in tumor location, and distribution of photosensitizer. Tissue oxygen imaging is performed by applying PALI, which relies on photoacoustic probing of oxygen-dependent, excitation lifetime of Methylene Blue (MB) photosensitizer. Lifetime information can also be used to generate image showing the distribution of photosensitizer. The level and penetration depth of PDT illumination can be deduced from photoacoustic imaging at the same wavelength. All images will be combined with ultrasound B-mode images for anatomical reference.

Atherosclerotic plaque at the carotid bifurcation is the underlying cause of the majority of ischemic strokes. Noninvasive imaging and quantification of the compositional changes preceding gross anatomic changes within the arterial wall is essential for diagnosis of disease. Current imaging modalities such as duplex ultrasound, computed tomography, positron emission tomography are limited by the lack of compositional contrast and the detection of flow-limiting lesions. Although high-resolution magnetic resonance imaging has been developed to characterize atherosclerotic plaque composition, its accessibility for wide clinical use is limited. Here, we demonstrate a fiber-based multispectral photoacoustic tomography system for excitation of lipids and external acoustic detection of the generated ultrasound. Using sequential ultrasound imaging of ex vivo preparations we achieved ~2 cm imaging depth and chemical selectivity for assessment of human arterial plaques. A multivariate curve resolution alternating least squares analysis method was applied to resolve the major chemical components, including intravascular lipid, intramuscular fat, and blood. These results show the promise of detecting carotid plaque in vivo through esophageal fiber-optic excitation of lipids and external acoustic detection of the generated ultrasound. This imaging system has great potential for serving as a point-ofcare device for early diagnosis of carotid artery disease in the clinic.

Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

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Brain imaging is becoming an important field in the frame of the neurophotonics in correlations with other medical ones in neuroscience studying functional and morphological aspects. In this presentation an overview on multi photon imaging of the brain will be presented, together with innovative aspects related to big area imaging and correlative microscopy approaches. Multiphoton imaging applications will be described together with methods to improve the penetration depth and obtain large area detection, or correlating functional aspects in vivo on single neuron with large area, even on whole brain, morphological aspects. Connecting super resolution features at the nanometer level with micro, meso and macroscopic architectures is in fact one of the challenging aspects to understand brain functioning.

Tape stripping on human skin induces mechanical disruptions of the epidermal barrier that lead to minor skin inflammation which leads to temporary changes in microvasculature. On the other hand, when mosquitoes probe the skin for blood feeding, they inject saliva in dermal tissue. Mosquito saliva is known to exert various biological activities, such as dermal mast cell degranulation, leading to fluid extravasation and neutrophil influx. This inflammatory response remain longer than the tape stripping caused inflammation. In this study, we demonstrate the capabilities of swept-source optical coherence tomography (OCT) in detecting in vivo microvascular response of inflammatory human skin. Optical microangiography (OMAG), noninvasive volumetric microvasculature in vivo imaging method, has been used to track the vascular responses after tape stripping and mosquito bite. Vessel density has been quantified and used to correlate with the degree of skin irritation. The proved capability of OMAG technique in visualizing the microvasculature network under inflamed skin condition can play an important role in clinical trials of treatment and diagnosis of inflammatory skin disorders as well as studying mosquito bite’s perception by the immune system and its role in parasite transmission.

Peritoneal carcinomatosis is metastatic stage aggravating digestive, gynecological or bladder cancer dissemination and the preoperative evaluation of lesions remains difficult. There is therefore a need for minimal invasive innovative techniques to establish a precise preoperative assessment of cancer peritoneal cavity. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of the microarchitecture of tissues during an endoscopy. The PERSEE project proposes new developments in robotics and pCLE for the exploration of the peritoneal cavity during laparoscopy. Two fluorescent dyes, Patent blue V and Indocyanine green have been evaluated on human ex vivo samples to improve the contrast of pCLE images. For a future implementation in clinical study, two topically staining protocols operable in vivo have been validated on 70 specimens from 25 patients with a peritoneal carcinomatosis. The specimens were then imaged by pCLE with an optical probe designed for the application. A histo-morphological correlative study was performed on 350 pCLE images and 70 standard histological preparations. All images were interpreted in a random way by two pathologists. Differential histological diagnostics such as normal peritoneum or pseudomyxoma could be recognized on fluorescence images. The statistical analysis of the correlative study is underway. These dyes already approved for human use are interesting for pCLE imaging because some micromorphological criteria look like to conventional histology and are readable by pathologist. Thus pCLE images using both dyes do not require a specific semiology unlike to what is described in the literature, for pCLE associated with fluorescein for the in vivo imaging of pancreatic cysts.

The development of Optical Coherence Tomography (OCT) systems started in the 1990's. The technology early found its application in Ophthalmology. Today more than 75% of the OCT market comes from the ophthalmic sector. However the growth of the ophthalmology OCT market has been slowing down for the last years. In this article, we present the results of our prospective study on the drivers of the OCT market growth in the coming years, based on bibliographical research and interviews with key players. In parallel of being used for ophthalmology, OCT has been developed and tested in new medical domains like cardiology, dermatology, gastroenterology, etc. OCT addresses key societal challenges such as the diagnosis of coronary artery diseases or skin cancer. There is a strong demand for fast, high-resolution, label-free and in vivo imaging tools in these fields. For the last 5 years, RD efforts have been focused on improving the performance and the compactness of OCT components and sub-systems. Advances in integrated photonics will enable the miniaturization of components and sub-systems and thus pave the way to Point-of-Care applications. Moreover the developments of new functionalities of OCT systems are undertaken to reach more complex diagnosis. OCT will no longer be a simple imaging device, it is on the verge of becoming a quantitative measurement tool. Our study shows that the emergence of new applications along with the improvements of components performance and the progress of functional OCT will drive the OCT market growth in the coming years.

We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20 kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment) enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime information. Intensity en face images over the four bands of the instrument were available within seconds whereas lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.

Objective: Over the past decade, fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have been applied in biomedical imaging in vitro and in vivo because of their fascinating optical properties. In this work, we investigated the application of CdTe QDs for tumor fluorescence in vivo imaging. Methods: The transparent dorsal skin fold window chamber (DSFC) was constructed on the 4~6 week-old BALB/c mice. The melanoma cells stably expressing green fluorescent protein ---ZsGreen were transplanted into the chamber and the melanoma DSFC model was established successfully. The water soluble CdTe QDs were synthesized and then administrated in the model through the tail intravenous injection. The fluorescent expression of B16 cells were assayed by fluorescent microscopy, the tumor growth, the blood capillaries distributions and its dynamic changes were observed by stereomicroscopy and laser scanning confocal microscopy. Results: The results demonstrated that the expression efficiency of ZsGreen was 41%, which met the experimental requirement. The tumors was visible inside the chamber after implantation of melanoma cells for 5~6 days, while no obvious changes in mice behaviors were found. After injection of the QDs, CdTe QDs accumulated at the invading edge of a range of solid tumor. We could also observe the tumor cells growth near the blood vessels, the angiogenesis occurred inside the tumor and the local blood capillaries increased. Conclusions: This work provided a new strategy for the tumor in vivo imaging and the development of targeted antineoplastic drugs.

Upconversion nanoparticles (UCNPs) have attracted much attention as novel contrast agents for low-background biomedical imaging and potential photosensitizer carriers for photodynamic therapy (PDT) in deep tissues. In this work, firstly we synthesized NaYF 4 :Yb 3+ ,Tm 3+ @NaYF 4 :Yb 3+ ,Er 3+ core-shell structured UCNPs with multiple emission peaks (e.g. 480, 539, 654 and 800 nm) by a seeded growth approach. By coating folate-modified amphiphilic chitosan (FASOC) on UCNPs, the as-prepared UCNPs were favorably endowed with good water solubility and the enhanced tumor-selectivity to cancer cells that overexpressed folate receptor. Then, water-insoluble photosensitizer zinc(II) phthalocyanine (ZnPc) was loaded into the FASOC-UCNPs via hydrophobic interactions for potential near-infrared light induced photodynamic therapy (PDT). Our results indicate that the multifunctional FASOC-UCNP-ZnPc nanoconstruct has efficient NIR-to-NIR upconversion luminescence and PDT abilities, which could be potentially employed as a theranostic platform for cancer treatment.

Herein we present recent improvements in system design and performance evaluation of near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system developed for small animal fluorescence tomography and installed within a commercial micro-CT/PET scanner. We improved system performance by increasing signal-to-noise ratio (SNR) through use of high powered rf modulation, novel data collection scheme, and data discrimination based on the associated noise levels. Noise characteristics show improvement with these techniques and are currently being employed to improve 3-D fluorescence for tomographic reconstructions in phantoms before incorporating into hybrid scanner.

The need to detect cancer at its early stages, as well as, to deliver chemotherapy to targeted site motivates many researchers to build theranostic platforms which combine diagnostic and therapy. Among imaging modalities, ultrasonography and Magnetic Resonance Imaging (MRI) are widely available, non invasive and complement each other. Both techniques often require the use of contrast agents. We have developed nanocapsules of perfluorooctyl bromide as dual contrast agent for both imaging modalities. The soft, amorphous polymer shell provides echogenicity, while the high-density perfluorinated liquid core allows detection by 19 F MRI. We have used a shell of poly(lactide-co-glycolide) (PLGA) since this polymer is biodegradable, biocompatible and can be loaded with drugs. These capsules were shown to be efficient in vitro as contrast agents for both 19 F MRI and ultrasonography. In addition, for in vivo applications a poly(ethyleneglycol) (PEG) coating promotes stability and prolonged circulation. Being stealth, nanocapsule can accumulate passively into implanted tumors by the EPR effect. We will present nanocapsule formulation and characterization, and will show promising in vivo results obtained for both ultrasonography and

Multi-photon imaging provides valuable insights into the continuous reshaping of neuronal connectivity in live brain. We previously showed that single neuron or even single spine ablation can be achieved by laser-mediated dissection. Furthermore, single axonal branches can be dissected avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Here, we describe the procedure to address the structural plasticity of cerebellar climbing fibers by combining two-photon in vivo imaging with laser axotomy in a mouse model. This method is a powerful tool to study the basic mechanisms of axonal rewiring after single branch axotomy in vivo. In fact, despite the denervated area being very small, the injured axons consistently reshape the connectivity with surrounding neurons, as indicated by the increase in the turnover of synaptic boutons. In addition, time-lapse imaging reveals the sprouting of new branches from the injured axon. Newly formed branches with varicosities suggest the possible formation of synaptic contacts. Correlative light and electron microscopy revealed that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites.

Hyperspectral imaging is a developing modality for cancer detection. The rich information associated with hyperspectral images allow for the examination between cancerous and healthy tissue. This study focuses on a new method that incorporates support vector machines into a minimum spanning forest algorithm for differentiating cancerous tissue from normal tissue. Spectral information was gathered to test the algorithm. Animal experiments were performed and hyperspectral images were acquired from tumor-bearing mice. In vivo imaging experimental results demonstrate the applicability of the proposed classification method for cancer tissue classification on hyperspectral images.

Fiber-optic based optical imaging is an emerging technique for studying brain activity in live animals. Here, we introduce a novel fluorescence fiber-optic microendoscopy approach to minimal invasively detect neural activities in a live mouse brain . The system uses a flexible endoscopic probe composed of a multi-core coherent fiber-bundle terminated with an approximately 1500-micron working distance objective lens. The fiber-optic neural interface is mounted on a 4-mm2 cranial window enabling visualization of glial calcium transients from the same brain region for weeks. We evaluated the system performance through in vivo imaging of GCaMP3 fluorescence in transgenic headrestrained mice during locomotion.

A novel x-ray fluorescence imaging setup for the in vivo detection of high-Z tracer distributions is investigated for its application in molecular imaging. The setup uses an energy resolved detection method based on a Bragg reflecting analyzer array together with a multiple scatter reducing radial collimator. The aim of this work is to investigate the potential application of this imaging method to in vivo imaging in humans. A proof of principle experiment modeling a partial setup for the detection of gold nano-particles was conducted in order to test the feasibility of the proposed imaging method. Furthermore a Monte Carlo simulation of the complete setup was created in order to quantify the dependence of the image quality on the applied radiation dose and on the geometrical collimator parameters as well as on the analyzer crystal parameters. The Monte Carlo simulation quantifies the signal-to-noise ratio per radiation dose and its dependence on the collimator parameters. Thereby the parameters needed for a dose efficient in vivo imaging of gold nano-particle based tracer distributions are quantified. However also a number of problems are found like the fluorescence emission as well as scatter from the collimator material obscuring the tracer fluorescence and the potentially large scan time.

Treating cancer is one of the major challenges of modern medicine. There is great interest in assessing tumor development in in vivo animal and human models, as well as in in vitro experiments. Existing methods are either limited by cost and availability or by their low accuracy and repr oducibility. Thermography holds the potential of being a non-invasive, low-cost, irradiative and easy-to-use method for tumor monitoring. Tumors can be detected in thermal images due to their relatively higher or lower temperature compared to the temperatur e of the healthy skin surrounding them. Extensive research is performed to show the validity of thermography as an efficient method for tumor detection and the possibility of extracting tumor properties from thermal imag es, showing promising results. However, deducing from one type of experiment to others is difficult due to the differences in tumor properties, especially between different types of tumors or different species. There is a need in a research linking different types of tumor experiments. In this research, parametric analysis of possible contributors to tumor thermal profiles was performed. The effect of tumor geometric, physical and thermal properties was studied, both independently and together, in phantom model experiments and computer simulations. Theoretical and experimental results were cross-correlated to validate the models used and increase the accuracy of simulated complex tumor models. The contribution of different parameters in various tumor scenarios was estimated and the implication of these di fferences on the observed ther mal profiles was studied. The correlation between animal and human models is discussed. Keywords: Thermal imaging, thermography, cancer, tumor monitoring, simulation, in vivo imaging, phantom experiments, parametric study.

To match recent clinical dual energy (DE) CT studies focusing on the lung, similar developments for DE micro-CT of the rodent lung are required. Our group has been actively engaged in designing pulmonary gating techniques for micro- CT, and has also introduced the first DE micro-CT imaging method of the rodent lung. The aim of this study was to assess the feasibility of DE micro-CT imaging for the evaluation of airway obstruction in mice, and to compare the method with micro single photon emission computed tomography (micro-SPECT) using technetium-99m labeled macroaggregated albumin (99mTc-MAA). The results suggest that the induced pulmonary airway obstruction causes either atelectasis, or air-trapping similar to asthma or chronic bronchitis. Atelectasis could only be detected at early time points in DE micro-CT images, and is associated with a large increase in blood fraction and decrease in air fraction. Air trapping had an opposite effect with larger air fraction and decreased blood fraction shown by DE micro-CT. The decrease in perfusion to the hypoventilated lung (hypoxic vasoconstriction) is also seen in micro-SPECT. The proposed DE micro-CT technique for imaging localized airway obstruction performed well in our evaluation, and provides a higher resolution compared to micro-SPECT. Both DE micro-CT and micro-SPECT provide critical, quantitative lung biomarkers for image-based anatomical and functional information in the small animal. The methods are readily linked to clinical methods allowing direct comparison of preclinical and clinical results.