Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions

New fluorophore-labelled G_M1 gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G_M1. Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.     

The mito::mKate2 mouse: A far‐red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo

Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission‐fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far‐red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R‐mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.     

Variations in slice sensitivity profile for various height settings in tomosynthesis imaging: Phantom study

Understanding the properties of slice sensitivity profile (SSP), or slice thickness, is crucial for an accurate and highly reproducible diagnosis using tomosynthesis imaging. The objectives of the present study are therefore to quantitatively evaluate how the SSP with the use of a small metal bead is affected by different settings of the height from the table and the height of the center of rotation (COR) in tomosynthesis imaging except for the digital breast tomosynthesis, and visually verify the effects on tomosynthesis images. The reconstruction filters used were three types of filtered back-projection and iterative reconstructions. The SSP was measured from the full width at half maximum (FWHM-SSP) of the profile curve of the bead in the perpendicular direction (z direction) relative to the table. Two types of anthropomorphic phantoms simulating the human body, with bones and soft tissues, were used to study the effects of different settings for the COR height. In all reconstruction filters, the FWHM-SSP changed as the height of the bead varied when the bead and COR were set to the same height from the table. If the bead and the COR were set to different heights, the FWHM-SSP increased (decreased) when the height of the bead was set to be greater (less) than the height of the COR. These changes were also confirmed on the anthropomorphic phantom images of the bones and soft tissues.     

Simulation of proton range monitoring in an anthropomorphic phantom using multi-slat collimators and time-of-flight detection of prompt-gamma quanta

Prompt-gamma (PG) imaging has the potential for monitoring proton therapy in real time. Different approaches are investigated. We focus on developing multi-slat collimators to image PG quanta, aiming at optimizing collimator performance to detect deviations in treatment delivery. We investigated six different multi-slat configurations, which have either optimal (analytical) intrinsic spatial resolution at fixed efficiency, or otherwise; at different distances from the proton pencil-beam axis (15 cm–35 cm). We used Geant4 to simulate irradiations of the head (energy: 130 MeV) and pelvis (200 MeV) of an anthropomorphic phantom, with and without physiologic/morphologic or setup changes of clinical dosimetric relevance. The particles escaping the phantom were transported through each of these multi-slat configurations and the gamma counts profiles were recorded at the collimator exit. Median filtering was applied to the registered PG-profiles to mitigate the effects of septa shadowing and statistical fluctuations. Time-of-flight discrimination was used to enhance the signal-to-background ratio, which appeared crucial for 200 MeV irradiations. Visual detection of the artificially introduced changes was possible by comparing the PG to the depth-dose profiles. Moreover, 2 mm range shifts could be detected in the head irradiation case using a simple linear regression fit to the falloff of the PG-profile. The influence of changes in complex, patient-like dose distributions on the PG-profiles obtained with multi-slat collimation is first studied in this work, which further gives insight on collimator design optimization and highlights its potential and simplicity for detecting proton treatment deviations over a wide range of Bragg peak positions.     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

Nonlinear optoacoustic readings from diffusive media at near‐infrared wavelengths

Optoacoustic (photoacoustic) imaging assumes that the detected signal varies linearly with laser energy. However, nonlinear intensity responses as a function of light fluence have been suggested in optoacoustic microscopy, that is, within the first millimeter of tissue. In this study, we explore the presence of nonlinearity deeper in tissue (~4 mm), as it relates to optoacoustic mesoscopy, and investigate the fluence required to delineate a switch from linear to nonlinear behavior. Optoacoustic signal nonlinearity is studied for different materials, different wavelengths and as a function of changes in the scattering and absorption coefficient of the medium imaged. We observe fluence thresholds in the mJ/cm² range and preliminary find that different materials may exhibit different nonlinearity patterns. We discuss the implications of nonlinearity in relation to image accuracy and quantification in optoacoustic tomography.      

Fiber‐based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label‐free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber‐optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen‐removed bovine pericardium. Fiber‐based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.      

In vivo blind‐deconvolution photoacoustic ophthalmoscopy with total variation regularization

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal‐to‐noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2‐fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well‐preserved blood vessels' edges simultaneously, which surpasses conventional Richardson‐Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.      

Chemical imaging of fresh vascular smooth muscle cell response by epi‐detected stimulated Raman scattering

An understanding of deformation of cardiovascular tissue under hemodynamic load is crucial for understanding the health and disease of blood vessels. In the present work, an epi‐detected stimulated Raman scattering (epi‐SRS) imaging platform was designed for in situ functional imaging of vascular smooth muscle cells (VMSCs) in fresh coronary arteries. For the first time, the pressure‐induced morphological deformation of fresh VSMCs was imaged with no fixation and in a label‐free manner. The relation between the loading pressure and the morphological parameters, including angle and length of the VSMCs, were apparent. The morphological responses of VMSCs to drug treatment were also explored, to demonstrate the capability of functional imaging for VSMCs by this method. The time‐course imaging revealed the drug induced change in angle and length of VSMCs. The present study provides a better understanding of the biomechanical framework of blood vessels, as well as their responses to external stimulations, which are fundamental for developing new strategies for cardiovascular disease treatment.