Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond

Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen – with sensitivity orders of magnitude higher than that currently attainable – can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as “FLIM + PLIM” as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences

Multimodal probes : superresolution and transmission electron microscopy imaging of mitochondria, and oxygen mapping of cells, using small-molecule Ir(III) luminescent complexes

We describe an Ir(III)-based small-molecule, multimodal probe for use in both light and electron microscopy. The direct correlation of data between light- and electron-microscopy-based imaging to investigate cellular processes at the ultrastructure level is a current challenge, requiring both dyes that must be brightly emissive for luminescence imaging and scatter electrons to give contrast for electron microscopy, at a single working concentration suitable for both methods. Here we describe the use of Ir(III) complexes as probes that provide excellent image contrast and quality for both luminescence and electron microscopy imaging, at the same working concentration. Significant contrast enhancement of cellular mitochondria was observed in transmission electron microscopy imaging, with and without the use of typical contrast agents. The specificity for cellular mitochondria was also confirmed with MitoTracker using confocal and 3D-structured illumination microscopy. These phosphorescent dyes are part of a very exclusive group of transition-metal complexes that enable imaging beyond the diffraction limit. Triplet excited-state phosphorescence was also utilized to probe the O2 concentration at the mitochondria in vitro, using lifetime mapping techniques

High-Throughput GoMiner, an 'industrial-strength' integrative gene ontology tool for interpretation of multiple-microarray experiments, with application to studies of Common Variable Immune Deficiency (CVID)

BACKGROUND: We previously developed GoMiner, an application that organizes lists of 'interesting' genes (for example, under-and overexpressed genes from a microarray experiment) for biological interpretation in the context of the Gene Ontology. The original version of GoMiner was oriented toward visualization and interpretation of the results from a single microarray (or other high-throughput experimental platform), using a graphical user interface. Although that version can be used to examine the results from a number of microarrays one at a time, that is a rather tedious task, and original GoMiner includes no apparatus for obtaining a global picture of results from an experiment that consists of multiple microarrays. We wanted to provide a computational resource that automates the analysis of multiple microarrays and then integrates the results across all of them in useful exportable output files and visualizations. RESULTS: We now introduce a new tool, High-Throughput GoMiner, that has those capabilities and a number of others: It (i) efficiently performs the computationally-intensive task of automated batch processing of an arbitrary number of microarrays, (ii) produces a human-or computer-readable report that rank-orders the multiple microarray results according to the number of significant GO categories, (iii) integrates the multiple microarray results by providing organized, global clustered image map visualizations of the relationships of significant GO categories, (iv) provides a fast form of 'false discovery rate' multiple comparisons calculation, and (v) provides annotations and visualizations for relating transcription factor binding sites to genes and GO categories. CONCLUSION: High-Throughput GoMiner achieves the desired goal of providing a computational resource that automates the analysis of multiple microarrays and integrates results across all of the microarrays. For illustration, we show an application of this new tool to the interpretation of altered gene expression patterns in Common Variable Immune Deficiency (CVID). High-Throughput GoMiner will be useful in a wide range of applications, including the study of time-courses, evaluation of multiple drug treatments, comparison of multiple gene knock-outs or knock-downs, and screening of large numbers of chemical derivatives generated from a promising lead compound

Genetic targeting of the endoderm with claudin-6CreER

A full description of the ontogeny of the β cell would guide efforts to generate β cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ERT2) cassette. Cldn6 null mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver

Hydra: A mixture modeling framework for subtyping pediatric cancer cohorts using multimodal gene expression signatures.

Precision oncology has primarily relied on coding mutations as biomarkers of response to therapies. While transcriptome analysis can provide valuable information, incorporation into workflows has been difficult. For example, the relative rather than absolute gene expression level needs to be considered, requiring differential expression analysis across samples. However, expression programs related to the cell-of-origin and tumor microenvironment effects confound the search for cancer-specific expression changes. To address these challenges, we developed an unsupervised clustering approach for discovering differential pathway expression within cancer cohorts using gene expression measurements. The hydra approach uses a Dirichlet process mixture model to automatically detect multimodally distributed genes and expression signatures without the need for matched normal tissue. We demonstrate that the hydra approach is more sensitive than widely-used gene set enrichment approaches for detecting multimodal expression signatures. Application of the hydra analysis framework to small blue round cell tumors (including rhabdomyosarcoma, synovial sarcoma, neuroblastoma, Ewing sarcoma, and osteosarcoma) identified expression signatures associated with changes in the tumor microenvironment. The hydra approach also identified an association between ATRX deletions and elevated immune marker expression in high-risk neuroblastoma. Notably, hydra analysis of all small blue round cell tumors revealed similar subtypes, characterized by changes to infiltrating immune and stromal expression signatures

Diffractive Dissociation of Alpha Particles as a Test of Isophobic Short-Range Correlations inside Nuclei

The CLAS collaboration at Jefferson Laboratory has compared nuclear parton distributions for a range of nuclear targets and found that the EMC effect measured in deep inelastic lepton-nucleus scattering has a strongly "isophobic" nature. This surprising observation suggests short-range correlations between neighboring $n$ and $p$ nucleons in nuclear wavefunctions that are much stronger compared to $p-p$ or $n-n$ correlations. In this paper we propose a definitive experimental test of the nucleon-nucleon explanation of the isophobic nature of the EMC effect: the diffractive dissociation on a nuclear target $A$ of high energy $\rm ^4He$ nuclei to pairs of nucleons $n$ and $p$ with high relative transverse momentum, $\alpha + A \to n + p + A' + X$. The comparison of $n-p$ events with $p-p$ and $n-n$ events directly tests the postulated breaking of isospin symmetry. The experiment also tests alternative QCD-level explanations for the isophobic EMC effect. In particular it will test a proposal for hidden-color degrees of freedom in nuclear wavefunctions based on isospin-zero $[ud]$ diquarks.Comment: 5 pages, references added, clarifications due to helpful referee comments (latexdiff for all changes). Accepted for publication in Physics Letters

Energetic Particles of Cosmic Accelerators I: Galactic Accelerators

The high-energy universe has revealed that energetic particles are ubiquitous in the cosmos and play a vital role in the cultivation of cosmic environments on all scales. Our pursuit of more than a century to uncover the origins and fate of these cosmic energetic particles has given rise to some of the most interesting and challenging questions in astrophysics. Energetic particles in our own galaxy, galactic cosmic rays (GCRs), engage in a complex interplay with the interstellar medium and magnetic fields in the galaxy, giving rise to many of its key characteristics. For instance, GCRs act in concert with galactic magnetic fields to support its disk against its own weight. GCR ionization and heating are essential ingredients in promoting and regulating the formation of stars and protostellar disks. GCR ionization also drives astrochemistry, leading to the build up of complex molecules in the interstellar medium. GCR transport throughout the galaxy generates and maintains turbulence in the interstellar medium, alters its multi-phase structure, and amplifies magnetic fields. GCRs could even launch galactic winds that enrich the circumgalactic medium and alter the structure and evolution of galactic disks. As crucial as they are for many of the varied phenomena in our galaxy, there is still much we do not understand about GCRs. While they have been linked to supernova remnants (SNRs), it remains unclear whether these objects can fully account for their entire population, particularly at the lower (approximately less than 1 GeV per nucleon) and higher (~PeV) ends of the spectrum. In fact, it is entirely possible that the SNRs that have been found to accelerate CRs merely re-accelerate them, leaving the origins of the original GCRs a mystery. The conditions for particle acceleration that make SNRs compelling source candidates are also likely to be present in sources such as protostellar jets, superbubbles, and colliding wind binaries (CWBs), but we have yet to ascertain their roles in producing GCRs. For that matter, key details of diffusive shock acceleration (DSA) have yet to be revealed, and it remains to be seen whether DSA can adequately explain particle acceleration in the cosmos. This White Paper is the first of a two-part series highlighting the most well-known high-energy cosmic accelerators and contributions that MeV gamma-ray astronomy will bring to understanding their energetic particle phenomena. For the case of GCRs, MeV astronomy will: 1) Search for fresh acceleration of GCRs in SNRs; 2) Test the DSA process, particularly in SNRs and CWBs; 3) Search for signs of CR acceleration in protostellar jets and superbubbles