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

Effects of replacing fishmeal with black soldier fly larval meal in the diets of grower-finishing guinea fowls reared under tropical conditions

The study was conducted with the view to determine the impact of replacing fishmeal with black soldier fly larval meal (BSFLM) on growing guinea fowls. BSFLM replaced fishmeal (3% in the control diet) in the ratios of 0, 20, 40, 60, 80, and 100% to produce six dietary treatments, which were iso-caloric and iso-nitrogenous. Two hundred and forty-eight-week old guinea fowls with mean live weight of 273.2 ± 10.9 g were tagged, weighted, and randomly assigned to 24 (6 × 4) floor pens; each pen was treated as a replicate. Feed and water were provided ad libitum during the entire period, which lasted 10 weeks. Feed consumption differed among the treatment groups (P = 0.0072) with the 100% fishmeal diets recording the lowest. Daily gain was significantly (P = 0.009) higher for birds fed high BSFLM diets compared to the control (100% fishmeal diet). The inclusion of BSFLM in the diets elicited positive linear effect on weight gains of the guinea fowls (R2 = 0.91) with increasing concentration resulting in higher live weight gains. The feed conversion ratio (FCR) also differed between treatments (P < 0.05) but similar for the 100% fishmeal (control) and 100% BSFLM diets. Organ and haematopoitic integrity were equally assured regardless of levels of the protein sources fed to the birds. Generally, meats from birds fed 60 to 100% BSFLM and from hens were more acceptable. A study to evaluate the economics of utilising BSFLM in guinea fowl production is recommended

Ultra weak photon emission—a brief review

Cells emit light at ultra-low intensities: photons which are produced as by-products of cellular metabolism, distinct from other light emission processes such as delayed luminescence, bioluminescence, and chemiluminescence. The phenomenon is known by a large range of names, including, but not limited to, biophotons, biological autoluminescence, metabolic photon emission and ultraweak photon emission (UPE), the latter of which shall be used for the purposes of this review. It is worth noting that the photons when produced are neither ‘weak’ nor specifically biological in characteristics. Research of UPE has a long yet tattered past, historically hamstrung by a lack of technology sensitive enough to detect it. Today, as technology progresses rapidly, it is becoming easier to detect and image these photons, as well as to describe their function. In this brief review we will examine the history of UPE research, their proposed mechanism, possible biological role, the detection of the phenomenon, and the potential medical applications

Time-resolved infra-red studies of photo-excited porphyrins in the presence of nucleic acids and in HeLa tumour cells: insights into binding site and electron transfer dynamics

Cationic porphyrins based on the 5,10,15,20-meso-(tetrakis-4-N-methylpyridyl) core (TMPyP4) have been studied extensively over many years due to their strong interactions with a variety of nucleic acid structures, and their potential use as photodynamic therapeutic agents and telomerase inhibitors. In this paper, the interactions of metal-free TMPyP4 and Pt(II)TMPyP4 with guanine-containing nucleic acids are studied for the first time using time-resolved infrared spectroscopy (TRIR). In D2O solution (where the metal-free form exists as D2TMPyP4) both compounds yielded similar TRIR spectra (between 1450–1750 cm−1) following pulsed laser excitation in their Soret B-absorption bands. Density functional theory calculations reveal that vibrations centred on the methylpyridinium groups are responsible for the dominant feature at ca. 1640 cm−1. TRIR spectra of D2TMPyP4 or PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP), double-stranded {d(GC)5}2 or {d(CGCAAATTTGCG)}2 contain negative-going signals, ‘bleaches’, indicative of binding close to guanine. TRIR signals for D2TMPyP4 or PtTMPyP bound to the quadruplex-forming cMYC sequence {d(TAGGGAGGG)}2T indicate that binding occurs on the stacked guanines. For D2TMPyP4 bound to guanine-containing systems, the TRIR signal at ca. 1640 cm−1 decays on the picosecond timescale, consistent with electron transfer from guanine to the singlet excited state of D2TMPyP4, although IR marker bands for the reduced porphyrin/oxidised guanine were not observed. When PtTMPyP is incorporated into HeLa tumour cells, TRIR studies show protein binding with time-dependent ps/ns changes in the amide absorptions demonstrating TRIR's potential for studying light-activated molecular processes not only with nucleic acids in solution but also in biological cells

Induction and processing of the radiation-induced gamma-H2AX signal and Its link to the underlying pattern of DSB: A combined experimental and modelling study

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular