Mapping Molecular Agents Distributions in Whole Mice Hearts Using Born-Normalized Optical Projection Tomography

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

Fluorescence microscopy tensor imaging representations for large-scale dataset analysis

Understanding complex biological systems requires the system-wide characterization of cellular and molecular features. Recent advances in optical imaging technologies and chemical tissue clearing have facilitated the acquisition of whole-organ imaging datasets, but automated tools for their quantitative analysis and visualization are still lacking. We have here developed a visualization technique capable of providing whole-organ tensor imaging representations of local regional descriptors based on fluorescence data acquisition. This method enables rapid, multiscale, analysis and virtualization of large-volume, high-resolution complex biological data while generating 3D tractographic representations. Using the murine heart as a model, our method allowed us to analyze and interrogate the cardiac microvasculature and the tissue resident macrophage distribution and better infer and delineate the underlying structural network in unprecedented detail

Image processing and speed-up improvement for real time Optical Projection Tomography: applications for high throughput ex -vivo lung and heart inflammatory diseases imaging in different mice models

L\u2019imaging clinico \ue8 largamente usato ai giorni nostri per fornire informazioni anatomiche, fisiologiche e metaboliche, ma generalmente non \ue8 in grado di fornire informazioni in merito ai sottostanti processi biologici legati a patologie. Recentemente l\u2019attenzione si \ue8 spostata verso lo studio dei processi molecolari che regolano l\u2019interazione tra differenti parti biologiche che contribuiscono al sistema complessivo. Questa nuova branca della medicina molecolare chiamata Imaging Molecolare usa sonde con elevate specificit\ue0 per fornire informazioni molto dettagliate. Il suo scopo \ue8 quello di fornire informazioni sui processi molecolari portando diagnosi sempre pi\uf9 precoci. L\u2019Imaging Molecolare ha il vantaggio di identificare segni di malattie nel loro stadio iniziale cio\ue8 quando modifiche a livello fisiologico o anatomico non hanno ancora avuto luogo. Diagnosi tempestive danno la possibilit\ue0 di ridurre notevolmente le dosi dei trattamenti medici e allo stesso tempo di ridurre al minimo gli effetti collaterali. Inoltre l\u2019Imaging Molecolare permette di sviluppare sistemi di monitoraggio in tempo reale per massimizzare la cura e controllare in modo efficace gli effetti della stessa. Esempi di applicazioni sono lo studio dei tumori, dei sistemi neurologico, cardiovascolare e respiratorio. All\u2019interno delle tecniche di Imaging Molecolare quella Ottica riveste un ruolo molto importante. Fino a pochi anni fa, il contributo dovuto allo scattering ha reso impossibile implementare sistemi ottici per questi studi. Infatti il potere di penetrazione della luce all\u2019interno di tessuti biologici era limitato a qualche centinaia di micron. Recentemente \ue8 stata sviluppata una tecnologia chiamata Optical Projection Tomography (OPT) in grado di superare tali impedimenti e di studiare campioni dello spessore anche di qualche centimetro come insetti, embrioni e interi organi di animali ad alta risoluzione e in modalit\ue0 ex-vivo. Le applicazioni dell\u2019OPT spaziano da analisi anatomiche e istologiche a studi di espressioni geniche e persino a studi di fenomeni infiammatori legati a particolari malattie. L\u2019OPT funziona in linea di principio come la tomografia assiale computerizzata (TAC) ma anzich\ue9 far uso di radiazione X utilizza quella luminosa approssimativamente nello spettro del visibile. L\u2019OPT \ue8 in grado di studiare fenomeni di fluorescenza che sono legati pi\uf9 che ad informazioni anatomiche a quelle funzionali. In quest\u2019ultimo caso la tecnica di ricostruzione \ue8 pi\uf9 complessa e pi\uf9 simile alla SPECT (single positron emission computed tomography) che alla TAC.Clinical imaging is largely used nowadays to provide anatomical, physiological or metabolical information but it cannot generally inform on the underlying molecular aberrations of the different diseases. Recently the attention has shifted towards investigating the molecular functions present in whole living systems focusing instead on the visualization of the molecular processes which regulate the interaction between the different biological parts that contribute to complex systems. This new Molecular Medicine branch, called Molecular imaging, exploits probes with high specificity and has the potential to provide more detailed information. Surface receptors, enzymes or structural proteins are the probes\u2019 targets that are used in order to achieve true molecular imaging. They are employed with the ambitious goal of gaining information on several distinct molecular processes, leading to both early detection and staging of diseases. Moreover Molecular imaging can promote tailoring of targeted therapies for individual patients. The advantage of using Molecular imaging techniques resides in the fact that it makes possible to look into early disease signs which are commonly not detectable by others medical imaging modalities. In fact anatomical and physiological modifications reveal just late disease information, making the disease treatments much harder. At the same time an early stage disease diagnosis allows for very small doses administrations or expositions minimizing the cure side-effects; by way of molecular imaging, it will be possible to implement a real-time patient\u2019s therapy response monitoring leading to a tailor-made and maximal efficiency cure design. Examples of application of Molecular imaging techniques are cancer, neurological, cardiovascular and respiratory diseases. Within the Molecular imaging techniques the Optical ones have recently increased in importance due to the overcoming of limiting technology hurdles. In fact tipical imaging is a key tool for the progress of biological sciences allowing for longitudinal unperturbed imaging of different biomarkers. Until recent years optical scattering contributions made impossible the implementation of optical imaging methodologies for the study of samples thicker than a few hundreds microns limiting their possible applications at the cellular level or for very small organisms\u2019 investigations. Unfortunately the price to pay in exchange of the deeper penetration is a substantial decrease in resolution. Optical projection tomography (OPT) is a recently introduced new imaging technique which allows to image small biological samples, such as small insects, embryos, or whole small animals organs ex vivo and at high resolution. Its applications span from anatomical and histological analysis to tissue proteins expression and distribution, from developmental biology to gene functions and recently to inflammation disease studies. One of the most significant features of OPT is its capability to image small biological samples up to a few centimeters in size with unprecedented resolution. To obtain such a degree of resolution, the samples under investigation are made optically transparent through a chemical clearing process. In this way its scattering and absorption properties are highly reduced, making the light diffusive contribution negligible. The sample is then illuminated with a light beam and absorption or fluorescence signal is acquired in transillumination mode by a CCD camera. Projection images of the cleared sample are taken in an X-CT analogue fashion and the reconstruction mathematical problem, at least for the case of absorption projections, can be solved in a similar fashion using the parallel beam filtered back projection algorithm. Fluorescence tomographic absorption reconstructions utilize instead a Born normalized method that relies on a normalized transillumination approach

High dynamic range fluorescence imaging

Fluorescence acquisition and image display over a high dynamic range is highly desirable. However, the limited dynamic range of current photodetectors and imaging charge-coupled devices impose a limit on the fluorescence intensities that can be simultaneously captured during a single image acquisition. This is particularly troublesome when imaging biological samples, where protein expression fluctuates considerably. As a result, biological images will often contain regions with signal that is either saturated or hidden within background noise, causing information loss. In this paper, we summarize recent work from our group and others, to extended conventional to high dynamic range fluorescence imaging. These strategies have many biological applications, such as mapping of neural connections, vascular imaging, biodistribution studies or pharmacologic imaging at the single cell and organ level

Improved intravital microscopy via synchronization of respiration and holder stabilization

A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy

Advanced Motion Compensation Methods for Intravital Optical Microscopy

Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions

Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement

Small molecule therapeutic drugs must reach their intended cellular targets(pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics).These processes are often difficult to measure in vivo due to their complexities andoccurrence within single cells. It has been particularly difficult to directly measure cellulardrug target binding

Fluorescence anisotropy imaging in drug discovery

Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting have proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital fluorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs