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Quantitative PA tomography of high resolution 3-D images: experimental validation in tissue phantoms
(2020)
Quantitative photoacoustic tomography aims recover the spatial distribution of absolute chromophore concentrations and their ratios from deep tissue, high-resolution images. In this study, a model-based inversion scheme based on a Monte-Carlo light transport model is experimentally validated on 3-D multispectral images of a tissue phantom acquired using an all-optical scanner with a planar detection geometry. A calibrated absorber allowed scaling of the measured data during the inversion, while an acoustic correction method was employed to compensate the effects of limited view detection. Chromophore- and fluence-dependent step sizes and Adam optimization were implemented to achieve rapid convergence. High resolution 3-D maps of absolute concentrations and their ratios were recovered with high accuracy. Potential applications of this method include quantitative functional and molecular photoacoustic tomography of deep tissue in preclinical and clinical studies.
We present a software-assisted workflow for the alignment and matching of filamentous structures across a 3D stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After an initial alignment, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labor. The software tool was developed to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite the problems related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time consuming. Furthermore, an interactive visualization of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialized, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. The key to our solution is a careful design of the visualization and interaction tools for each processing step to guarantee real-time response, and an optimized workflow that efficiently guides the user through datasets.
Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. Here, we report direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains.
We show that different acidification-sensing cargo probes are sorted into distinct classes of degradative ‘‘hub’’ compartments for synaptic vesicle proteins and plasma membrane proteins at axon terminals. Sorting and degradation of the two cargoes in the separate hubs are molecularly distinct. Local sorting of synaptic vesicle proteins for degradation at the axon terminal is, surprisingly, Rab7 independent,
whereas sorting of plasma membrane proteins is Rab7 dependent. The cathepsin-like protease CP1 is specific to synaptic vesicle hubs, and its delivery requires the vesicle SNARE neuronal synaptobrevin. Cargo separation only occurs at the axon terminal, whereas degradative compartments at the cell body are mixed. These data show that at least two local, molecularly distinct pathways sort membrane cargo for degradation specifically at the axon terminal, whereas degradation can occur both at the terminal and en route to the cell body.
In this paper, we present a software-assisted workflow for the alignment and matching of filamentous structures across a stack of 3D serial image sections. This is achieved by a combination of automatic methods, visual validation, and interactive correction. After an initial alignment, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. This is supported by a quality assessment that visualizes regions that have been already inspected and, thus, allows a trade-off between quality and manual labor.
The software tool was developed in collaboration with biologists who investigate microtubule-based spindles during cell division. To quantitatively understand the structural organization of such spindles, a 3D reconstruction of the numerous microtubules is essential. Each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The sections then need to be stitched, i.e. non-rigidly aligned; and the microtubules need to be traced in each section and connected across section boundaries. Experiments led to the conclusion that automatic methods for stitching alone provide only an incomplete solution to practical analysis needs. Automatic methods may fail due to large physical distortions, a low signal-to-noise ratio of the images, or other unexpected experimental difficulties. In such situations, semi-automatic validation and correction is required to rescue as much information as possible to derive biologically meaningful results despite of some errors related to data collection.
Since the correct stitching is visually not obvious due to the number of microtubules (up to 30k) and their dense spatial arrangement, these are difficult tasks. Furthermore, a naive inspection of each microtubule is too time consuming. In addition, interactive visualization is hampered by the size of the image data (up to 100 GB). Based on the requirements of our collaborators, we present a practical solution for the semi-automatic stitching of serial section image stacks with filamentous structures.
Quantitative photoacoustic tomography aims to recover maps of the local concentrations of tissue chromophores from multispectral images. While model-based inversion schemes are promising approaches, major challenges to their practical implementation include the unknown fluence distribution and the scale of the inverse problem. This paper describes an inversion scheme based on a radiance Monte Carlo model and an adjoint-assisted gradient optimization that incorporates fluence-dependent step sizes and adaptive moment estimation. The inversion is shown to recover absolute chromophore concentrations, blood oxygen saturation and the Grüneisen parameter from in silico 3D phantom images for different radiance approximations. The scattering coefficient was assumed to be homogeneous and known a priori.
Mitotic and meiotic spindles are microtubule-based structures to faithfully segregate chromosomes. Electron tomography is currently the method of choice to analyze the three-dimensional (3D) architecture of both types of spindles. Over the years, we have developed methods and software for automatic segmentation and stitching of microtubules in serial sections for large-scale reconstructions. 3D reconstruction of microtubules, however, is only the first step toward biological insight. The second step is the analysis of the structural data to derive measurable spindle properties. Here, we present a comprehensive set of techniques to quantify spindle parameters. These techniques provide quantitative analyses of specific microtubule classes and are applicable to a variety of tomographic reconstructions of spindles from different organisms.
In oocytes of many organisms, meiotic spindles form in the absence of centrosomes [1–5]. Such female meiotic spindles have a pointed appearance in metaphase with microtubules focused at acentrosomal spindle poles. At anaphase, the microtubules of acentrosomal spindles then transition to an inter- chromosomal array, while the spindle poles disappear. This transition is currently not understood. Previous studies have focused on this inter- chromosomal microtubule array and proposed a pushing model to drive chromosome segregation [6, 7]. This model includes an end-on orientation of microtubules with chromosomes. Alternatively, chromosomes were thought to associate along bundles of microtubules [8, 9]. Starting with metaphase, this second model proposed a pure lateral chromosome-to-microtubule association up to the final meiotic stages of anaphase. Here we applied large-scale electron tomography [10] of staged C. elegans oocytes in meiosis to analyze the orientation of microtubules in respect to chromosomes. We show that microtubules at metaphase I are primarily oriented laterally to the chromosomes and that microtubules switch to an end-on orientation during progression through anaphase. We further show that this switch in microtubule orientation involves a kinesin-13 microtubule depolymerase, KLP-7, which removes laterally associated microtubules around chromosomes. From this we conclude that both lateral and end-on modes of microtubule-to-chromosome orientations are successively used in C. elegans oocytes to segregate meiotic chromosomes.
In oocytes of many organisms, meiotic spindles form in the absence
of centrosomes [1–5]. Such female meiotic spindles have a pointed appearance in metaphase with microtubules focused at acentrosomal spindle poles. At anaphase, the microtubules of acentrosomal spindles then transition to an inter- chromosomal array, while the spindle poles disappear. This transition is currently not understood. Previous studies have focused on this inter- chromosomal microtubule array and proposed a pushing model to drive chromosome segregation [6, 7]. This model includes an end-on orientation of microtubules with chromosomes. Alternatively, chromosomes were thought to associate along bundles of microtubules [8, 9]. Starting with metaphase, this second model proposed a pure lateral chromosome-to-microtubule association up to the final meiotic stages of anaphase. Here we applied large-scale electron tomography [10] of staged C. elegans oocytes in meiosis to analyze the orientation of microtubules in respect to chromosomes. We show that microtubules at metaphase I are primarily oriented laterally to the chromosomes and that microtubules switch to an end-on orientation during progression through anaphase. We further show that this switch in microtubule orientation involves a kinesin-13 microtubule depolymerase, KLP-7, which removes laterally associated microtubules around chromosomes. From this we conclude that both lateral and end-on modes of microtubule-to-chromosome orientations are successively used in C. elegans oocytes to segregate meiotic chromosomes.
Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic \textit{Caenorhabditis elegans} spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.