Rhodopseudomonas palustris is a phototrophic purple nonsulfur bacterium that adapts its photosystem to allow growth at a range of light intensities. It does this by adjusting the amount and composition of peripheral light-harvesting (LH) antenna complexes that it synthesizes. Rhodopseudomonas strains are notable for containing numerous sets of light-harvesting genes. We determined the diversity of LH complexes and their transcript levels during growth under high and low light intensities in 20 sequenced genomes of strains related to the species Rhodopseudomonas palustris. The data obtained are a resource for investigators with interests as wide-ranging as the biophysics of photosynthesis, the ecology of phototrophic bacteria, and the use of photosynthetic bacteria for biotechnology applications. Rhodopseudomonas palustris produces different types of LH complexes under high light intensities (LH2 complex) and low light intensities (LH3 and LH4 complexes). There are multiple pucBA operons that encode the α and β peptides that make up these complexes. However, low-resolution structures, amino acid similarities between the complexes, and a lack of transcription analysis have made it difficult to determine the contributions of different pucBA operons to the composition and function of different LH complexes. It was also unclear how much diversity of LH complexes exists in R. palustris and affiliated strains. To address this, we undertook an integrative genomics approach using 20 sequenced strains. Gene content analysis revealed that even closely related strains have differences in their pucBA gene content. Transcriptome analyses of the strains grown under high light and low light revealed that the patterns of expression of the pucBA operons varied among strains grown under the same conditions. We also found that one set of LH2 complex proteins compensated for the lack of an LH4 complex under low light intensities but not under extremely low light intensities, indicating that there is functional redundancy between some of the LH complexes under certain light intensities. The variation observed in LH gene composition and expression in Rhodopseudomonas strains likely reflects how they have evolved to adapt to light conditions in specific soil and water microenvironments.Many photosynthetic bacteria have peripheral light-harvesting (LH) antenna complexes that increase the efficiency of light energy capture. The purple nonsulfur photosynthetic bacterium IMPORTANCERhodopseudomonas palustris is a phototrophic purple nonsulfur bacterium that adapts its photosystem to allow growth at a range of light intensities. It does this by adjusting the amount and composition of peripheral light-harvesting (LH) antenna complexes that it synthesizes. Rhodopseudomonas strains are notable for containing numerous sets of light-harvesting genes. We determined the diversity of LH complexes and their transcript levels during growth under high and low light intensities in 20 sequenced genomes of strains related to the species Rhodopseudomonas palustris. The data obtained are a resource for investigators with interests as wide-ranging as the biophysics of photosynthesis, the ecology of phototrophic bacteria, and the use of photosynthetic bacteria for biotechnology applications. Purple nonsulfur bacteria (PNSB) have served as models for studies of photosynthesis because they have much simpler systems of light energy conversion than do green plants or cyanobacteria. PNSB generate ATP by cyclic photophosphorylation in which electrons that have been energized by light leave a bacteriochlorophyll (Bchl)-containing reaction center, are cycled through a proton-pumping electron transport chain, and are then returned to the reaction center. This cyclic flow of electrons does not generate oxygen, and the process is not obligatorily linked to carbon dioxide fixation. Light-harvesting (LH) antenna complexes act in conjunction with reaction centers to increase the efficiency of light energy capture by PNSB.Rhodopseudomonas palustris. Most PNSB, including R. palustris, have peripheral LH complexes known as LH2 that are involved in capturing light and transferring that energy to the LH1 complex, which surrounds the reaction center to form a core photoconversion complex. Both types of LH complexes are circular oligomers of a protomer composed of hydrophobic polypeptides α and β that interact with the light-absorbing pigments bacteriochlorophyll (Bchl) and carotenoids (Car). The LH1 complex from R. palustris contains 15 pairs of the α and β peptides that bind one Car and two molecules of Bchla, which are tightly coupled and absorb light maximally at a wavelength of 875 nm , and two more tightly coupled Bchla molecules that absorb light at 850 nm (B850)  are compared to those of cells grown under low light (LL) intensities (4 µmol photons/m2/s). Under HL, LH2 is the predominant peripheral LH complex in Rhodopseudomonas membranes, although BisA53 also makes an additional LH2 complex known as LH2′ that absorbs light at wavelengths of 800 nm and 840 nm  with R. palustris CGA009  intensities. Taken together, these data likely reflect how Rhodopseudomonas strains have diversified to occupy specific microenvironments in terrestrial soil and water environments that have different levels of exposure to light.Since they contain so many sets of light-harvesting genes, s CGA009  and thustris see . Four ad10.1128/mSystems.00006-15.1Table S1 Rhodopseudomonas strains grown under high light intensity (30 µmol photons/m2/s) and low light intensity (4 µmol photons/m2/s) under nitrogen-fixing conditions. Download Table S1, XLSX file, 63 KB.Relative transcript levels for all LH genes and growth rates of 20 Copyright © 2015 Fixen et al.2015Fixen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pucBA, with pucA encoding the α peptide and pucB encoding the β peptide of a light-harvesting complex monomer. The α peptides of the LH2-like complexes are 60 to 65 amino acids in length and can be distinguished based on the identities of two or three amino acids, which result in differences in Bchl binding and thus their light absorption properties. The β peptides are approximately 50 amino acids in length but are almost identical in their amino acid sequences, with greater than 90% identity. Thus, we used the amino acid sequences of the predicted α peptides to determine which types of LH complexes are encoded in each genome. As shown in Rhodopseudomonas strains have multiple sets of LH2 genes, which we categorized as LH2A, LH2B, and LH2E based on their amino acid identities to LH2 genes in our reference strain CGA009 , one operon that encodes the LH4 complex (pucBAd), and one operon, pucBAc, which would encode an LH3 complex except that pucAc is a pseudogene due to a 2-bp insertion in the coding sequence. All locus numbers for each of the pucBA homologs from each strain are listed in Rhodopseudomonas strains that are most closely related to CGA009 (≥99.5% identity in 16S rRNA sequence), 11 strains had all three operons encoding LH2, one operon encoding LH4, and an intact operon encoding an LH3 complex, and this likely reflects the most prevalent combination of pucBA genes contained in Rhodopseudomonas strains closely related to CGA009  have a greater diversity in the number and type of LH peptides that they encode. BisA53 stands out as having the most LH complexes, and unlike the other strains, only 7 of its 8 pucA genes are found in tandem with a pucB gene  have more divergent pucB sequences  to measure the transcript levels of the LH genes in the 20 Rhodopseudomonas strains grown under the same HL and LL conditions. The LH genes are the most highly expressed genes in the genomes of each of the strains under both HL and LL conditions. While the majority of genes in cells grown under HL and LL have relative transcript levels of between 50 and 250 rpkm (reads per kilobase per million uniquely mapped reads), the LH genes can have relative transcript levels upwards of 200,000 , the LH4 operon (ΔpucBAd), or in both operons (ΔpucBAa ΔpucBAd) in strain CGA009 and measured the growth rates of the mutants under HL and LL. To verify that these deletions affect LH complex synthesis under LL, the absorption spectra of intact cells were determined. As expected when grown under LL, the ΔpucBAd strain was unable to produce any LH4 (pucBAa) showed a slight decrease in absorbance at 860 nm when grown under LL, and cells that were unable to produce either the LH2A or LH4 complex (ΔpucBAa ΔpucBAd) showed the greatest decrease in absorption and produced the smallest amount of LH complex under LL (2/s), the LH4 mutant grew more slowly than both the wild type and the LH2A mutant  (2/s) from a 60-W halogen light bulb  and then diluted twice into PM medium without ammonium sulfate and supplemented with 20 mM acetate, Wolfe’s vitamins (2/s) from a 15-W halogen light bulb , or extremely low light (<1 µmol photons/m2/s) from a 15-W halogen light bulb controlled by a dimmer switch. Escherichia coli S17-1  at 100 µg/ml. E. coli cultures were supplemented with Gm at 20 µg/ml.For routine maintenance, ium (PM)  supplemevitamins , and 10 li S17-1  was growGenomic DNA was extracted using the Qiagen Genomic-tip 500/G kit and sequenced using paired-end sequencing with Illumina HiSeq 2000 . SequencpucBAa and pucBAd were created by PCR using the Phusion high-fidelity DNA polymerase (New England Biolabs) to amplify 1 kb of DNA upstream of the second codon in the coding region for each of these genes and 1 kb of DNA downstream of the stop codon for each of these genes. These fragments were then incorporated into PstI-digested (pucBAa) or NotI/PstI-digested (pucBAd) pJQ200SK suicide vector . Because pucB homologs have a high sequence identity, they were categorized as pucBa, pucBb, pucBc, pucBd, and pucBe based on the pucA homolog contained in the same operon. The evolutionary history was inferred using the neighbor-joining method . The Genomes Online Database (GOLD) analysis project IDs for each strain are as follows: strain 0001L, Ga0011343; strain 1a1, Ga0011382; strain AP1, Ga0011400; strain ATCC 17007, Ga0011373; strain BIS3, Ga0011374; strain CEA001, Ga0011372; strain DCP3, Ga0011383; DSM126, Ga0011344; strain DSM8283, Ga0011384; strain KD1, Ga0011330; strain NCIB8288, Ga0011345; strain RCH350, Ga0011355; strain RCH500, Ga0011356; strain RSP24, Ga0011420; strain TIE-1, Ga0030132; strain CGA009, Ga0030129; strain DX-1, Ga0030130; strain BisA53, Ga0030126; strain BisB18, Ga0030127; strain BisB5, Ga0030128. All transcriptomic data are available as raw sequencing reads deposited in NCBI Gene Expression Omnibus under the accession number GSE59544.The DNA sequence reads for assemblies of the genomes are available in the NCBI Short Read Archive under ID number


Generation of complex Poincaré beams is enabled by amplitude and phase modulation accompanied by simultaneous spatially polarization structuring. A holographic system to tailor complex light fields and optical angular momentum structures forecasts promising applications in quantum communication and optical trapping. Experimental results are presented together with simulations of complex Poincaré beams embedding different types of polarization singularities. Additionally, parameters of the dynamic polarization modulation system are discussed and analyzed to demonstrate the enormous capability of the method. Moreover, Poincaré beams are strongly connected to singular optics4 due to the appearance of different types of vectorial singularities within light fields revealing structured polarization5. Different techniques have been proposed to generate inhomogeneous distributions of states of polarization as e.g. the superposition of spatial modes7, the application of polarization holograms8, q-plates10, or plasmonic metasurfaces11 and dynamic techniques using spatial light modulators16 and digital micro-mirror devices17. Recently the combination of polarization modulation with amplitude or phase modulation has been investigated21 more intensively. However, a dynamic technique to modulate extended polarization structures is essential for investigations of advanced polarization singularity formations.Polarization is one of the most fundamental yet fascinating properties of transverse waves, as light waves, gravitational waves, and sound waves in solids. For polarization studies, the polarization of light waves is a representative being accessible easily to experimental demonstrations, and allows characterizing as well as manipulating polarization in all its features. While homogeneously distributed states of polarization have been investigated comprehensively, recent research focuses on spatially inhomogeneous distributions of polarization, i.e. the coexistence of several states of polarization in complex transverse light fields. These so-called Poincaré beams22.In this paper, we present the experimental realization of dynamically modulated Poincaré beams. Primarily, the polarization of Laguerre-Gaussian (LG) beams is designed according to the structure of its amplitude distribution. Furthermore, different combinations of spin and orbital angular momentum are modulated in a helical LG beam. Finally, polarization singular structures are enabled which is demonstrated by a spiraling Poincaré beam containing various kinds of polarization singularities. All results are experimentally implemented by a dynamic modulation technique which is discussed within the method section. It is realized by a single spatial light modulator which is in combination with a pair of wave plates capable of generating all kinds of fully-polarized polarization states. As we use a pixelated display, various combinations of coexisting states of polarization become possible in a spatially highly resolved pattern. To verify high modulation quality of extended polarization patterns, a spatially resolved measurement of Stokes parameters (see method sections) is employed25, an explicit superposition and alignment of orthogonally polarized modes is not necessary. This avoids propagation instabilities of (singular) polarization fields due to misalignment and gives the opportunity to investigate the longitudinal evolution of such structures.The advantage of our approach is the automatic alignment of horizontal and vertical polarization components due to the dynamic modulation technique. Opposed to previous studies26 of a parallel aligned nematic liquid crystal spatial light modulator (SLM) as depicted in Fig. 28 allows the realization of e.g. higher order laser modes as Hermite-, Laguerre- or Ince-Gaussian beams. However, the method is not restricted to these modes but enables to modulate arbitrary light fields. In the second step, a dynamic polarization modulation system (DPMS) is implemented using the second half of the SLM (see Methods below) to realize spatially polarization structured Poincaré beams. Furthermore, our system enables to demonstrate orbital and spin angular momentum coupling as well as to create and study polarization singularities as V-points, C-points, L-lines and C-lines as discussed in the following.To access simultaneous holographic modulation of amplitude, phase as well as polarization, we employ a two step method experimentally implemented by a split-screen configurationn,l) modes and simultaneously modulate polarization states spatially29. In Fig. ψ   are shown. Therefore the total amount of angular momentum ψ of polarization states  and orbital (OAM) angular momentum. We explore this opportunity by investigation of a higher order helical LG beam, which is visualized in Fig. tes Fig.  as well tes Fig. .Figure 231.The given example demonstrates the capability to tailor optical angular momentum structures by spatial phase and polarization modulation. Beside a general interest of singular optics to use this opportunity to investigate fundamental properties of such light fields, especially domains sensitive to optical angular momentum will profit from our investigations, as quantum communication and optical tweezers and its applications in optofluidics and biophotonics33. In Fig. 34. It is surrounded by intertwining lines of linear and left- or right-handed circular polarizations, so called L- and C-line singularities. In case of L- or C-line singularities the handedness or orientation of polarization ellipses is undefined, respectively. In between two adjacent line singularities elliptical polarization states form a smooth transition required for a continuous polarization flow field. To distinguish between different polarization singularities, they are sketched by green (L-line), red (right-handed C-line) and blue (left-handed C-line) lines and a black dot (V-point) in Fig. 2,3-plane . Experimental results of the spiraling polarization field resulting from the phase hologram ΔϕSLM in Fig. 32, namely the Poincaré field index ϕ2,3 = arctan(S3/S2) of the Poincaré field shown in Fig. ϕ1,2 = arctan(S2/S1) is shown in Fig. 1, S2, S3) as well as ellipticity and orientation of polarization ellipses .Holographic polarization modulation not only allows for spatial variation of different polarization states, but also gives the opportunity to create certain polarization structures which reveal a continuous polarization flow field including polarization singularities35 become realizable. For example, higher order singularities in form of polarization flowers and webs as well as hybrid polarization structures can be generated36. In addition, beside transverse appearance and characteristics, especially dynamics of complex light fields, i.e. their longitudinal propagation, reveal a broad range of interesting findings as the evolution of phase and polarization singularities. As already phase as well as polarization singularities reveal interesting evolutions if considered independently, we expect distinctive dynamics if evolving polarization singularities are combined with naturally unfolding phase singularities. Dynamics provoked by customized light fields, generated by our dynamic holographic method, will be investigated in future.The direct modulation of spatial structures of polarization states as demonstrated in this example, allows to study complex Poincaré beams and polarization singular light fields. Auto-alignment of polarization components enables high modulation quality and enhanced stability compared to interferometric methods. This facilitates good comparability of experimental results and simulations. In particular, complex polarization singularity structuresA÷ = Av/Ah and phase shift Δϕ = ϕv − ϕh between these two components. A polarization sensitive device which only affects one of both components can be used to alter A÷ and/or Δϕ and therefore change the polarization of light. For this purpose, nowadays liquid crystal spatial light modulators (SLM) are the device of choice as they allow for a computer controlled and spatially resolved (pixelwise) variation of the complex field27. While the polarization component parallel to the liquid crystal director (P||) is shifted in amplitude and phase, its orthogonal component (P⊥) is not altered for a phase-only and parallel aligned nematic-liquid-crystal SLM as used here34. The relative amplitude and phase also depend on the input polarization  to influence the amplitude ratio A÷, a spatially resolved phase shift ΔϕSLM (both induced by the SLM), retardations of the two wave plates Γ1,2(n) = λ/n with  in the direction of propagation. Exemplary, an orientation angle of θ = −45° is depicted in Fig. A rotatable wave plate is able to transform linear polarized light into elliptical or circular polarization states in dependence of the orientation angle 1, S2, S3) with all fully-polarized states located on its surface. Linear polarization states can be found on the equator, while right circular and left circular polarization states mark the north and south pole, respectively. All possible output polarizations For a comprehensive analysis of accessible subspaces and influence of parameters, the resulting states of polarization are represented on a Poincaré sphere which is a unit sphere spanned by the Stokes parameters  = 1) the subspace of the DPMS can be described as the intersection of a plane and the surface of the Poincaré sphere which gives a circle in a three dimensional space, as illustrated in Fig. r, ϑ, φ) = (r(θ1), ϑ(θ2), φ(θ2)) from the origin of the sphere to the center C of the circle in spherical coordinates and its radius R(θ1). In many applications a 1:1 ratio of P|| and P⊥  used as WP1 shows lower angle sensitivity which simplifies its handling in an experimental setup. In Fig. θ1 are shown for a combination of two half and two quarter wave plates , respectively. Although in both cases WP1 is rotated about 45°, the circles are distributed over the whole Poincaré sphere for the half wave plates, while for the quarter wave plates only one half is covered, resulting in lower aberrations per angle uncertainty. In general, it is possible to access every polarization state by at least one combination of wave plates. An overview of all parameters used in Fig. 2) is used. Its retardation determines the trajectory of n2 and θ2. For a half wave plate used as WP2 this trajectory is identical with the equator. Comparable to the longitudes of a sphere, Fig. R = 1 (θ1 = 45°) for a variation of θ2 in case of WP2 being a half wave plate. If WP2 is replaced by a quarter wave plate, 2 enables to shift R = 1 a rotation of WP2 (=quarter wave plate) leads to a variable inclination of the circle’s plane with the equatorial plane, as presented in (e). As polarization states can be part of several circles, the flexibility of the DPMS is very high to choose between different combinations of parameters to spatially control the state of polarization of a light field. Especially a variable wave plate used as WP2 allows shifting θ1 = −1° while θ2 is varied for n2 = {2, 3, 4}. Advantages of our setup compared to previous methods are the two independently rotatable wave plates which allow to tilt a subspace (circle) around the Poincareé sphere . It can be seen, that our measurements match theoretical predictions very well. In (h) WP2 is varied while WP1 is fixed. Here some deviations can bee seen which can be explained by an aberration of the retardation of the wave plate (within the retardance accuracy of λ/200 of the multi-order wave plate). This is verified by the measurement in (i) where 3 axis for θ2 = ±45° as expected for WP2 being a quarter wave plate, as well. As the retardation of a wave plate typically cannot be influenced, we recommend to use high quality wave plates, only.Sub- Fig. ϕ = ϕv − ϕh and amplitude ratio A÷ = Av/Ah. Both parameters can be influenced homogeneously for the whole beam profile by setting the orientation of the WPs. In addition, the SLM allows for a spatially resolved modification of phase shift of polarization components by introducing ΔϕSLM as discussed in the previous section. By this method already a plethora of polarization structures can be realized as for example all possible combinations of linear polarizations, i.e. vector beams. However, it is desired to influence the amplitude ratio spatially as well, giving access to almost any polarization structure.In general, any state of polarization is describable as combination of a linear horizontal and a linear vertical polarization state and is explicitly defined by their phase shift ΔA÷ of perpendicular polarization components is implemented. We developed a new method, which is related to combined amplitude and phase modulation27, but now applied to polarization modulation. More specific, spatially resolved variation of phase Δϕ and amplitude A÷ ratio of polarization components is realized by a phase-only SLM. Therefore, the before mentioned phase ΔϕSLM is accompanied by an additional amplitude factor Therefore, we upgrade our method to not only allow polarization states represented on single rings on the Poincaré sphere but to enable polarization structures formed by polarization states given by continuous segments of the surface of the Poincaré sphere. For this purpose, spatial modulation of the amplitude ratio φB to diffract portions of one polarization component to a higher diffraction order (i.e. off-axis propagation) to affect the amplitude ratio A÷ of modulated and non-modulated polarization components P⊥ and P|| within the zeroth order.The idea is to employ a blazed grating The zeroth order transmission function of the phase-only function ASLM, ASLM. To eliminate the additional phase term, one has to multiply by its complex conjugate. Employed within the DPMS this technique directly effects the amplitude ratio1,2(n), θ1,2, ΔϕSLM and ASLM.If we choose entclass1pt{minimaλ = 532 nm). A half wave plate  measurements behind a rotating quarter wave plate (rotation angle θ3) and a fixed linear polarizer are related to the corresponding Stokes vector of the polarization state22 by Fourier analysis:N > 8 is the number of measurements (minimum given by sampling theorem). To yield normalized Stokes parameters S1,2,3 have to be divided by S0. Within the experimental setup, we employed a camera to enable spatially resolved Stokes parameter measurements. While for homogenous polarization distributions it is possible to take average values of several camera pixels, for the complex spatially structured Poincaré beams a pixel wise evaluation of Stokes parameters is performed. Opposite to other methods frequently used for vector beam analysis, as e.g. intensity observations behind a rotating polarizer, here beside orientation also ellipticity information of polarization states is recorded.In the experiment a reflective SLM is illuminated under a small angle by a linearly polarized laser  and their analysis are of general importance as singular structures are not only present in optics but (among others) also occur in astronomy or Bloch walls of magnetic systems. Although already several polarization singularities have been modulated with the DPMS, our system will lead to experimental realizations of even more complex polarization structures36 combining several types of singularities to whole polarization networks. Additionally, we expect distinctive dynamics if (unstable) polarization singularities are combined with (unfolding) phase singularities. Finally, capabilities of the DPMS have been evaluated within the method section. It has been characterized with respect to the phase of the SLM, the retardation of the wave plates and their orientation to demonstrate the enormous flexibility of the method.Simultaneous modulation of spatial phase, amplitude, and polarization values by a holographic method enables to tailor complex Poincaré beams, advanced optical angular momentum structures and various kinds of polarization singularities. To show the potential of the method, we demonstrate the generation of higher order LG modes combined with spatial modulation of polarization states. However, our holographic modulation system (DPMS) is not restricted to LG beams, but allows generating any light mode together with linear, elliptic and circular states of polarization in various combinations. Furthermore, spatial combination of spin and orbital angular momentum, as shown for a higher order helical LG beam, can be employed in quantum communication and classical entanglement


Sciaenops ocellatus). Red drum is an economically and ecologically important species native to coastal regions and estuaries of the Gulf of Mexico – habitats that routinely experience pronounced hypoxic events. Using a transcriptomic approach, we demonstrate that red drum red blood cells express 7 and 5 Hbα and Hbβ isoforms, respectively. Phylogenetic analysis grouped these isoforms into distinct isoHb clades, and provided evidence of lineage specific expression of particular isoHbs. In normoxia, three isoHbs predominated . A three-week hypoxia acclimation (48 mmHg) resulted in significant up-regulation of Hbα-2, Hbα-3.2, and Hbβ-3.1, effectively switching the predominantly expressed isoforms. Changes in subunit expression were correlated with a decrease in non-stripped hemolysate P50. Similarly, hypoxia acclimation resulted in a 20% reduction in whole animal critical oxygen threshold (Pcrit). Hypoxia acclimation was not associated with changes in gill morphology, hematocrit, or relative ventricular mass. Overall, these data provide support for the hypothesis that Hb isoform switching can provide a physiological benefit to counteract environmental stress in fishes.Hemoglobin (Hb) multiplicity is common in fish, yet despite its ubiquitous nature, the functional significance is unclear. Here we explore the hypothesis that Hb multiplicity plays a role in hypoxia tolerance using the red drum ( Hb isoforms (isoHbs) in teleosts are classified into three broad categories8. Class I Hbs are electrophoretically anodal and exhibit sensitivities to phosphates, temperature, and pH (e.g. Platessa platessa and Platichthys flesus)9. Class II Hbs found in Oncorhynchus mykiss10, Anguilla sp.11, and morays13 contain both electrophoretically anodal and cathodal components. The anodal components are similar to Class I Hbs, and are typically characterized by marked Bohr (decreased Hb-O2 affinity with a decreased pH) or Root effects (decreased Hb-O2 affinity and O2 carrying capacity with decreased pH), whereas the cathodal components are relatively insensitive to pH and temperature, and may have intrinsically higher oxygen affinities. Class III Hbs are sensitive to pH but not temperature, and are found in a few active, warm-blooded fish such as tuna14. Thus, it has been suggested that fish might be able to respond to environmental challenges such as hypoxia and long-term temperature changes by altering isoHbs5.Hemoglobin (Hb) multiplicity is a well-documented phenomenon in fishMugil cephalus)6 and gilt-head bream (Sparus aurata)15 distinct isoHbs have almost identical functional properties. Conversely, Japanese eel (Anguilla japonica) express at least two functionally distinct Hb pools7, and acclimation to varying temperatures has also been demonstrated to differentially regulate isoHbs in several species17. These findings suggest that Hb multiplicity may confer a physiological advantage under specific environmental conditions, which is supported by similar findings in tetrapod vertebrates19.While Hb multiplicity in fish is well known and wide spread, the physiological implication of multiple isoHbs is unclear. In species such as the striped mullet  ishmaeli demonstrated differential expression of isoHbs when reared under normoxia and hypoxia5. The hypoxia reared fish Hbs also exhibited higher Hb-O2 affinities, suggesting that Hb multiplicity may constitute part of a regulatory mechanism that protects oxygen uptake under adverse conditions.Of the myriad environmental challenges that fish experience, hypoxia profoundly impacts oxygen uptake. Fish are capable of maintaining oxygen uptake under hypoxic conditions through a variety of mechanisms that include hyperventilation, increased blood perfusion through the gills, and enhanced Hb-OSciaenops ocellatus) is an economically important species native to the coastal regions of the Gulf of Mexico (GoM). Like many estuarine species, red drum regularly encounter hypoxia in their natural environment, and this is compounded by the occurrence of wide scale oxygen minimum zones in the northern GoM. Importantly, this species has been shown to exhibit phenotypic plasticity in response to a wide array of environmental perturbations23. These factors make red drum an ideal species to explore the capacity of a marine fish to acclimate to prolonged hypoxia through phenotypic plasticity, and whether Hb multiplicity may play a role in this plasticity. As such, the first objective was to explore the prevalence of Hb multiplicity in red drum in relation to other teleosts using a combination of transcriptomic and phylogenetic analyses. A second objective sought to explore the effect of prolonged environmental hypoxia on Hb expression, blood oxygen affinity and whole animal critical oxygen thresholds (Pcrit). Additionally, these studies also addressed the potential roles of branchial plasticity in hypoxia acclimation through the analysis of gill surface area and diffusion distance.Red drum . In this case the majority of clades contained all three superorders. The lone exception was clade II, which only contained two sequences and was not expressed by red drum.i.e. 64-times greater expression; data not shown).Of the 12 Hb subunit isoforms identified in the red drum transcriptome, 10 exhibited detectable expression via real-time PCR. The relative expression values of the detectable Hbα subunit isoforms revealed that Hbα-3.1 was the predominantly expressed isoform, with the Hbα-3.2 isoform the second most expressed at 12 ± 7% relative abundance . With respect to the β subunits, Hbβ-3.1 was the most abundant in both normoxia and hypoxia treatments, with expression levels more than two orders of magnitude higher than the other β subunits (data not shown).Prolonged hypoxia exposure resulted in significant up-regulations of Hb subunits in an isoform specific manner Fig. . Express50 for non-stripped hemolysates was significantly lower (P < 0.05) in hypoxia acclimated fish compared to the normoxia acclimated fish. Note that there was no interaction between pH and hypoxia on P50. Bohr coefficients were not different between the two treatments  account for the majority of observed expression in normoxia. Both of these subunit isoforms have theoretical isoelectric points above 8.0 (data not shown), which indicates that the primary Hb isoforms in red drum are cathodal. However, this should be viewed with caution as these are only theoretical subunit isoelectric points based on amino acid composition.25. Our Hbβ phylogeny conforms to this paradigm, but our analysis of the Hbα subunits has both expanded on the phylogeny and provided new insight into the diversity of subunits across different fish lineages. The Hbα phylogeny identified all previously described groups (I-IV and short), but also identified a distinct group (clade V) closely related to clade II. Also of note was the loose collection of sequences with unresolved identities, which included two red drum sequences. We have tentatively named these sequences Hbα-6.1 and -6.2; however, this group may become more resolved as sequence information from additional species becomes available. Several other trends became apparent when the species taxonomy within the respective clades was considered. Only clade III and the “short” clade appear to be widely found among teleosts. Conversely, clade I, II, IV and V are clearly not found across all teleost lineages, with clades I and IV not found in red drum or any other Acanthopterygii. As such, it is important to consider species phylogeny when attempting to understand the functional significance of Hb multiplicity. Interestingly, the most highly expressed Hbα subunits in red drum were both from clade III, which contains isoforms from all tested fish lineages. This may suggest a primary functional role of this particular group of subunits, while other subunits offer specialized or redundant functions.The currently accepted phylogeny for Hb subunits in fish suggests that the α-subunits are divided among five distinct clades, while there are four β-subunit clades27.The Hbα-short isoform was also found to be widespread across fish lineages, but was only expressed at very low levels in sub-adult red drum red blood cells. The conservation of this truncated subunit in all fishes suggests an important physiological role; however, its low expression indicates it isn’t critical for oxygen delivery in developed fish under normal conditions. It is possible that this subunit is important at an earlier life stage; however, currently accepted phylogenetic relationships and the available evidence from rainbow trout suggests that the embryonic subunits are a duplication of Hbα-150 of red drum non-stripped hemolysates fell within the range typically found in teleosts in both normoxia and hypoxia treatments; however, prolonged hypoxia acclimation was associated with a significantly lower non-stripped hemolysate P50 (i.e. increased Hb-O2 affinity). The greatest reduction was observed at lower pH; however, the interaction between P50 and pH was not statistically significant. The Bohr coefficient for Hb was also not impacted by hypoxia acclimation for either pH range. Interestingly, work on cichlids showed the strongest effects of hypoxia acclimation on P50 at pH ≥ 7.6 for both stripped and NTP saturated lysates. While our data were derived from non-stripped lysates, to preserve any hypoxia driven changes in NTP content, the magnitude of observed changes between studies is comparable, with Rutjes et al.5 reporting hypoxia driven reductions of 1 to 4 mmHg for stripped and saturated lysates. But it is important to remember that P50 values from hemolysates are generally lower than intact red cells28. This is owed in part to dilution of hemoglobin, NTP and other allosteric modifiers, although the ratio of these parameters remained consistent. Nonetheless, these data give strong evidence that hypoxia acclimation results in an increased blood P50 in red drum, which should increase the ability of these animals to extract oxygen from the environment.The P29. Unfortunately, limitations on sample volume precluded measurement of NTP concentration in our experiment. We instead chose to explore the hypothesis that differential regulation of specific Hb components may coincide with increased oxygen affinity during hypoxia acclimation. It has been previously hypothesized that species with multiple Hbs acting in concert would be better suited to tolerate environmental perturbation32; however, the physiological support for this is mixed30. But previous work has primarily focused on temperature, with relatively less work focusing on prolonged hypoxia acclimation34. Here we provide evidence that specific Hb isoforms are selectively up-regulated in response to hypoxia stress, which is also correlated with a decrease in hemolysate P50. It is tempting to suggest that the selective up-regulation is promoting an isoHb with higher oxygen affinity, but this cannot be concluded without additional studies that involve purification and protein sequencing  has been linked to whole blood Hb-O2 binding affinity38. Interestingly, red drum exhibited a significant 22% decrease in Pcrit, from 36.2 ± 3.3 to 28.1 ± 2.1 mmHg, after hypoxia acclimation. Normoxia exposed fish showed no reduction over the same time period. Also note that there was no effect of hypoxia acclimation on SMR, which has also been correlated to Pcrit in some fish species39, although not red drum40. Similar findings in response to hypoxia exposure have been shown in goldfish and southern catfish 42, with goldfish capable of decreasing Pcrit by 50% following hypoxia acclimation41. It is important to stress that even small reductions in Pcrit are of substantial ecological importance. When animals are exposed to ambient oxygen levels below Pcrit they must augment aerobic metabolism with anaerobic metabolism. While animals can vary substantially in their ability to survive on anaerobic metabolism, this strategy is inherently unsustainable over prolonged time scales. By lowering Pcrit, red drum reduce the risk associated with hypoxic environments. Based on our previous work, the 48 mmHg PO2 treatment used in this study equates to an approximately 90% reduction in aerobic scope43. It would be interesting to assess whether acclimation also increases the available aerobic scope to animals in hypoxia.An increase in Hb oxygen affinity can play an important role in the ability of fish to maintain oxygen uptake, and aerobic metabolism, as ambient oxygen levels decline . This was performed to simplify analysis and graphical display to emphasize deeper phylogenetic trends. Similarly, targeted Blast search efforts were made to ensure proper coverage of the Acanthopterygii, Protacanthopterygii, and Otocephala superorders across the various Hb subunit isoforms. Final analyses were performed using maximum likelihood methods on protein sequences with the following options: JTT model, NNI ML heuristic method and complete deletion of gaps/missing data. Phylogenies were tested using bootstrap methods with 1000 iterations. All sequence identifiers within the final analysis are included in Fig. All experimental procedures were approved by the University of Texas at Austin Institutional Animal Care and Use Committee (IACUC), and all experiments were performed in accordance with the approved IACUC protocols and methodologies. Unless specified all chemicals were obtained from Fisher Scientific. Red drum were obtained from Copper Shoals Red Drum aquaculture facility , and held on-site at the University of Texas Fisheries and Mariculture Laboratory. Sub-adult red drum  were held in recirculating in-door 150 L tanks supplied with filtered natural seawater maintained at 22 °C. The recirculation system was equipped with a common biological filter tank to control ammonia levels. Fish were fed daily with commercial fish pellets  and tanks were siphoned daily to remove debris. All fish were acclimated to the laboratory setting for at least two weeks prior to experimentation.crit measurement. Oxygen consumption rate (2) of the fish was measured using computerized intermittent-flow respirometry  for SMR calculations47 at 24 °C for at least 20 h. Each 2 measurement cycle lasted 6 min, consisting of a 3 min flushing period followed by a 3 min closed period. 2 was calculated from the decline in PO2 in the last 2 min of the closed period according to Eqn 2 is oxygen uptake (μmol kg−1 min−1), δPO2 is the slope of the decline in water oxygen tension (mmHg min−1) during a closed respirometer cycle, αO2H2O is the solubility of oxygen in the water at the relevant temperature (μmol L−1 mmHg−1), Vchamber is the volume of the respirometer (L), and Mf is the mass of the fish (kg). Values for 2 were omitted when the R2 of the linear regression for the decline in PO2 was less than 0.95. Background tests were conducted for 0.5 h-2 h both before the fish was placed into the respirometer chamber and after the fish was removed from the respirometer chamber. Background 2 was subtracted from the calculated 2, by assuming a linear increase in O2 consumption rate over the measurement interval. The lowest 10% 2 values obtained from the measurement period was averaged to determine SMR for comparison with previous work conducted in our lab on red drum40. Directly following SMR measurement, the fish was subjected to a Pcrit trial whereby the respirometer was placed on a closed circuit until PO2 within the chamber dropped below 8% of air saturation (12.7 mmHg). 2 was calculated from the decline in PO2 in 2 min intervals during the closed period. Pcrit was then determined as the PO2 where 2 was reduced below the SMR43.Food was withheld for at least 48 h prior to measurements. Standard metabolic rate (SMR) of fish was measured in advance of Pmentclass2pt{minimO2 = 48 ± 8 mmHg) and the other at normoxic condition (PO2 ≥ 151 mmHg). Hypoxia was maintained using an automated nitrogen bubbling system, which consisted of a fiber optic oxygen electrode and associated meter (PreSens) interfaced to a gas flow solenoid, which was controlled using AutoResp software (Loligo Systems). Tanks were monitored daily for salinity and pH, and temperature in both tanks was maintained at 22 °C. Fish were sub-dermally tagged with VI alpha tags , according to the manufacturer guidelines, 1 week prior to experimentation. Individuals were then randomly assigned to either hypoxia or normoxia treatments, with 7 fish per treatment. Pcrit of all experimental fish was measured before subjecting them to their respective treatments and at the end of the 2-week acclimation period. Fish were fed ad libitum. After the final Pcrit measurement, fish were placed back into their respective treatments for 1 week to recover from the Pcrit test, after which individuals were euthanized and sampled as described below.Exposures were performed in two 150 L tanks, with one maintained at hypoxic condition (P3) followed by spinal transection. Body mass was recorded, and blood was taken by caudal puncture using a 23-gauge needle pre-rinsed in heparinized saline (50 units/mL). Hematocrit was measured and blood samples were centrifuged at 13,000 rcf for 1 min to separate the plasma. Packed red blood cells were preserved at −80 °C for further analysis. The heart was excised and the ventricle was separated and weighed. The first gill arch on the left side of the fish was sampled for gill surface area and gas diffusion distance analysis. Gill samples were placed immediately in zinc formalin fixative , stored overnight, and subsequently transferred to 70% ethanol for long-term storage.Fish were euthanized by full immersion in a MS-222 bath  as an internal control50. The relative abundance of Hbα transcripts within each sample was also assessed using a rearrangement of the numerator from the delta-delta ct formula, which allowed proper incorporation of the respective PCR efficiencies (EHbα-3.1ct/Enct). In this case, relative abundance was calculated relative to the dominant transcript in normoxia, Hbα-3.1. Successful DNase treatment was verified using a no reverse transcriptase control for each treatment.Total RNA was extracted using TRI Reagent  according to manufacturer guidelines, with homogenization performed using a 18 gauge and then a 23 gauge needle. Total RNA was quantified using an ND-1000  spectrophotometer at a wavelength of 260 nm and sample purity was assessed using 260:280 ratios. cDNA synthesis was performed using RevertAid reverse transcriptase (Thermo Scientific) according to the manufacturer specification, using 1 μg of DNase I (Thermo Scientific) treated total RNA as template. Relevant Hbα and Hbβ gene sequences were identified from a commercially developed  RNA-Seq transcriptome library for red drum. Primer sets were designed using Primer32 equilibria experiments were performed at the University of British Columbia (UBC), Vancouver, Canada. Isolated red blood cells were delivered to UBC on dry ice and subsequently stored at −80 °C until use. For analysis, red blood cells were thawed on ice and the Hb concentration was quantified using the Drabkin’s method51. Note that owing to the small sample volume (50–80 µl) the sample was mixed with 40 µl of deionized water prior to quantification, which both maximized sample utility and pipetting accuracy during quantification. Methemoglobin concentration was assessed spectrophotometrically and used to correct total Hb concentration to ensure a functional Hb4 concentration of 0.6 mM. There was no significant difference in methemoglobin levels between control and hypoxia acclimated fish. Hemolysates were prepared in HEPES buffer (100 mM) of nominal pH 7, 7.4 and 7.8 supplemented with 40 mM KCl and 4 mM ATP. Hb-O2 equilibria were constructed using a previously described custom built gas equilibration chamber52 placed within a plate spectrophotometer (Molecular Devices). Gas flow was controlled by a Wösthoff DIGAMIX® gas mixing pump . Assays consisted of equilibration at 0, 0.05, 0.1, 0.15, 0.25, 0.5, 0.75, 1, 1.5, 2, 5, 8, 12, 21 and 100% O2 balanced with N2, and the absorbance plateaus at each O2 tension were assessed manually by the operator. All assays were performed at 22 °C. The absorbance at each O2 tension was used to generate Hill plots, log([oxyHb]/[deoxyHb]) vs logPO2, and thereby estimate P50 – the partial pressure that results in 50% Hb-O2 saturation. Each plot was assessed manually to verify proper dose response relationships with respect to absorbance and O2 level. P50 was estimated from Hill plots using the following formula: All Hb-O53. Briefly, the total number of filaments on the first gill arch was counted and the length of every tenth filament was measured under a dissecting microscope. The linear spacing between lamellae along the filament was measured along at least 8 lamellae at 100X magnification under a light microscope (Nikon Eclipse TE2000-U) at the base, midsection and tip of 5 filaments evenly distributed along the gill arch using digital image capture software (QED Capture). An image of a stage micrometer was also digitally captured to calibrate the measurements.Measurements of gill surface area were carried out according to methods outlined by Hughes® as a mounting medium after the slides were dehydrated.Approximately 10 filaments from the midsection of the gill arch were dehydrated first in an ethanol series , then in butanol for 1 h, and finally left in butanol overnight (12–16 h). Filaments were then treated twice with Histochoice clearing reagent  for 90 min, followed by two 60 min washes in Paraplast Plus embedding media  at 58 °C. The samples were embedded in casting cubes at room temperature and stored at 4 °C until sectioned. Samples were sectioned at 6 μm using a microtome and mounted onto Superfrost Plus slides . At least 5 sections were mounted onto each slide. Slides were then deparaffinized with two 5 min washes in Histochoice clearing agent and rehydrated in an ethanol series . Slides were stained with hematoxylin solution, gill no. 2  for 120 s, soaked in 0.3% HCl-EtOH solution for 60 s, blued in Scott’s tap water substitute  for 45 s and counterstained with eosin y solution, alcoholic (Sigma-Aldrich) for 60 s. Slides were rinsed with tap water in between the different solutions and soaked additionally for 30 s in 95% ethanol before counterstaining. Coverslips were then applied with PermountL is the total filament length (mm), f is the number of lamellae per millimeter on both sides of the filament, and B is the average bilateral surface area of a single lamella (mm2). Surface area was standardized to the mass of the fish for comparisons between different fish.Stained slides were viewed under a light microscope (Nikon Eclipse TE2000-U) at 400X magnification. Digital images of 20 lamellae were captured for each fish to quantify lamellar area. All lamellae were selected from the midsection of the filament and no more than 4 lamellae were chosen from a single filament. Images were assigned random numbers before surface areas were scored to minimize bias. Total surface area was calculated as Fifteen digital images of lamellae were then taken for each fish under 1000X magnification and 10 measurements of lamellar blood-to-water diffusion distance were taken to estimate the lamellar blood-to-water diffusion distance of each fish. Similar to surface area measurements, images were also assigned random numbers before diffusion distances were scored.50 data was analyzed using a two-way ANOVA with pH and acclimation condition as the two independent factors. Note that a significant interaction between factors was not observed, thus treatment effects at a specific pH could not be assessed. Pcrit from hypoxia acclimation experiment was analyzed using one-tailed paired t-test, under the a priori assumption that dynamic Hb expression and reduced hemolysate P50 should reduce Pcrit.Data are presented as means ± SE. Gene expression levels and morphometric data collected after the acclimation experiment were analyzed using unpaired t-test to identify differences between the normoxia and hypoxia group. Statistical significance was assumed at P < 0.05. Blood P


Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application. This emerging class of probes offers photo-physical properties that differ to a range of commercially available organic probes, and is enabling a new range of technologies to be explored. Notably, recent advances in optics and electronics, combined with the long emission lifetimes typical for transition metal complexes (from hundreds of nanoseconds to microseconds), has resulted in the emergence of multiphoton microsecond lifetime mapping techniques, such as Phosphorescence Lifetime Imaging Microscopy (PLIM)5 and Time-Resolved Emission Imaging Microscopy (TREM)4.The use of metal complexes as dyes and probes for emission-based cellular imaging has developed into a vibrant area of research over the past decade10. One application of phosphorescent emitters in biological imaging is in oxygen detection, where more sensitive, non-invasive methods are in demand. The combination of phosphorescence quenching and high-resolution lifetime imaging is a powerful approach for non-invasive, real-time hypoxia detection and oxygen quantification.Luminescent transition metal complexes typically emit from a triplet excited state. Although transitions between states of a different spin are formally forbidden, intersystem crossing to the triplet state and subsequent relaxation to the ground state via phosphorescence are facilitated in transition metal complexes by the high spin orbit coupling constant associated with the heavy metal atom. The forbidden nature of the phosphorescence transition, results in a slow rate of emission; hence phosphorescence lifetimes are typically the order of hundreds of nanoseconds to microseconds. It is well documented that molecular oxygen quenches such triplet emitters and that the rate of quenching is dependent on oxygen concentration7; in determining oxygen gradients across 2D monolayers, 3D neurospheres and spheroids using a combination of single photon time correlated single photon counting (TCSPC) and PLIM; in measuring in-vivo oxygen concentration changes in the blood stream of live animals, using bespoke multi-photon microscope13.Oxygen quantification in biological systems is primarily focused on the use of large platinum and palladium porphyrins. These compounds display long emission lifetimes , which typically decreases upon exposure to oxygen. They have been successfully used for measuring bulk intracellular oxygen concentrations in a range of models. Thus they have been used in an intensity-based, high-throughput method utilizing a time-resolved fluorescence plate readerEfficient cellular uptake can be problematic with porphyrins, due to their large size. For some applications, such as intra-cellular and blood oxygen concentrations, this is a positive attribute as it prevents the porphyrin from sequestering into the surrounding cells or tissue. For intercellular oxygen determination and penetration into larger or deeper samples, it is a major drawback. This problem has been overcome in some cases by the addition of cell-penetrating moieties, specific cell markers or encapsulation in nanoparticles, however, these systems are complex and require long multi-step syntheses.16. To obtain a detailed relationship between cellular location of the probe and its emission lifetime (and therefore oxygen concentration) excellent spatial and temporal resolution is required. Intensity based imaging using wide field illumination will only give a general picture of the oxygen concentration as the intensity information will be averaged in the ‘z’ direction. Furthermore, intensity based images can be affected by background emission, probe concentration, imaging parameters and photobleaching. Lifetime mapping using a point-by-point scanning method, such as that used in confocal imaging, with time correlated single photon counting, offers a more accurate method that circumvents many of the drawbacks associated with intensity imaging. Yet there is a hurdle: the longer the lifetime of the O2 probe, the longer the image acquisition time. To establish a lifetime decay at each pixel , with a good signal-to-noise, the microscope scan head must remain at each pixel for a period of time that is 5 to 6 times longer than the emission lifetime of the probe, and over tens or hundreds of frame scans. Therefore, porphyrins with very long emission lifetimes require very long acquisition times, over which sample drifting can be a problem. One solution is to sacrifice spatial resolution by using a smaller number of larger pixels. Alternatively, using an oxygen responsive probe with a shorter emission lifetime can reduce sampling time.Phosphorescence quenching offers a direct, real-time and quantitative method for oxygen determinationSmall molecule transition metal complexes with emission lifetimes on the order of hundreds of nanoseconds to microseconds provide an alternative that requires less of a trade-off between resolution and speed, with simpler synthetic procedures. Some transition metal complexes can penetrate living cells without the need for elaborate synthetic modification and in some cases demonstrate specificity to particular organelles, which can be desirable when investigating oxygen changes as a response to external stimuli.nCl for in vitro live cell imaging and two-photon lifetime mapping using TCSPC18. This family of complexes benzene) based ligand) , high emission quantum yields (c.a. 70%)19, excellent photostability and low cytotoxicity. Previous studies have investigated the mechanism of entry across human fibroblast and melanoma cells in vitro, and report on properties that enable passage across intact cell membranes via diffusion after incubation with PtLnCl for 5 minutes, permitting imaging thereafter17. Moreover, these PtLnCl complexes have a relatively short synthetic pathway, which can be accelerated by microwave-assisted methods20. We have already demonstrated that the emission lifetime of complexes of this type varies with exposure to oxygen inside living cells (CHO cell cytoplasm from 95% N2/5% Co2 from 1.49 µs to 6 µs18). Cytosol staining appears uniform under two-photon excitation and exhibits the lowest level of protection from collisional quenching, making these complexes an excellent choice for oxygen detection via phosphorescence quenching. Another key advantage of such small Pt(II) molecules is their diffusion controlled intracellular accumulation without an active intake pathway, which makes them ideal as probes for experimental systems such as 3D tumour spheroid models.We have previously reported on the application of a series of small luminescent platinum (II) complexes PtL22. These solid aggregates resemble an avascular tumour23. The model provides an assay to study the tumour microenvironment where the outermost layers of cells receive nutrients by diffusion and discard waste products into culture medium. However, the innermost cells outgrow the natural diffusion range of nutrients, oxygen and waste products, leading to formation of hypoxia and necrosis within the inner ‘core’ areas. The extent of these features can be controlled experimentally as they are determined by the type of cells in culture, the size of the MCTS, time in culture, together with the proliferation rate of the cancer cells in question.Multicellular tumour spheroids (MCTS) involve culturing cancer cells in a non-adherent environment, which consequently stimulates them to adhere with one another25. Therefore, 3D MCTS models can be exploited as a structural model with a necrotic central core due to: (i) deficiency of nutrients and metabolites; (ii) variation in oxygen concentration; (iii) accumulation of waste products and (iv) low pH. These are some characteristics related to the phenomena seen in vivo of a solid tumour over a distance >100 µm from a blood supply27.It has been established that not all cell types have the ability to form spheroids, and some cell types form irregular shaped aggregates rather than spheroids. The heterogeneity of cells and the metabolic gradients  become apparent in spheroids only when they are larger than 150 µm. The appearance of a necrotic and hypoxic central region becomes apparent only after a diameter of ~500 µm18, herein we use a human melanoma multi-cellular tumor spheroid model in a longitudinal study and investigate the small molecule Pt dye for variations in oxygen tension. This progresses previous single melanoma cell reports to an organoid study of physiological context. We demonstrate the first example of a small-molecule platinum (II) complex for oxygen mapping using one-photon phosphorescence lifetime imaging microscopy, which provides a non-invasive imaging modality for oxygen distribution across live human melanoma tumour spheroids at various stages of development.Extending upon previous studies using small molecule Pt dyesin vivo solid tumour, such as, heterogeneous proliferative and necrotic cells and varied metabolic gradients of oxygen, nutrients and waste product28. These tumour microenvironments exhibit three distinct regions of oxygenation30, simulating the oxygen gradient found across a solid tumour; where the majority of tumour cells located close to a blood vessel are proliferative while the inner area (far from the blood vessel) is made up of hypoxic and anoxia cells29. Using an in vitro melanoma C8161 tumour spheroid model (between 100 µm and 1000 µm in diameter) we successfully characterized these three physically distinct regions: (a) a proliferative viable region of human melanoma cells, (b) an inner hypoxic region and (c) a central necrotic core, using complementary methods of light microscopy, histology, immunohistochemistry and phosphorescence lifetime mapping.To mimic solid tumour conditions, we used a melanoma multi-cellular tumour spheroid model (MCTS). MCTS models exhibit characteristics of an in vitro, using an established liquid overlay technique21. C8161 melanoma cells were the most successful, forming compact MCTS within first 1–2 days of culture with a very high yield of 90 to 100%. A seeding density of 12,000 cells was selected as the optimal starting point for the C8161 MCTS model  were initially investigated for their ability to form multi-cellular tumour spheroids (MCTS) 0 Figure . The siz0 Figure  showed i0 Figure . This wa0 Figure  revealed0 Figure , which iThe radius of the unstained necrotic region increased in size from 65.6 µm (±22.5) on day 4 to 338.2 µm (±11.8 µm) (n = 10) on day 10, whereas the proliferative area reduced in size from 186.4 µm (±31.3) to 109.7 µm (±5.7) µm Fig. . After 82 of 10mm Hg at 37 °C31. In MCTS at day 4 a central hypoxic area was observed  molecule O2 sensor and PLIM34.Hypoxic regions within MCTS across different growth stages were detected using pimonidazole hydrochloride with immuno-labelling of protein adducts of reductively activated pimonidazole as evidence of hypoxia. A commercially available kit (Hypoxyprobe™) was employed using the pimonadizole as an oxygen marker that binds only to cells with an oxygen saturation less than pOsCl and lifetime mapping, we first assessed the penetrative ability of the PtLsCl in to the spheroids by steady-state microscopy under two-photon excitation (λex = 800 nm and λem = 500–550). Previous studies  on 2D cell monolayers demonstrated that PtLsCl is rapidly taken up by living cells after just 5 minutes incubation at 100 µM. In contrast, at the same concentration, we found that PtLsCl required 12 hours at 37 °C to completely penetrate though a C8161 melanoma spheroid of <500 µm in diameter. (Higher concentrations of label (up to 1000 μM) for 5 minutes incubation did not penetrate significantly beyond the outer cell layer, and was similar to the depth observed using 100 μM). PtLsCl uptake and distribution was uniform across spheroids of different sizes at different growth stages . Previous studies  also show that the probe remains sensitive to oxygen when inside a living cell; exemplified by the variation in emission lifetime across a single cell where τnucleoli ~ 7 µs > τnucleus ~ 5.6 µs > τcytosol ~ 4.0 µs  to show the lifetime variation (and distinct regions) across the melanoma spheroids. The decay kinetics of PtLsCl emission can be readily quantified in different cells and in different regions of a cell and tissue  time correlated single photon counting (TCSPC) module, coupled to a confocal microscope and a photomultiplier, was used for photon detection. In a one-photon set-up the pixel clock, which controls the scan head of the microscope, acts as the trigger for the TCSPC module enabling the emission decay of each pixel in the 256 × 256 array to be analysed independently of one another. To construct lifetime maps, decay traces for all pixels across the 256 × 256 array (or those selected as a region of interest) were fitted to a double exponential model, with a bin (averaging parameter) of 2, and τsue Figs  and SI 3sCl in MCTS could be classified in to three distinct ranges; the shortest lifetime  were observed at the outer edges of the MCTS, which increased to c.a 4–6.5 µs (green) then ca. 6.5–12 µs (blue) with increased proximity to the centre of the spheroid   Figs  and 4. Tes) Figs . This diThis is the first example of real-time oxygen mapping across a 3D human spheroid model using one-photon PLIM imaging, at high resolution. The data has provided a spatial-lifetime across the section of the spheroid, with enhanced resolution in the x, y plane revealing a clear cellular structure at the edges of the spheroid where the cells are intact and proliferating normally, and a lack of structure at the centre of the spheroid where there is mostly cell debris. Z-stacking data obtained by PLIM Fig.  did reve36. The measurement of oxygen is important for assessing cellular activity and in clinical diagnosis, tumour chemo-sensitivity, in pharmacological drug screening and for environmental toxicant screening. Furthermore, changes in oxygen levels during tumour development can form the basis for the identification of neoplastically-transformed cells as micro-tumours27.Intracellular oxygen concentrations play a critical role in many physiological and pathological processessCl lifetime mapping using PLIM on MCTS of different sizes and growth stages cumulatively showed large spheroids having a significant lifetime-oxygen gradient, while smaller and earlier growth stage spheroids showed no such gradient. This heterogeneity in lifetime was dependent on spheroid culture conditions. Finally, pixel-by-pixel measurements of lifetime in these three distinct areas allowed demographic quantification of average lifetimes values in spheroids across the z-axis, illustrating a sensitive, real-time, quantification technique.PtLsCl type, and diffusion-limited properties through a multi-layered tumour spheroid, where a longer incubation time using the same concentration as for single cell imaging was required. This property, in combination with a high emission quantum yield and moderately long emission lifetime, which is strongly oxygen sensitive, offers a simple imaging procedure. This has potential in terms of PLIM acquisition times where subsequent excitation pluses must be separated by at least 4 to 5 times the length of the emission lifetime of the phosphorescent probe. Following on from this study we aim to investigate the full optical slicing ability of two-photon PLIM with PtLsCl, to assess applicability  in keratinized cancer skin models for broader clinical applications of melanoma diagnosis.Moreover, we observed a diffusion limited cell permeability property of the small molecule PtLThe present study demonstrates a novel approach for the detection of relative oxygen levels in a melanoma tumour tissue mass, based on the ability to combine PLIM and single-photon excitation in combination with an oxygen sensitive platinum label. Given that solid tumours display varied oxygen levels, this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations and we therefore present this as a novel optical method. The data shows one of the first examples of non-invasive oxygen mapping across a melanoma tumour spheroid using one-photon PLIM, revealing a variation in lifetime that correlated with oxygen concentration. These measurements were quantitative and enabled real time oxygen mapping with enhanced spatial resolution.38. Our PtLsCl probe along with confocal PLIM provides information that may be useful in selecting patients who would benefit from new hypoxic- targeted therapy39. We suggest that the development of a non-invasive 3D mapping of the tumour microenvironment with high resolution will open up new avenues for therapeutic interventions. For example, investigating the hypoxic environment of a tumour in vivo would be of value in the surgical management of melanoma or in the use of high precision irradiance modulation to achieve selective dose escalation in chemo/radio resistant hypoxic cells within the tumour microenvironment. The study of this family of probes benefits from access to multiphoton microscopy.There is experimental data that suggests that hypoxic cells within tumour microenvironments are less sensitive to chemotherapy and radiotherapy. In addition, it has been suggested that tumour hypoxia may also relate to malignant progression40. The direction of research is that it should prove possible to use fibre optic probes for use in the clinic, which would allow the interrogation of tumours within the skin up to a depth of 0.5 mm, sufficient to detect surface, deep and metastasising melanoma leaving the primary tumour. Thus, one can excite the compound with 400 nm light, and emission is such that fibre optic excitation with long or short wave light will still lead to a desired measurement. We therefore suggest PLIM incorporated with PtLsCl probes may be developed as a technology and optical tool for the detection of both physiological and pathological oxygen levels in live tumour tissue mass, with the potential for broader clinical application.Parallel developments in dyes and imaging technology are also being studiedsCl probe was synthesised as described by us previously19. Other imaging dyes and stains were purchased from commercial suppliers (as indicated in the SI) and used as per the suppliers instructions, all other reagents were purchased from Sigma Aldrich .The PtL16. Briefly, 100 µL of cells (1.2 × 105 per mL) were added to each well of a 96-well plate previously coated with type V agarose (1.5% (w/v)) in the growth medium (EMEM). The cells were incubated at 37 °C, 5% CO2 and monitored overtime for spheroid production. Cell culture medium was refreshed every 2–3 days by replacing 100 µL with fresh medium. MCTS was imaged every day for 12 days using phase contrast light microscopy to detect spheroid formation  and allowed to settle overnight at 37 °C, 5% (v/v) CO2 in full EMEM medium. Spheroids were then incubated with PtLsCl 100 µM for 12 hours . Before imaging, spheroids were washed with PBS (x3) and then immersed in PBS.Staining with PtLex = 405 nm, λem = 500–550 nm, respectively. The diode laser was ‘on/off’ controlled for PLIM imaging by the Becker and Hickl FLIM module. The lifetime imaging window was set to 32 µs, which included a laser on rise time of 2 μs. The field of view was made up of a 128 × 128-pixel array.Measurements were performed at the Central Laser Facility, Rutherford Appleton Laboratory, STFC, Oxford, UK. The set-up comprised a Nikon microscope connected to a Becker and Hickl DCS-120 confocal scanning FLIM system and a Becker and Hickl 405 nm diode laser (BDL-405-SMC). Excitation and emission filters used were λsCl phosphorescence lifetime was measured at three intercellular positions: (1) nucleus, (2) cytoplasm and (3) nucleoli with 14 independent sample points for each region, averaged and plotted against standard deviation  and lifetime emission images were stacked together using ImageJ software (version 1.4 G) for a volume 3D image of the whole construct. Furthermore, PtLsCl phosphorescence lifetime values were measured at three regions of the spheroid: (1) outer; (2) middle and (3) central, which were averaged and plotted (n = 5 independent areas using xy coordinates for each spheroid area) against standard deviation. In order to check for any statistically significant differences, a paired t-test was performed. Results with p-values of ≤0.05 were considered statistically significant. All data was analysed using Graphpad Prism software, version 6.0 . Decay kinetics were best fit to a double exponential approximation on a pixel-by-pixel basis. Pixel binning and threshold function were kept constant for all measurements. Lifetime data collected on single cells were taken from three independent regions of interest for each cell line. PtLn Figure . PtLsCl .89. DecaSupplementary information


The evolutionarily conserved Wnt signaling pathway provides temporal and spatial cues during early embryonic development and during homeostatic function of numerous adult tissues . Wnt sigAPC mutant CRC cell lines, have emerged as a promising class of novel therapeutics. However, Tankyrase inhibitors exhibit overt on-target gastrointestinal (GI) toxicity in mouse CRC models due to effects on the Wnt-dependent intestinal stem cells that maintain normal GI homeostasis [Wnt ligands are palmitoylated in the endoplasmic reticulum by the membrane bound O-acyltransferase, Porcupine, and palmitoylation of Wnt ligands is necessary for secretion of the active protein as well as high affinity binding to the Frizzled receptor . To dateeostasis . Such a in vitro. Interestingly, pyrvinium was previously approved by the FDA and used successfully and safely for many decades in the clinic as an anti-pinworm medication. The bioavailability of pyrvinium was limited to the GI tract upon oral administration, a positive attribute for an anthelmintic drug, but one that limited its repurposing as a Wnt inhibitor for CRC patients. In a recent study, we described the development and characterization of a second-generation pharmacological activator of CK1α, SSTC3, which exhibited significantly improved bioavailability compared to pyrvinium [in vitro and prolonged the survival of a mouse CRC tumor mouse model. Taking advantage of the improved pharmacokinetics of SSTC3, we showed for the first time the efficacy of CK1α activators in attenuating the growth of CRC in vivo using both cell lines and patient-derived CRC xenografts (including a lung metastatic cancer harboring a KRas mutation that was derived from a patient). We found that SSTC3 exhibited greater efficacy than a Tankyrase inhibitor in reducing CRC growth and demonstrated significantly less on-target GI toxicity in mice. We showed that this increased therapeutic index of SSTC3 was due to the differential abundance of CK1α, the direct target of SSTC3, in normal GI tissue relative to that in tumor tissue  withoutDespite the potential clinical advantage of CK1α activators, several mechanistic questions regarding their observed therapeutic index remain to be elucidated. One such question is understanding how CK1α pharmacological activators function mechanistically. While CK1 was originally proposed to be constitutively active, it is now clear that there are multiple CK1 family members and that their activities are highly regulated . Our datCK1α gene) and methylation of the CK1α promoter have been observed. Non-coding RNA, microRNA-155, and specific transcription factors/co-factors  have also been reported to modulate CK1α expression. Finally, the stability of CK1α protein has recently been shown to be regulated by the chemokine SDF-1 as well as the E3 ligase CRL4(CRBN). Although these highlighted mechanisms have already been linked to Wnt signaling, their role in regulating CK1α levels in CRC remains unclear. Regardless, reduced steady-state levels of CK1α render its overall activity rate limiting [A major question is how constitutive Wnt activity modulates CK1α abundance. There are many potential ways CK1α levels might be regulated in this context, a number of which have already been previously reported. At the genomic level, loss of regions of chromosome 5 (encompassing the limiting , providi


The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100–500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells. The mammalian genome is partitioned into topological chromatin domains (CDs) in the living cell nuclei. Gene expression is highly regulated within CDs according to their structure, whereas chromatin itself is highly dynamic. This raises the following question: how is the CD structure in such dynamic chromatin? We developed a conceptual framework that unifies chromatin dynamics and structure. Using a polymer model with a fractal domain structure in a viscoelastic medium, we analytically show that nucleosome movement is subdiffusive and depends on CD structure. Hence, structural information can be extracted based on nucleosome movement in living cells with single-particle tracking experiments. This framework provides physical insights into the relationship between dynamic genome organization and gene expression. Genomic DNA is packed and folded three-dimensionally in the cell nuclei. In the nuclei of eukaryotic cells, the nucleosome is a basic unit consisting of an approximately 147-bp DNA wrapped around core histones –7. On thBy virtue of recent developments of chromosome conformation capture (3C) techniques, the genome-wide chromosome organization has been revealed by detecting the physical contact frequencies between pairs of genomic loci . More reDapp is the apparent diffusion coefficient with dimension m2/sβ. This means that nucleosome movement must be affected by restrictions from some factors but thermal noise. Therefore, there must be a way that the dynamic aspect is consistent with aspects of the genome organization. A theory is required to relate the dynamic aspects described by Dapp and β to the structural features of CDs. To date, the subdiffusive exponent β has been considered to depend on the folding structure of nucleosome fibers 2〉∼tα, has been observed 2〉, is expressed asCp(t)≡〈Xp(t) ⋅ Xp(0)〉. Multiplying Xp(0) and averaging with 〈gp(t) ⋅ Xp(0)〉 = 〈gp(t)〉⋅〈Xp(0)〉 = 0, we can derive that the correlation function for p ≥ 1 satisfiesp = 0 in the RHS of α  is the Mittag-Leffler function. According to the polymer physics  for x ≪ 1, and the conversion of the sum into the integral, we obtain for t ≪ τdf,αOn the other hand, the second term in the RHS of  physics  for t ≪ periment  is calcuIn order to apply our model to living human cells, single-particle imaging of nucleosomes was performed by observation of PA-mCherry labels  attachedt up to 0.5 s, are shown in Here, to evaluate the state of CDs according to their position in the nucleus, we focused on the nuclear interior and periphery (or surface) , and calt0.44μm2 and 0.013 t0.39μm2 for the interior and the periphery, respectively.As we analytically derived the subdiffusive MSD , the expβ and Dapp are calculated asR〉CD and df. Since β and Dapp can be determined by the fitting in our experiments, we can therefore estimate 〈R〉CD and df, inversely.Comparing Eqs Dapp,periphery < Dapp,interior and βperiphery < βinterior, reflects the fact that the CDs near the periphery are in a more compact conformational state and are smaller in size than those at the interior: df,periphery > df,interior and 〈R〉CD,periphery < 〈R〉CD,interior. This property is consistent with the conventional distribution of heterochromatin: the CDs in the heterochromatin-rich nuclear periphery are more compact than those in the euchromatin-rich interior  (see Eq. S34 in α) is within the range 1 ≤ 1/Γ(1 + α)<1.13 for 0 < α ≤ 1, the friction coefficient of EGFP in the nucleus can be approximately regarded as the diffusion coefficient as γα → 1,EGFP = kBT/DEGFP. The hydrodynamic radius of a nucleosome bead with an H2B-PA-mCherry is assumed to be approximately quadruple for the EGFP. This means that the friction effect is also 4 times larger ; ti ≡ i × 50 ms. Several representative trajectories of fluorescently tagged single nucleosomes are shown in Local nucleosome fluctuation was observed ( u-track  was usedti) was sufficiently small. The number of trajectories Mi and the SEM of MSD(ti) are summarized in According to observed regions, we calculated the ensemble-averaged MSD of single nucleosomes: S1 Text(PDF)Click here for additional data file.S1 Figμm.Illumination laser (green) and focal plane (red) in the living cells are shown. Note that the two different focal planes were precisely verified by nuclear surface labeling with Nup107 (a nuclear pore component)-Venus (a bright yellow fluorescent protein) . The nuc(TIF)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Figα has only a slight effect on Cdf,α.(EPS)Click here for additional data file.S1 TableThe measurements at each region were performed using 10 cells.(PDF)Click here for additional data file.