Combining multicolor FISH with fluorescence lifetime imaging for chromosomal identification and chromosomal sub structure investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes

Single- and multi-photon excited fluorescence from serotonin complexed with B-cyclodextrin

The fluorescence of serotonin on binding with B-cyclodextrin has been studied using both steady-state and time-resolved methods. Steady state fluorescence intensity of serotonin at 340 nm showed ~ 30% increase in intensity on binding with Ka ~ 60 dm3 mol 1 and the fluorescence lifetimes showed a corresponding increase. In contrast, the characteristic green fluorescence (‘hyperluminescence’) of serotonin observed upon multiphoton near-infrared excitation with sub-picosecond pulses was resolved into two lifetime components assigned to free and bound serotonin. The results are of interest in relation to selective imaging and detection of serotonin using the unusual hyperluminescence emission and in respect to recent determinations of serotonin by capillary electrophoresis in the presence of cyclodextrin. The results also suggest that hyperluminescence occurs from multiphoton excitation of a single isolated serotonin molecule

Arabidopsis thaliana myosin XIK is recruited to the Golgi through interaction with a MyoB receptor

Perico et al. use co-expression analysis and a FRET-FLIM approach to show that the Arabidopsis MyoB myosin receptor, MRF7, triggers the relocation of Myosin XI-K to the Golgi. As such, this study provides evidence for plant myosin recruitment and control of organelle movement

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

Anticancer phototherapy using activation of E-combretastatins by two-photon–induced isomerization

The photoisomerization of relatively nontoxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one- and two-photon excitations at 340 and 625 nm, respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygen- independent and active in hypoxic environments such as in tumors. The use of red or near-infrared (NIR) light for two-photon excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes nondiscriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of two-photon red or NIR excitation for deeper tissue penetration

The antibacterial activity of a photoactivatable diarylacetylene against Gram-positive bacteria

The emergence of antibiotic resistance is a growing threat to human health, and therefore, alternatives to existing compounds are urgently needed. In this context, a novel fluorescent photoactivatable diarylacetylene has been identified and characterised for its antibacterial activity, which preferentially eliminates Gram-positive over Gram-negative bacteria. Experiments confirmed that the Gram-negative lipopolysaccharide-rich outer surface is responsible for tolerance, as strains with reduced outer membrane integrity showed increased susceptibility. Additionally, bacteria deficient in oxidative damage repair pathways also displayed enhanced sensitivity, confirming that reactive oxygen species production is the mechanism of antibacterial activity. This new diarylacetylene shows promise as an antibacterial agent against Gram-positive bacteria that can be activated in situ, potentially for the treatment of skin infections

A signal motif retains Arabidopsis ER-α-mannosidase I in the cis-Golgi and prevents enhanced glycoprotein ERAD

The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is a novel amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1