Correlated Multimodal Imaging in Life Sciences:Expanding the Biomedical Horizon

International audienceThe frontiers of bioimaging are currently being pushed toward the integration and correlation of several modalities to tackle biomedical research questions holistically and across multiple scales. Correlated Multimodal Imaging (CMI) gathers information about exactly the same specimen with two or more complementary modalities that-in combination-create a composite and complementary view of the sample (including insights into structure, function, dynamics and molecular composition). CMI allows to describe biomedical processes within their overall spatio-temporal context and gain a mechanistic understanding of cells, tissues, diseases or organisms by untangling their molecular mechanisms within their native environment. The two best-established CMI implementations for small animals and model organisms are hardware-fused platforms in preclinical imaging (Hybrid Imaging) and Correlated Light and Electron Microscopy (CLEM) in biological imaging. Although the merits of Preclinical Hybrid Imaging (PHI) and CLEM are well-established, both approaches would benefit from standardization of protocols, ontologies and data handling, and the development of optimized and advanced implementations. Specifically, CMI pipelines that aim at bridging preclinical and biological imaging beyond CLEM and PHI are rare but bear great potential to substantially advance both bioimaging and biomedical research. CMI faces three mai

Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy

Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time

Optical Near-Field Electron Microscopy

Imaging dynamical processes at interfaces and on the nanoscale is of great importance throughout science and technology. While light-optical imaging techniques often cannot provide the necessary spatial resolution, electron-optical techniques damage the specimen and cause dose-induced artefacts. Here, Optical Near-field Electron Microscopy (ONEM) is proposed, an imaging technique that combines non-invasive probing with light, with a high spatial resolution read-out via electron optics. Close to the specimen, the optical near-fields are converted into a spatially varying electron flux using a planar photocathode. The electron flux is imaged using low energy electron microscopy, enabling label-free nanometric resolution without the need to scan a probe across the sample. The specimen is never exposed to damaging electrons

Clusters of polymersomes and Janus nanoparticles hierarchically self-organized and controlled by DNA hybridization

The combination of "hard", structurally well-defined particles with "soft", functional compartments bears great potential to produce structurally intricate hybrid nanomaterials that promote a multitude of applications that require multimodal agents and that permit the production of molecular factories. However, the co-assembly of "hard" and "soft" components in a programmable and directional manner is challenging due to the strongly differing mechanical properties of such disparate entities. Here, a versatile strategy to generate clusters by the directional and controlled self-organization of "hard" Janus nanoparticles (JNPs) with "soft" polymersomes is described. The hybridization of complementary ssDNA strands bound to the components drives cluster formation, while the asymmetry of the JNPs governs the directionality of the self-organization. Various factors have been explored to simultaneously preserve the integrity of the polymersomes and program the cluster formation. Differently loaded polymersomes on each lobe of the JNPs preserved their architecture in the clusters which, were shown to be non-toxic when interacting with cell lines. The architecture of the clusters, as a molecular factory where each component can be separately controlled bears great promise for use in advanced medical applications, including theranostics and correlative imaging

Medical physics and imaging - a timely perspective

Radiolog

Medical physics and imaging-a timely perspective

Radiolog

Characterisation of the antibiotic profile of Lysobacter capsici AZ78, an effective biological control agent of plant pathogenic microorganisms

Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agricultur

A Potential Therapeutic Target in PSMA-negative and Neuroendocrine Prostate Carcinoma

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2021.8. Keon Wook KangGi Jeong Cheon.막횡단 단백질인 전립선특이막항원 (prostate-specific membrane antigen, PSMA)은 전립선암의 영상 및 치료를 위한 유망한 표적이다. PSMA는 대부분의 전립선암에서 과발현되며 PSMA 표적 양전자방출단층촬영 (positron emission tomography, PET) 프로브를 사용하여 임상적으로 영상화 할 수 있다. 현재 글루타메이트-우레이도-리신(Glutamate-Ureido-Lysine, GUL) 유도체 방사성 리간드는 PSMA 표적 PET 영상을 위한 선도적인 프로브이다. 신경내분비 전립선암 (neuroendocrine prostate cancer, NEPC)과 같이 높은 사망률, 치료 내성을 보이는 특정 전립선암 부분집단에서는 PSMA 발현이 낮은 편이다. 하지만 GUL 기반 PSMA 표적 프로브는 NEPC 전이성 종양 병변을 잘 검출하는 경우가 보고되고 있다. 이러한 PSMA 발현이 낮은 종양에서 GUL 기반 프로브는 PSMA가 아닌 비표적 단백질에 결합할 것으로 예상된다. 본 연구에서는, NEPC를 포함한 진행성 전립선암에서 PSMA 유사 아미노펩티다제 NAALADaseL 및 대사성 글루타메이트 수용체 (mGluR)의 높은 발현을 확인하였다. 또한, mGluRs의 발현 수준이 PSMA 발현과 역상관 관계가 있으며 불량한 임상 예후와 관련이 있음을 확인하였다. 컴퓨터 계산적 방법을 통해 GUL 기반 프로브의 NAALADaseL 및 mGluRs에 결합 능력을 예측하였고 in-vitro 형광 프로브를 활용하여 GUL 기반 프로브의 PSMA, NAALADaseL 및 특정 mGluRs에 대한 우수한 친화성을 검증하였다. 종합하여 볼 때 mGLuR 및 NAALADaseL은 GUL 기반 프로브의 표적이 될 수 있으며 특히, PSMA 발현이 낮은 NEPC와 같은 치료저항성 암에 대한 새로운 진단 및 치료 표적이 될 수 있다.Prostate-specific membrane antigen (PSMA), a transmembrane protein, is a promising target for imaging and therapy of prostate cancer. PSMA is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-positron emission tomography (PET) probes. Development of small molecules for targeting PSMA is important for PSMA-PET and Glutamate-Ureido-Lysine (GUL)-derivative radioligands are currently leading probes for PSMA-PET. PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based probes still sometimes identify NEPC metastatic tumours. These probes may bind unknown proteins associated with PSMA-suppressed cancers. In this Ph.D. dissertation, the upregulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in advanced prostate cancers including NEPC was identified. This work shows that the expression levels of mGluRs inversely correlate with PSMA expression and are associated with poor survival outcome. Computationally predicting that GUL-based probes bind well to these targets, a fluorescent probe used to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL and specific mGluRs.1. Introduction １ 1.1. Study Background １ Prostate cancer １ Prostate-specific membrane antigen (PSMA) １ Molecular biology of PSMA expression ４ Role of preclinical imaging in development and reassessment of radioligands ９ 1.2. Purpose of Research １２ 2. Materials and Methods １３ 2.1. Biological materials and methods １３ Cell culture １３ Quantitative real-time PCR analysis １５ Immunoblotting and immunocytochemistry １６ Cy3-GUL cytotoxicity evaluation １６ Cytometric analysis １７ Cy3-GUL in vitro imaging １７ 2.2. Data mining analysis １８ The survival data and pairwise-correlations of gene expression ２１ 2.3. Animals and PDX models ２２ 2.4. Synthesis of a novel Cy3-GUL probe ２３ General Experimental ２３ Protected Cy3-GUL (12) ２６ Cy3-GUL ２７ 2.5. Computational Analyses ２８ Initial structural similarity assessment ２８ Docking simulations ２８ Protein preparation ２８ Ligand preparation ２９ Rigid receptor docking studies ２９ Induced-fit docking study ３０ Homology modeling ３０ Molecular dynamics protocol ３１ 2.6. Statistical analysis ３２ 3. Results and Discussions ３３ 3.1. F-GUL and Ga-GUL are predicted to have high affinity for PSMA. ３３ 3.2. F-GUL and Ga-GUL bind NAALADaseL1 and mGLuR8. ３９ 3.3. Aminopeptidase NAALADaseL1 is elevated in NEPC. ５５ 3.3. mGluRs are upregulated during progression to NEPC. ５９ 3.4. A novel synthetic fluorescent Cy3-GUL probe is predicted to bind to all three proteins. ６４ 3.5. Cy3-GUL binds to PSMA in vitro. ６９ 3.6. Cy3-GUL Probes are selectively taken up by mGLuR and NAALADaseL1. ７１ 4. Conclusion ７８ References ８１ Acknowledgements ８９ 국문 초록 ９１박

Radiolabeling of Theranostic Nanosystems

In the recent years, progress in nanotechnology has significantly contributed to the development of novel pharmaceutical formulations to overcome the drawbacks of conventional treatments and improve the therapeutic outcome in many diseases, especially cancer. Nanoparticle vectors have demonstrated the potential to concomitantly deliver diagnostic and therapeutic payloads to diseased tissue. Due to their special physical and chemical properties, the characteristics and function of nanoparticles are tunable based on biological molecular targets and specific desired features (e.g., surface chemistry and diagnostic radioisotope labeling). Within the past decade, several theranostic nanoparticles have been developed as a multifunctional nanosystems which combine the diagnostic and therapeutic functionalities into a single drug delivery platform. Theranostic nanosystems can provide useful information on a real-time systemic distribution of the developed nanosystem and simultaneously transport the therapeutic payload. In general, the diagnostic functionality of theranostic nanoparticles can be achieved through labeling gamma-emitted radioactive isotopes on the surface of nanoparticles which facilitates noninvasive detection using nuclear molecular imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), meanwhile, the therapeutic effect arises from the potent drug released from the nanoparticle. Moreover, some radioisotopes can concurrently emit both gamma radiation and high-energy particles (e.g., alpha, beta, and Auger electrons), prompting the use either alone for radiotheranostics or synergistically with chemotherapy. This chapter provides an overview of the fundamentals of radiochemistry and relevant radiolabeling strategies for theranostic nanosystem development as well as the methods for the preclinical evaluation of radiolabeled nanoparticles. Furthermore, preclinical case studies of recently developed theranostic nanosystems will be highlighted.Peer reviewe