Genetically encoded fluorescent reporters enabled high-resolution imaging of stromal cells and tumour cell migration. Sunitinib treatment impaired RT2 tumour angiogenesis and growth, while overexpression of the vascular endothelial growth factor (VEGF)-B increased tumour angiogenesis though tumour growth was impaired. In conclusion, we present a novel platform for intravital high-resolution and 3D imaging of PNET biology and cancer drug assessment.Preclinical trials of cancer drugs in animal models are important for drug development. The Rip1Tag2 (RT2) transgenic mouse, a model of pancreatic neuroendocrine tumours (PNET), has provided immense knowledge about PNET biology, although tumour progression occurs in a location inaccessible for real-time monitoring. To overcome this hurdle we have developed a novel platform for intravital 3D imaging of RT2 tumours to facilitate real-time studies of cancer progression. Pre-oncogenic islets retrieved from RT2 mice were implanted into the anterior chamber of the eye (ACE) of host mice, where they engrafted on the iris, recruited blood vessels and showed continuous growth. Noninvasive confocal and two-photon laser-scanning microscopy through the transparent cornea facilitated high-resolution imaging of tumour growth and angiogenesis. RT2 tumours in the ACE expanded up to 8-fold in size and shared hallmarks with tumours developing  The Rip1Tag2 (RT2) transgenic mouse model represents a widely used model of pancreatic neuroendocrine tumours (PNET)3. In this model, the simian virus 40 large T-antigen (Tag) oncogene is expressed under the control of the rat insulin gene promoter (Rip) leading to multifocal development of insulin-producing beta cell carcinoma (insulinoma) in the pancreatic islets3. Although all beta cells express the Tag oncogene at birth, stochastic events will add further genetic and epigenetic changes that are required for successful stepwise carcinogenesis4. The RT2 model has proven highly instrumental for preclinical validation of eligible biological or pharmacological anti-angiogenic compounds, some of which have been subsequently successfully implemented into clinical practice2. Recent, cross-species comparison between human PNETs and the RT2 model has also revealed that this mouse model shares many similarities with human PNETs5. Despite successful application of the RT2 model, current investigations are limited by the lack of techniques supporting temporal and high-resolution studies of PNET progression.Preclinical trials of cancer drugs in animal models are an important step in the drug development process where genetically engineered mouse models represent a valuable platform that may inform about success or failure in humans6. Clinical imaging modalities, such as PET, MRI, CT, etc., allow a view of whole organs in a noninvasive fashion. However, the low spatial resolution of these modalities prevents visualization of the earliest stages of disease onset and determination of cause-and-effect relationships among cells during tumour progression6. Intravital microscopy by confocal and multiphoton imaging in combination with sophisticated genetic models and animal preparations  can overcome these limitations and provide high-resolution visualization of cellular and sub-cellular structures7. Hence, intravital microscopy has successfully enabled detailed and dynamic investigations on the cellular level in multiple tumour preparations, including pancreatic cancer9. However, intravital imaging of internal organs presents several challenges that arise from both the inaccessibility and the constant motion of these organs. This applies to the pancreas and especially to the pancreatic islets, embedded microorgans associated with many pathologies, including cancer and diabetes.The application of imaging techniques in both medicine and research has proven to be crucial for the understanding of the structure and function of many tissues in health and disease11. Here, we have developed this platform further by establishing a protocol for implantation of pre-oncogenic RT2 tumours to the ACE, and by developing a protocol for 3D vascular quantification. Hence we present a platform for noninvasive intravital 3D imaging of RT2 tumour progression. Pre-oncogenic islets where isolated from young RT2 mice prior to the onset of oncogenic growth and implanted into the ACE of recipient mice. The RT2 islets engrafted on the iris where they recruited blood vessels and commenced to expand. Noninvasive imaging facilitated repetitive high-resolution 3D visualization and quantification of tumour growth, by label free imaging of scattered light, and of tumour angiogenesis, by imaging of systemic administered fluorescent dextrans. The application of fluorescent reporter mice facilitated visualization of distinct cell populations within the tumour microenvironment, and imaging of tumour cell migration. Treatment with Sunitinib caused vessel regression and impaired expansion of RT2 tumours located in the ACE, validating functional assessment of drug efficacy. Intravital imaging of individual RT2 tumours overexpressing the vascular endothelial growth factor (VEGF)-B revealed that VEGF-B strongly repressed tumour growth, while tumour vascularization was increased. Taken together, we present a novel platform for noninvasive intravital imaging of RT2 tumour progression that provides 3D, high-resolution and dynamic assessment of PNET biology and cancer drug efficacy.We have previously developed a platform for intravital imaging of pancreatic islets transplanted to the anterior chamber of the eye (ACE), which facilitates noninvasive confocal and multiphoton imaging of pancreatic islet revascularization and beta cell function+/− transgenic mice3 were isolated and cultured for two days, before implantation into the anterior chamber of the eye (ACE) of syngeneic recipients  pancreatic islets where also implanted into the same eye. Confocal microscopy facilitated imaging of scattered light, i.e. non-fluorescent light dispersed by the tissue13, and thereby enabled visualization of non-labelled tumour cells  mice17. Tumour blood vessels could be visualized in great detail by EYFP expressed by the endothelial cells, and repeated imaging facilitated 3D monitoring of the intratumoural vasculature network during tumour expansion , and obtained by crossing Tie2-Cre miceion Fig. . Adminision Fig. . After tion Fig. , arrows.ion Fig. . These c21 and R26R-Tomato(Ai14)17 mice to generate RT2-Tomato mice, characterized by expression of the fluorescent tdTomato protein by tumour cells. Imaging of RT2-Tomato tumours in the ACE following implantation facilitated visualization of tumour cells at a greater detail and depth compared with imaging of scattered light , or control treatment with vehicle 34, further support the application of the ACE for intravital imaging. Here, we present a novel platform for noninvasive intravital high-resolution imaging of PNET progression and cancer drug efficacy. This platform opens up a new window of opportunities to study complex cellular processes in tumour progression and treatment strategies by longitudinal and high-resolution live imaging.Noninvasive intravital imaging in the ACE has successfully been applied to study multiple aspects of beta cell physiology3, Tie2-Cretg/wt16, R26R-Ai3-EYFPfl/wt17, RIP-CreTg/Wt21, R26R-Ai14-Tomatofl/wt17, RIP-VEGF-B25 and maintained on C57Bl/6 J background.All animal procedures were performed in accordance with relevant guidelines and regulations and approved by the Stockholm North Ethical Committee on Animal Research, Stockholm, Sweden. The following mouse strains were used: RIP1-Tag2 (RT2)For Sunitinib treatment, Sunitinib (SelleckChem) was dissolved at 80 mg/ml in 100% DMSO (Sigma), allocated and frozen at −21 °C for up to 1 week. Pure DMSO was similarly allocated and frozen. 200 μl DMSO, with Sunitinib or without (vehicle), were freshly mixed with 400 μl PEG400 (Sigma) and 400 μl DPBS (Sigma) and injected subcutaneous within 30 min. Every mouse received 50 μl/day for 14 consecutive days of the Sunitinib solution (treatment) or vehicle (sham treatment).14. Briefly, the pancreas was filled with 1 mg/mL collagenase  and kept on ice until digestion at 37 °C, between 8–12 min. The digestion was stopped with ice-cold HBSS supplemented with 1% BSA/25 mM Hepes, and the islets were purified by hand picking. Implantation of islets into the anterior chamber of the eye (ACE) was conducted as described in detail12.Islet isolation was performed as described12. Briefly, the mice were anesthetized using 2% isoflurane anaesthesia and injected with the indicated vascular tracer and placed in a custom designed head holder that to allow stabilization of the eye. Imaging was performed using an upright Leica TCS SP8 MP microscope, equipped with a 25×/1 NA long working distance immersion lens . Two-photon microscopy was conducted with external Coherent Cameleon VisionS laser and HyD or PMT detectors. Fluorophores were excited at 820 nm or 960 nm. Fluorescence was collected using the following filter cubes: 447/60 and 525/50 (blue/green), 607/70 and 697/75 (orange/red) with a LP560 main beam splitter, and. Scattered light, i.e. non-fluorescent light dispersed by the densely granule packed hormone producing cells, was collected by confocal imaging of as described12. Images were registered in bidirectional non-resonant scanning mode with a step size of 1 μm for stacks, using the minimal necessary laser power . Images were collected in 8-bit format and pseudo coloured as indicated with black corresponding to the lowest intensity level. All images are maximum projections of image Z-stacks and are displayed close to the original intensity. For recording of Z-stacks, the signal in the upper image planes was set close to saturation, while the signal gradually became weaker with increased imaging depth, as illustrated in Supplement Movie Intravital imaging of islets in the ACE was conducted as described in detail35. Skeletonization and manual correction was performed in Imaris software (Bitplane), and was followed by extraction of parameters of 3D vascular network. Definition of vascular parameters; Vascular length: total length of vasculature per (10000 µm³) of islet volume; Vascular volume: total volume of vasculature per (%) of islet volume; Branching points: total number of branching points per (100000 µm³ = 0.0001 mm³) of islet volume; Mean Diameter: weighted arithmetic mean of filaments diameter weighted by the length of filaments.Image processing  and visualization was performed with Imaris Software (Bitplane) and Fiji/ImageJ (NIH). Each individual image was registered if necessary for correction of mouse movement during the image acquisition and filtered by Median 3D filter (Fiji). Manual 3D cropping to remove the iris vasculature was performed with use of Fiji Segmentation Editor in user developed macro. A binary 3D image of blood vessels was obtained by thresholding the image previously enhanced by Tubeness pluginp values below 0.05 were considered significant. In all figures, data points are presented on scatter plots with means +/− SD.Statistical analysis was performed with the GraphPad Prism 6.0 . All tests of statistical significance were two-sided. Unpaired Student’s t-test was used to study differences between two groups. In cases of unequal variance, Welch’s t-test was used. Supplement InformationSupplement Movie 1Supplement Movie 2Supplement Movie 3Supplement Movie 4


Fluid flow behaviour in paper is of increasing interest due to the advantages and expanding use of microfluidic paper-based analytical devices (known as µPADs). Applications are expanding from those which often have low sample fluid volumes, such as diagnostic testing, to those with an abundance of sample fluid, such as water quality testing. The rapid development of enhanced features in μPADs, along with a need for increased sensitivity and specificity in the embedded chemistry requires understanding the passively-driven fluid motion in paper to enable precise control and consistency of the devices. It is particularly important to understand the influence of parameters associated with larger fluid volumes and to quantify their impact. Here, we experimentally investigate the impacts of several properties during imbibition in paper, including geometry (larger width and length) and the surrounding conditions (humidity and temperature) using abundant fluid reservoirs. Fluid flow velocity in paper was found to vary with temperature and width, but not with length of the paper strip and humidity for the conditions we tested. We observed substantial post-wetting flow for paper strips in contact with a large fluid reservoir. Mic10−18 L) ,5,6, and10−18 L) . µPADs c10−18 L) , heavy m10−18 L) , bacteri10−18 L) , airborn10−18 L) ,15, and 10−18 L) ,18,19,20µPADs are ideal for applications in low-resource settings due to their low cost, ease of use, and passively-driven flow that allows for independence from auxiliary equipment ,21. A viThe analytical capacity of µPADs has been increasing rapidly due to the development of enhanced features such as multi-dimensional device designs ,27,28,29In this paper, we characterize fluid flow in paper-based devices using an experimental analysis to determine which parameters influence imbibition and need to be considered when predicting flow behaviour in µPADs when there is an abundance of sample fluid volume available. Specifically, we perform experiments to investigate the influence of surrounding temperature and humidity levels, paper machine direction, strip length, and strip width on the wicking behaviour of fluid in a paper strip when the source of the fluid is a large reservoir. This analysis of influential parameters can be incorporated in design considerations for paper-based devices when larger fluid volumes are available for a range of climate conditions.We used pure cellulose chromatography paper  for the paper strips. Experiments were conducted in a temperature and humidity controlled chamber measuring 51.5 cm × 41.5 cm × 41 cm, as shown in The paper strip  was folded and placed in a 10 cm diameter petri dish filled with a 12.5 mmol aqueous solution of Allura red food colour dye  and to keep the strip level the opposite end was supported by an inverted petri dish as shown in Experiments were conducted to study the influence of the surrounding temperature and humidity levels, the paper machine direction, and the dimensions of the paper strips including strip length and strip width on the wicking behaviour of fluid in a paper strip fed from a large fluid reservoir.fT) was what influenced the wicking behaviour rather than the air temperature (aT). It can be observed in versus the inverse root of viscosity at varying fluid temperatures. This correlation between wicking distance and viscosity follows the Washburn model Equation (1), where the wicking distance is inversely proportional to the root of viscosity. The corresponding dependence of wicking distance and the viscosity term on temperature shown in H), at a fixed air temperature of 20 °C, and the experimental results are shown in Wicking in paper strips with the same dimensions as the temperature tests (10 mm width and 45 mm length) were tested in humidity conditions ranging from 30% to 85% relative humidity  of 10 mm. It can be seen that fluid flow in the machine direction is faster than in the cross direction and this observation becomes more distinct as wicking distance increases. In The machine direction is labelled in the SEM micrograph inset of L) were tested to ascertain if fluid flow behaviour in paper is influenced by the length of the strip. Paper-based devices can be used for analytical testing in situations where there is an abundance of sample fluid volume, for example in water quality testing, and longer paper strip lengths can be exploited for a number of functions including additional reaction steps or to increase the concentration of an analyte by flowing more fluid over a reaction zone. To examine the impacts of paper strip dimensions on fluid wicking, strips of varying lengths  or speeding up the flowing fluid (with decreased width/area) ,26. It iom above . Hong anom above . The sloet al. [Bohm et al.  also obsOur most unexpected observation was fluid flow in the paper strip after wetting was complete. The paper strips remained in contact with the reservoir after the liquid front had travelled the full length of the strip. Though no additional fluid transport was expected due to capillary forces, since wetting was complete, further flow, or post-wetting flow, was observed in the paper strip. Post-wetting flow caused an increase in the amount of dye in the paper strip, and was detected visually as a darkening in the colour of the paper strip. ImageJ software was used to analyze images of post-wetting flow to quantify our observations and rule out other causes such as evaporation or diffusion.To determine the post-wetting flow behaviour as a function of time we analyzed the colour intensity of the paper strip after wetting was complete. The maximum value for red pixels is the numerical value assigned by the software to the brightest red pixel in the image. This value was measured every minute after saturation, where a lower value represents a darker red colour corresponding to an increase in dye concentration, and these values are plotted in et al. observed that the bulk filling of pores is not the primary flow mechanism in paper, rather it is flow along the channels caused by fibre overlap, in other words, capillary driven film flow. The cellulose fibres also contain pores within the fibre, known as intrafibre pores [A close look at the fibre structure of paper, as seen in the SEM micrograph in re pores ,38. Fillre pores ,43,44. WFor design of precise µPADs when using a large fluid reservoir, it will be important to consider the post-wetting effects in relation to the intensity of colorimetric signals by controlling the time of submersion and elapsed time prior to test readout by the user. This phenomenon is of particular interest for paper-based microfluidic applications where high sample volumes are readily available.In this paper, we investigated the impact of surrounding conditions such as temperature and humidity, paper machine direction, strip length, and strip width on fluid flow behaviour in paper strips fed from a large fluid reservoir. Using experimental data, we determined that length and humidity do not have a significant impact on flow behaviour in paper strips for the conditions we tested. We noticed quicker wicking for flow parallel to the machine direction of the paper compared to the cross (perpendicular) direction. Our experimental results indicated that the wicking time decreases as temperature increases in proportion to decreases in the inverse root of the fluid viscosity. Additionally, we observed that wicking time also decreases with increasing width for thin (1 CHR) paper strips, a dependency which diminishes as width increases. Width was not found to influence the wicking behaviour for the thicker paper strips (17 CHR). We also observed post-wetting flow when the paper strips remained in contact with a fluid reservoir, which should be considered during design of µPADs used in conjunction with large sample volumes, for example, in environmental testing applications.


Angus Dalgeish's degree should also have appeared as FRCP.In the article, “Case report on the role of radiofrequency-assisted spleen-preserving surgery for splenic metastasis in the era of check-point inhibitors”


How and when a newborn is first colonized by microbes continues to be of great interest due to its broad implications on human health and disease. Payne et al. express their opinion about our recent study in which we characterized the virome and bacterial microbiota of amniotic fluid from 24 uncomplicated term pregnancies. We conducted additional validation studies and respond to their comments. We conclude that in amniotic fluid from healthy term pregnancies, the bacterial microbiota is indistinguishable from contamination controls, and there is no evidence of a core virome. How and when a newborn is first colonized by microbes continues to be of great interest due to its broad implications on human health and disease . Payne ePayne et al. comment that our findings contradict two prior studies on the amniotic fluid bacterial microbiome , 3. Howeg for 5 min at 4 °C   the sample collection was biased against bacterial cells, (B) a SYBR green 16S qPCR is more appropriate, (C) the presence of human DNA may confound 16S qPCR results, and (D) samples with fewer than 5000 16S rRNA gene sequencing reads should not have been omitted. To demonstrate that the brief, low-speed centrifugation (1620 g for 5 min at 4 °C) does not bias against bacterial cells, we prospectively obtained amniotic fluid from two women with full term gestations undergoing planned C-section prior to the onset of labor. The amniotic fluid was obtained in a sterile fashion at the time of C-section by aspirating through intact amniotic membranes as described in the original study [Payne et al. state that “the authors have only included OTUs in their analyses that originated from bacterial reads which were detected in the amniotic fluid samples and not in the blank extraction or PCR controls, regardless of the levels at which they were detected ”. On the contrary, the data in the 16S rRNA gene analyses shown in Fig. 2a–c  of [In summary, our original paper , a recen


Tumour responses to radiotherapy are currently primarily assessed by changes in size. Imaging permits non-invasive, whole-body assessment of tumour burden and guides treatment options for most tumours. However, in most tumours, changes in size are slow to manifest and can sometimes be difficult to interpret or misleading, potentially leading to prolonged durations of ineffective treatment and delays in changing therapy. Functional imaging techniques that monitor biological processes have the potential to detect tumour responses to treatment earlier and refine treatment options based on tumour biology rather than solely on size and staging. By considering the biological effects of radiotherapy, this review focusses on emerging functional imaging techniques with the potential to augment morphological imaging and serve as biomarkers of early response to radiotherapy. The introduction of cross-sectional anatomical imaging using computed tomography (CT) and magnetic resonance imaging (MRI) in the 1970s revolutionised clinical oncology by permitting non-invasive determination of tumour burden that is essential for diagnosis and staging and assessing treatment responses. To standardise characterisation of tumour responses that were frequently incomplete and heterogeneous and for comparison between clinical trials the World Health Organisation defined the first response criteria in 1979 ,3, are rIn this review we will focus on emerging functional imaging techniques that exploit the biological changes in tumours following radiation therapy and have the potential to improve the early detection of treatment response. Many of the functional imaging techniques discussed can also be applied to delivering intensity-modulated or stereotactic body radiation therapy using increasingly sophisticated methods, a subject that is beyond the scope of this review but has been extensively reviewed elsewhere ,8. •OH), superoxide (O2−) and hydrogen peroxide (H2O2)) produced by ionisation of adjacent molecules ..13C-labe13C2)fumarate, which is hydrated to malate by fumarase. During cell death an increase in membrane permeability results in leakage of fumarase into the extracellular space and an increased rate of malate production following hyperpolarised fumarate injection which, in preclinical studies, has been shown to be a sensitive indicator of cell death [The other readily translatable hyperpolarised substrate is  is taken into cells and phosphorylated by thymidine kinase, the first step of the thymidine salvage pathway essential for DNA synthesis. Thus, 18F-FLT preferentially accumulates in cells undergoing proliferation with potentially greater tumour specificity than 18F-FDG. Several systematic reviews have concluded that  [18F-FLT uptake did not correlate with pathological response, a discrepancy attributed to changes in perfusion following radiotherapy [Several PET tracers have been designed as biomarkers of proliferation. 3′-deoxy-3′-survival ,146. In vs. 30%) . Howeverotherapy .18F-FDG is a particular problem in neuroradiology where there is high background uptake from normal brain tissue and radiation necrosis is often hypermetabolic [18F-FDG for detection of recurrence and differentiation from pseudoprogression. Brain tumours often have increased uptake of amino acids relative to normal brain and several have been labelled with 11C and 18F for PET imaging [11C-methionine has been the most widely used amino acid PET tracer. It can differentiate tumours from normal brain with an accuracy of 94% versus 80% for 18F-FDG [11C-labelled substrates will always be limited by the short half-life (20 min) requiring onsite production of the tracer. Therefore, several alternatives have been developed including 18F-fluoro-ethyl-tyrosine, an artificial amino acid that is not incorporated into proteins but has increased uptake into tumours [18F-FDG for distinguishing recurrence from radiation necrosis include 11C- and 18F-labelled choline , an analog of the dopamine precursor L-DOPA . Quantification is seen as a major strength of functional imaging but a lack of consensus over the vast number of imaging biomarkers to use significantly limits the comparison of findings and meta-analysis. Unfortunately, in clinical practice, quantitative metrics do not necessarily perform better than simple qualitative analysis . Further


Chow et al., Chem. Sci., 2015, 6, 4148–4159.Correction for ‘Assessing the exchange coupling in binuclear lanthanide( A grant from the European Community's Seventh Framework Programme was inadvertently omitted in the Acknowledgements of this manuscript. The following information should be added to the Acknowledgements section:“The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement number 611488.”The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Nano Neuro Net, or N3-MEAs. Material choice, dimensions, and room for further upgrade, were carefully considered when designing such probes in order to cover the widest application range possible. Proof of the operation principle of these novel probes is shown in the manuscript via the recording of extracellular signals, such as action potentials and local field potentials from cardiac cells and retinal ganglion cells of the heart tissue and eye respectively. Reasonably large signal to noise ratio (SNR) combined with effortless operation of the devices, mechanical and chemical stability, multifunctionality provide, in our opinion, an unprecedented blend. We show successful recordings of (1) action potentials from heart tissue with a SNR up to 13.2; (2) spontaneous activity of retinal ganglion cells with a SNR up to 12.8; and (3) local field potentials with an ERG-like waveform, as well as spiking responses of the retina to light stimulation. The results reveal not only the multi-functionality of these N3-MEAs, but high quality recordings of electrogenic tissues.In the current work, we introduce a brand new line of versatile, flexible, and multifunctional MEA probes, the so-called  The current state-of-the-art in neural technology has advanced in numerous and different approaches. A vast variety of devices have been designed and proposed in the last decades, creating unique tools to address specific tasks, such as penetrating and surface electrodes for the brain   are used as tools to interface electrogenic organs in vitro studies to further real in vivo measurements. And the problems are technological. Most of the in vitro based devices are planar -probe, that can solve the problem and play a role of a transient tool between in vitro and in vivo recordings. As shown in 3-MEAs compared to the common planar MEAs: the latter are used to grow the cell on top of them, which might take days, weeks, or even month of time in incubator. In the case of N3-MEAs, however, it is possible to use a single probe, and a number of different cell lines, tissues, that are cultured separately, and make multiple recording with one device for more valid comparative measurements.In this work we propose a novel tool, 3-probes reported in the work are shown to successfully record action potentials from heart tissue cells, spontaneous activity of retinal ganglion cells, and local field potentials with an ERG-like waveform as a proof of principle. Yet, we believe the N3-probes have potential to cover a much larger set of application, including chronic applications. The proposed N3-probes are multifunctional and can further be upgraded toward measurements from in vitro cell culture to ex vivo recordings of electrogenic tissues and even more complex 3D cultures of neurons or any other cell type. Moreover, the shape, dimensions, and functionality of the N3-probes allow to think beyond typical neuronal recordings toward the application of neural prostheses, such as μECoG on-dura brain tissue or retinal implants. An important remark on the fabrication of such tool has to be made, as our fabrication routine does not require e-beam lithography [as opposed to reference  on the wafers with the rate of 5,000 rpm, that is later soft baked at 120°C for 4 min  and hard baked in a convention oven filled with nitrogen atmosphere at 350°C . Such process ensures a smooth and pinhole free polyimide layer of ~2.5 μm in thickness. Further, a layer of Ti/Au (10/200 nm) is formed on top via an e-beam assisted evaporation and is shaped into the desired structure by means of lift-off process through a double layer of nLOF-2020  and LOR-3B photoresists (Microchem). A second layer of polyimide is then spin-coated, soft-baked and hard baked in exactly the same manner as explained above with only additional step of spin-coating the adhesion promoter, VM-652 (HD Microsystems), beforehand in order to enhance the interlayer adhesion. Later, two reactive ion etching (RIE) steps through two different masks were performed in order to etch away: (i) the electrode openings (only top 2.5 μm PI), (ii) the desired shape of the probes (~5 μm of PI). Both steps are performed by using AZ-9260 as a thick protective photoresist and O2/CF4 as gas combination to etch the polyimide. The AZ-9260, spin-coated at 3,000 rpm results in approximately 9 μm thick layer, that is thick enough to ensure complete etching of the 5 μm PI layer. The RIE etch was performed in the mixture of O2/CF4 (36/4 sccm) gases with RF power of 50W, ICP power of 500 W, and pressure of 0.007 Pa. As we determined experimentally, these conditions result in the etch rate of the most polymers, such as polyimide, parylene-C and most of the common photoresists of 700–800 nm/min. Therefore, the etching times of 4 and 8 min were used to etch the passivation openings and shapes, correspondingly. The wafer is further diced into smaller clusters in order to simplify the Cr etching and probe release process. See The N2 flow. Important to note that the PI-based probes of only 5 μm are not just stable enough to sustain the process, but can be hardly broken if specifically intended to  for 2 h. Typical etch rate of Cr/Au/Cr layer with only sidewall access was found to happen at approximate rate of 1.5 mm/hour. When the etching is finished, the probes are washed thoroughly in water, and dried with Nd to see .3-probes are given in The fabricated probes have dimensions of 15 mm in width and 3 mm in length of the back-side (that is later connected to a PCB carrier), while the active part is only ~2.5 mm in diameter, where the electrodes are located radially on the nodes. The diameter of the electrodes as well as their distribution is selected to fit the wide range of applications and varies from 30 to 80 μm. Details of the design can be found in the Electrical impedance spectroscopy study is performed on VSP-300 (BioLogic Science Instruments) potentiostat. The measurements are performed in a simplified three electrode schematics with Ag/AgCl pellet used as both reference and counter electrode since there is no expected current flow in the system. The spectra are taken in a frequency range from 1 Hz to 100 kHz in 1x PBS solution, with 10 mV AC potential applied.The extracellular recordings are performed with the in-house developed biological multifunctional amplifier system (BioMAS), which is capable of recording up to 64 channels simultaneously with a sampling rate of 10 kHz per channel, as well as allows a maximum signal amplification of 1010   ending with a set of pins. The long connectors do possibly introduce more noise into the recordings, yet as will be shown later, the measurements are robust enough to not be significantly affected by the noise.The BioMAS system is a multi-unit system that, however initially was built for 3-probes. The carrier is placed on a hotplate (180°C), and a solder paste  is dispensed on top of the contact pads. When the flux is evaporated and the excess alloy is removed, only small amounts of alloy is left on top of the carrier's contact pads. Then, the N3-probe is placed on top of the carrier and aligned under the microscope. When cooled down, any remaining flux is removed in ethanol, and the probe is carefully glued with medical grade epoxy (EPO-TEK 302-3M). Optical images of the N3-probes as well as zoom-in into the PCB-probe assembly is given in The probes, in their turn, are flip-chip bonded to a custom made printed circuit board (PCB) carrier of approximately 10 cm long and 15 mm wide where the inner pads match the contact pads design of the N3-probes were thoroughly washed with DI water followed by a 1-h soak in Terg-a-zyme solution in order to eliminate any organic residue and washed with DI water again.After each electrophysiological measurement, the N2 and decapitated. The pups were then removed and also decapitated under sterile conditions. The heart of an embryo was quickly isolated, washed in Hank's balanced salt solution (HBSS), then stored and measured in supplemented Claycomb medium. More details of the heart tissue preparation can be found elsewhere . The animal was anesthetized with isoflurane  and killed by decapitation. This procedure was carried out in accordance to the German Law for the Protection of Animals and after approval was obtained by the regulatory authorities, the Forschungszentrum Jülich, and the Landesamt für Natur, Umwelt und Verbraucherschutz of the land North-Rhine Westfalia. The eyeballs were then enucleated and perfused with oxygenated Ames' medium (Sigma-Aldrich) at a pH of 7.4 at room temperature. The cornea, the lens, and the vitreous body were first removed for each eye, followed by the isolation of the retina from the eyecup. The tissue was then cut into halves and each piece was mounted on top of a millipore filter paper  with a precut hole of 1.5 mm in the middle. In this way, the filter paper was used as a carrier to transfer the retina onto the device, and the precut hole allowed the retina, with the ganglion cell layer (GCL) facing downwards, to contact the electrodes. Furthermore, a glass ring glued the lid of a petri dish was used to create a reservoir of the physiological solution, and a silicone rubber pillow served as support for the NDuring the experiments, carbogen gas  was used to constantly oxygenate the medium, and a perfusion system was used to maintain the tissue vital with fresh medium. In order to observe physiological responses of the retina, optical stimulation was carried out with a light-emitting diode (LED) using 500 ms light pulses every 20 s. In addition, to confirm the responses of the retina evoked by light, the electrical activity of the tissue in a hypoxic/ischemic state was performed after stopping the perfusion of fresh oxygenated medium to the tissue. At last, to confirm the spiking activity of RGCs, the electrical activity was recorded when the tissue was on top of the electrodes and when the tissue was being removed.3-16 and N3-32. In both of the probes, the electrodes are arranged radially through the web-like net of polyimide that is made specifically to be strong and stable in all directions. However, the N3-16 probes feature electrodes with openings of 30, 50, and 70 μm in diameter, and one on-probe reference electrode of 200 μm diameter .The recordings from 3-probes toward multifunctional electrophysiology, we started with possibly the most robust ex vivo electrogenic tissue: heart tissue. Prepared as explained in the methods section of the manuscript, the embryonic heart tissue is kept floating in the supplemented Claycomb solution.In order to show the applicability of the N3-probe, bonded to a PCB carrier and connected through a wire cord to the pre-amplifier  exposing an electroretinogram (ERG)-like waveform were captured in response to light stimulation see . Such poFurthermore, in order to confirm that the spikes and LFPs elicited during light stimulation were not artifacts induced by light but a physiological response of the retina to light, the same explanted retina was stimulated with the same light stimuli after 1 h and 20 min without fresh oxygenated medium. During the hypoxic/ischemic state of the retina, most electrical activity was gone, however spontaneous APs were still captured from the retina in one channel. In addition, some low frequency waves were seen during light stimulation, however these LFPs were low in amplitude (~ ±10 μV) and in the range of the background noise. Therefore, neither visible spiking reactions, nor ERG-like signal responses to the stimuli were captured see . These r3-MEA probes that are introduced in this work are shown to be advantageous for manifold electrophysiological measurements. The unique physical structure of the N3-MEA allows a seamless integration with neuronal cells and tissues, resulting in low noise and large SNR recordings. We have shown ability to record electrical signal from heart tissue cells with SNR up to 13.2 as well as from retinal ganglion cells with SNR up to 12.8.The novel multifunctional, ultrathin, robust and versatile N3-probes can play the role of a transient tool between in vitro and in vivo recordings. The application range is not limited to the two applications reported in the manuscript, i.e., recordings from heart tissue and retinal tissue. In contrary, we believe that the proposed design, shape, dimensions, and functionality of the N3-probes allow to go beyond such neuronal recordings toward the application of neural prostheses as μECoG on-dura brain tissue or retinal implants. In order to perform the chronic ECoG measurements, or to measure electrical activity of 3D neuronal cell culture, the PCB connector board can be simply substituted with a flat flexible cable (see proposed schematic in 3-probes, therefore improving the contact with tissue. Second advance of the probes we envision to come from utilizing other materials instead of simple gold, such as CNT, Pt black, PEDOT:PSS, porous graphene or single layer graphene. Moreover, usage of graphene, in our opinion, will bring the third advance to the future work: intrinsic flexibility, conductivity and transparency opens interesting prospects for optogenetics and retinal applications. Thinning down the probe thickness as well as use of graphene or other two-dimensional materials will bring us closer to the term nano claimed in the name of the N3-probes. Stacking multiple N3-probes in z-direction or twisting/rolling single probes makes them useful for advanced 3D neuronal measurements and peripheral nerve measurements and stimulation. Optical transparency and physical mesh structure allows to further increase the potential application range of the N3-MEAs toward three-dimensional cell culture. In the latter case, the N3-probe can be immersed into hydrogel-based solution before the solidification, allowing neuronal cells afterwards to grow freely through the culture and through the probe, and allowing a real three-dimensional electrophysiological recording from these 3D cell cultures without disturbance (see We believe that these Nance see .The experiments are done with the approval of the Landesumweltamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany, number 84-02.04.2015.A173.DK and AO conceived the study and designed fabrication protocols. DK, VM, and KS fabricated the devices. DK and JS performed electrical impedance characterization of electrodes. DK performed heart tissue experiments. DK and VM performed retina experiments. All authors contributed to the writing and agreed to the submission of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the Funding statement, the following grant numbers are missing from the Carlsberg Foundation: CF15-0672 and CF16-0551.


In this research, we reveal the transient behavior of capillary pressure as the fluid-fluid interface travels across the juncture between a converging and uniform capillary, via high-resolution CFD  simulations. Simulations were performed at different wetting conditions (strong-wet and intermediate-wet) and capillary wall convergence angles. Our results demonstrate that as the angle of convergence increases, capillary pressure at the junction decreases commensurately. Moreover, in contrast to strong-wet conditions, the profile of capillary pressure at the converging-uniform capillary juncture under intermediate-wet conditions is highly non-monotonic, being characterized by a parabola-like form. This non-monotonic behavior is a manifestation of strong inertial forces governing dynamic fluid-fluid interface morphology. This yields conditions that promote the advancement of the fluid-fluid interface, as inertial forces partially nullify the capillary pressure required for the immiscible interface to enter the uniform capillary. In addition to numerical analysis detailed above, a novel theoretical stability criteria that is capable of distinguishing between stable (capillary dominated) and unstable (inertia dominated) interfacial regimes at the converging-uniform capillary juncture is also proposed. In summary, this fundamental study offers new insights into the interface invasion protocol, and paves the way for the re-evaluation of capillary junction controlled interfacial dynamics. Mason and Morrow7 performed a theoretical study to highlight the effect of cross-sectional angularity of channel on the capillary pressure behavior. They demonstrated that under perfectly-wet conditions the capillary pressure required for the interface to displace the residing fluid increases as the angularity of channel enhances. Later Ma et al.8 used the Mayer and Stowe-Princen9 (MS-P) theory of drainage to investigated the behavior of interface in angular capillaries under partial-wet conditions. They revealed instability in capillary pressure profile under partial-wet conditions, which was associated with the complex relationship between the channel angularity and the contact angle between the fluid-fluid interface and solid surface. While the focus of the majority of previous work has been on the uniform capillary, some studies11 have also investigated the behavior of interface in a capillary channel with non-uniform cross-section. Rabbani et al.10 performed CFD simulation to examine the capillarity of the converging-diverging channel, illustrating that under intermediate-wet conditions there is a shift in the direction of capillary forces as interface traverses from converging to uniform capillary that consequently results in a change in displacement mechanism from drainage to imbibition. Although the underlying physics and theoretical tools to quantify capillary pressure inside uniform capillaries12 and capillaries with axial variation (converging or diverging capillary)11 are well known, the behavior of capillary pressure at the juncture between capillary channels of discordant orientation, and fundamental principles controlling such phenomena have, hitherto, remained elusive. Given the commonly disparate and disordered geometry of many natural and manufactured pore networks, the behavior of capillary pressure at the capillary junction merits further investigation.Defined as the pressure difference across a fluid-fluid interface, capillary pressure is a first-order control over immiscible fluid displacement within numerous porous materials, and is fundamental to the study of the multiphase flow central to a wide variety of natural and industrial processes such as cardiac transcapillary exchangeThe objective of this work is to delineate the behavior of capillary pressure at the juncture between converging and uniform capillaries under dynamic conditions. To achieve this aim, we performed high-resolution two-phase flow simulations through converging-uniform capillaries using computational fluid dynamics (CFD) based modelling at various wetting conditions and capillary wall orientation angles (i.e. angles of convergence). The capillary pressure profile was computed as the fluid-fluid interface enters from converging to uniform capillary. The invasion protocol within this study is limited to drainage conditions (invading fluid is non-wetting) only.Porous media composed primarily of packed particulate  typically contain pore networks comprising of intergranular pore bodies connected to comparatively smaller pore throats formed between adjacent grain contacts via converging capillary channels Fig. .Figure 1Textural disparities in both natural and synthetic particulate columns, owing to variations in grain size, grain shape, sorting and packing configuration, give rise to intrinsic variability in the angles of convergence between interconnected pore bodies and pore throats see Fig. . Despite15. Our study rationalizes the underlying physics of such phenomena indicating that the synergistic impact of increase in convergence angle and contact angle dampens the capillary resistive forces acting along the interface, further promoting the fluid invasion in porous medium. This insight is potentially transformative towards for reservoir engineering, geoengineering and hydrogeology/soil science applications , as well for chemical engineers in managing water flooding issues in fuel cells. In addition to aforementioned porous media, an understanding of the immiscible fluid flow characteristics at capillary junctures also play a key role in the design and optimization of microfluidic devices (e.g. droplet generators).It is known from the literature that the efficiency of immiscible displacement enhances as the wetting conditions of porous media changes from strong to intermediate15, and therefore will not be repeated here.We performed Computational Fluid Dynamics (CFD) simulations using C++ library OpenFOAM (Open source Field Operation And Manipulation). InterFoam: a solver of OpenFoam, was utilized to compute the Navier-Stokes equations which are coupled with the volume of fluid method to track the fluid-fluid interface. The details of the numerical formulation are described elsewhere in detaild at an angle of orientation β  and the fluid-fluid interface resides in the converging channel close to the junction. The inlet of converging capillary supplies the invading fluid (viscosity −3) for the displacement at a constant flow rate −3·s−1, while the outlet of the uniform capillary is maintained at atmospheric pressure. The interfacial tension between the fluids is σ = 70 × 10−3 N·m−1. The static contact angle θ, which corresponds to the angle between the defending fluid and solid surface is recognized as the direct measure of the wettability of system and was included as input parameter in the solver.The cross-section of both capillaries is square with half corner angle 45° Fig. . Initialβ = 21.8°, 38.7° and 50.2°. The β values were varied by changing the diameter of uniform capillary d as 0.8 × 10−3 m, 0.6 × 10−3 m and 0.4 × 10−3 m in accordance to Eq. θ values 30° (strong-wet) and 60° (Intermediate-wet) were considered in this research. As the simulation begins, the fluid-fluid interface traverses from the converging capillary to the uniform capillary.Series of 3D numerical simulations were performed with 16.Our simulations do not take into account contact angle hysteresis and dynamic contact angle effects. Moreover, since the fluid densities are identical, gravity forces do not play a role in the immiscible displacement. Visualization and post-processing of the numerical results were performed using ParaViewsw at the cross-section of the junction of converging-uniform capillary  fluid pc as a function of defending fluid saturation sw under strong-wet conditions (θ = 30°) and intermediate-wet conditions (θ = 60°) is displayed in Fig. pc decreases with increasing β (and thus decline in size of the uniform capillary d), indicating reduction in the resistance offered by capillary forces, and therefore less energy required by the fluid-fluid interface to advance into the uniform capillary. The results presented in Fig. pc and d. It is important to note that we performed additional simulations where β was varied by changing the inlet diameter D while keeping the diameter of uniform capillary d constant, the results are shown in Fig. pc at junction strongly depends upon the converging angle of the flow domain and the wettability that results in et al.10 which can also be ascertained from Fig. The behavior of capillary pressure ions θ = 0° and inions θ = 0° and inions θ = 0° and inions θ = 0° and inθ changes from 30° to 60°, the capillary pressure profile starts to display highly non-monotonic behavior  at β = 38.7° and 50.2°, w displays marked enhancement as the fluid-fluid interface enters the converging-straight capillary juncture: behavior that is notably absent from other simulated cases. It is clear from Fig. θ increases, inertial effects on fluid-fluid interfacial dynamics are enhanced. This in turn results in a decline in pc, which ultimately engenders the non-monotonic (parabolic) form of the pc trend displayed in Fig. It is apparent from Fig. 21, the exact nature of how inertia contributes towards dynamic interface behavior have remained, hitherto, unresolved. Our numerical results unravel this phenomena, rationalizing the underlying physics. It can be clearly seen from Figs sw), the relative influence of inertial forces is enhanced (increase in w), which in turn, promotes the reduction in curvature of fluid-fluid interface decreasing the pc required to enter the channel. Thus, the narrower the juncture is relative to the converging capillary, the larger the influence of inertia at the juncture will be, and the greater ease with which the fluid-fluid interface will enter the capillary.Though it is known from the literature that inertia plays a prominent role in governing the motion of interface23 that are based on pore filling rules proposed by Lenormand et al.24 may require revision to incorporate the effect of the capillary junction on invasion protocols. Moreover, inertial forces are commonly ignored in pore network modelling approaches25. Our results suggest that this can be a reasonable assumption for strong-wet conditions, but under intermediate wet conditions the localized impact of inertia on interface invasion is important to capture, as demonstrated in Figs Figures w*, that can be used as a diagnostic tool to distinguish between an inertia controlled interfacial regime and a capillary dominated interfacial regime at the juncture between a converging-uniform capillary channel. The new modified Weber number w* is shown as Eq. In this section we will be proposing a new modified Weber number: sw = 0 to sw = 1 for each simulated case against β.For an inertia controlled interfacial regime θ = 60°), β = 38.7° and 50.2°, while for all other cases w*  simulations with high spatial and temporal resolution to delineate capillary pressure behavior at the juncture between and converging and uniform capillary channel. The studied capillary model consisted of a converging capillary, connected to a uniform capillary via an obtuse junction, with the sense of displacement from the converging channel into the uniform capillary under drainage conditions. The simulations were conducted under different wetting conditions (strongly water-wet and intermediate wet) and angles of convergence, with capillary pressure computed as the fluid-fluid interface enters the junction.It can be concluded from our results that as the orientation angle increases, capillary pressure required to enter the uniform capillary decreases. In addition, we have demonstrated that, in contrast to strong-wet conditions, the nature of capillary pressure curve under intermediate-conditions is highly non-monotonic, which is related to strong, localized inertial control on the interface morphology at the junction. In addition, we also proposed a novel theoretical model (stability criterion) that is capable of distinguishing between inertia and capillary dominated interfacial regimes at the junction of converging-uniform capillary.Inertia Controlled Capillary Pressure at the Juncture between Converging and Uniform Channels


The electroosmotic flow (EOF) of viscoelastic fluid in a long nanoslit is numerically studied to investigate the rheological property effect of Linear Phan-Thien-Tanner (LPTT) fluid on the fully developed EOF. The non-linear Poisson-Nernst-Planck equations governing the electric potential and the ionic concentration distribution within the channel are adopted to take into account the effect of the electrical double layer (EDL), including the EDL overlap. When the EDL is not overlapped, the velocity profiles for both Newtonian and viscoelastic fluids are plug-like and increase sharply near the charged wall. The velocity profile resembles that of pressure-driven flow when the EDL is overlapped. Regardless of the EDL thickness, apparent increase of velocity is obtained for viscoelastic fluid of larger Weissenberg number compared to the Newtonian fluid, indicating the shear thinning behavior of the LPTT fluid. The effect of the Weissenberg number on the velocity distribution is less significant as the degree of EDL overlapping increases, due to the overall decrease of the shear rate. The increase (decrease) of polymer extensibility (viscosity ratio) also enhances the EOF of viscoelastic fluid. Over recent decades, nanofluidics has undergone significant development due to the advances in nanofabrication and its promising applications in (bio)nanoparticle sensing and detection ,2,3, manMost of the theoretical studies on electroosmotic flow (EOF) in the literature assume that the fluid follows the Newtonian model. However, in real nanofluidic applications, complex solutions such as polymer and DNA solutions are frequently involved, and they impart distinctively different characterization from the Newtonian fluid ,24, inclWi), viscosity ratio and polymer extensibility parameter on EOF velocity profile and dynamic viscosity are examined. The rest of this paper is organized as follows. The problem under consideration is physically described and the governing equations are presented in Most of the studies on EOF of viscoelastic fluid in a microchannel assume relatively small surface potential and thin EDL so that the Poisson-Nernst-Planck equations can be simplified. The condition with EDL thickness comparable to the channel height, which is typical in modern nanofluidics ,13,36,37+ and Cl− in a long channel of length L, height H and width W under externally applied potential difference V0 across the channel. We assume that the channel height is much smaller than both the length and the width  is the trace of the conformation tensor c. The evolution of the conformation tensor + (Cl−) ions, respectively. As shown in R and T are, respectively, the gas constant and the absolute temperature; and ith ionic species. The distribution of the ionic concentration is governed by the Nernst-Planck equation,The set of governing equations can be normalized by selecting p’, and In the above, The boundary conditions are given as following.On the charged wall,At the Anode (or Cathode),As the problem is symmetric, at the centerline of channel https://openfoam.org). For numerical simulation of viscoelastic fluid flow, the so-called high Weissenberg number problem (HWNP), due to the hyperbolic nature of the additional equations, the loss of symmetric positive definite (SPD) property and the unfaithful evaluation of conformation tensor c, significantly impedes its accuracy and stability at high Wi [c [c by solving its logarithm instead of solving it directly, thereby, guaranteeing its SPD property automatically. Meanwhile, the deviation between polynomial fitting and exponential variation profiles of the conformation tensor c is eliminated. The above coupled Equations (7)–(11) are solved by a new solver implemented in an open source CFD software OpenFOAM  for the conformation tensor upferman  and Zhanupferman . After ΨTo improve the convergence and the stability of the calculation, the convection terms in Equations (8), (11) and (16) are discretized by QUICK , Gauss lL = 300 nm. For comparison with the sPTT model in the reference, the solvent viscosity is set to 0, i.e., y-component velocity distribution in the middle of the channel at Wi = 0) and viscoelastic fluid at Wi. As the EDL is relatively thin, the velocity profile is plug-like, and increases with higher Wi. It is clearly seen that our numerical results agree well with analytical solutions of Afonso et al. [Wi. To check the validity of the developed code, we first compare the numerical predictions with the analytical results of Afonso et al. , who dero et al.  for bothWi, the extensibility parameter L = 5 μm and height H = 100 nm is considered, which is long enough to eliminate the end effect. The fully developed EOF for different Wi is simulated under different EDL conditions: C0 = 0.01, 0.1, and 10 mM, corresponding to The validated solver is then applied to investigate the effects of y-component velocity profile at different Wi at Wi = 3 is 2.50 times of that for Newtonian fluid. The variation of flow rate with Wi is shown in inset graph of Wi increases, the flow rate demonstrates a monotonous growth over the whole range of Wi investigated. y-component velocity profile for different Wi at Wi, the velocity keeps increasing from the wall to the channel center. As the EDL is almost overlapping under this condition, the whole channel is filled with more counter-ions, so the velocity is not uniform even near the channel centerline. The maximum velocity at the channel center for viscoelastic fluid at Wi = 3 is 2.05 times of that for Newtonian fluid. The flow rate also monotonously increases with Wi as shown in inset graph of Wi = 3 is 1.75 times of that for Newtonian fluid. The inset figure depicts the dependence of flow rate on Wi. Wi = 3. Comparing the three cases, the enhancement of maximum velocity at the centerline for viscoelastic fluid decreases as the EDL thickness increases. This is because the velocity is increasing slowly across the whole channel instead of increasing sharply near the wall as EDL becomes overlapped, thus the overall shear rate is smaller, especially near the wall. As the mechanism of the velocity increase is due to the shear thinning effect, it is expected that the viscoelasticity has larger effect on the case that has larger shear rate. This trend is more obvious for the condition with apparent EDL overlap when more counter-ions are accumulated within the channel , as can The distribution of the total dimensionless shear stress for different values of After analyzing the velocity profile and the shear stress, the shear viscosity is calculated fromWi under different values of Wi increases, a more apparent decrease is observed. This is the shear thinning characteristics of the viscoelastic fluid, leading to the increase of the velocity. Comparing the variation of shear viscosity Wi = 2 and the EDL thickness unchanged at The EOF of viscoelastic fluid is dependent on the rheological parameter of the fluid. Numerical study for the EOF of viscoelastic fluid in a long nanochannel is conducted to investigate the effects of rheological properties of LPTT fluid on the fully developed EOF. The non-linear Poisson-Nernst-Planck (PNP) equations are adopted to describe the electric potential and ionic concentration distribution within the channel without using the assumptions of low surface (or zeta) potential and thin EDL. EDL overlapping is considered in this study due to the use of the PNP equations. When the EDL is not overlapped, the velocity profiles for both Newtonian and viscoelastic fluid of different Weissenberg number are plug-like with a rapid increase within the EDL. Apparent increase of velocity is observed for viscoelastic fluid compared to the Newtonian fluid, and this is due to the shear thinning effect. The increase of maximum velocity at the center of the channel is less significant for thicker EDL. EOF velocity increases with an increase in the polymer extensibility (


Wide tumour excision is currently the standard approach to surgical treatment of solid cancers including carcinomas of the lower genital tract. This strategy is based on the premise that tumours exhibit isotropic growth potential. We reviewed and analysed local tumour spreading patterns in 518 patients with cancer of the uterine cervix who underwent surgical tumour resection. Based on data obtained from pathological examination of the surgical specimen, we applied computational modelling techniques to simulate local tumour spread in order to identify parameters influencing preferred infiltration patterns and used area-proportional Euler diagrams to detect and confirm ordered patterns of tumour spread. Some anatomical structures, e.g. tissues of the urinary bladder, were significantly more likely to be infiltrated than other structures, e.g. the ureter and the rectum. Computational models assuming isotropic growth could not explain these infiltration patterns. Introducing ontogenetic distance of a tissue relative to the uterine cervix as a parameter led to accurate predictions of the clinically observed infiltration likelihoods. The clinical data indicates that successive infiltration likelihoods of ontogenetically distant tissues are nearly perfect subsets of ontogenetically closer tissues. The prevailing assumption of isotropic tumour extension has significant shortcomings in the case of cervical cancer. Rather, cervical cancer spread seems to follow ontogenetically defined trajectories. All of these cancers are solid tumours and surgery or (chemo-) radiotherapy are currently the only available options for curative loco-regional treatment5. Generally, the goal of surgery should be to minimize loco-regional recurrence rates without increasing operative morbidity unnecessarily. To accomplish these objectives, the surgeon is faced with the following central questions: (I) Which tissues are at risk for both visible or occult tumour infiltration and need to be removed? (II) Which tissues can be safely spared, thus minimizing treatment-related morbidity? (III) Which locally advanced tumours can still be submitted to surgical treatment? Conventionally, the answer to these questions has been based on two assumptions: First, that local tumour growth is unpredictable and therefore can occur in any direction, and second, that a microscopically invisible tumour front precedes the identifiable tumour margin. These dogmas of local tumour growth are reflected in the surgical treatment strategy of wide excision. Hereby, a metrically defined circumferential safety margin of healthy tissue is excised around the tumour with the goal of removing all (occult) tumour cells that are thought to permeate this region. Even though a single malignant neoplasm usually does not display isotropic growth, it is thought to have isotropic growth potential. As the actual direction of local cancer growth in a given case is assumed to be stochastic, the average growth pattern of multiple tumours is expected to be isotropic. The tissue at risk for infiltration of occult tumour cells is therefore currently defined as an isotropic tissue rim which is removed by wide excision.Cancer of the breast, prostate, colorectum, lung, and cervix uteri are among the most commonly diagnosed malignant tumours worldwide7. Furthermore, the width of resection margins would be expected to be one of the most important determinants of local tumour control. However, clinical data does not support this concept as has been demonstrated for several solid tumours, and reports from multiple investigations on resection margins provide conflicting results with some studies indicating that the width of resection margins is an important prognostic indicator while others fail to show an effect on local tumour recurrence and survival11. Imaging modalities are currently not capable of identifying single occult tumour cells that might precede the cancer invasion front. What surgeons need to know, therefore, is which tissues are at risk for potential infiltration of occult tumour cells and need to be removed to ensure oncological safety. Likewise, tissues that have a low probability of tumour infiltration should be spared with the goal of decreasing treatment-related morbidity. As a proxy for other solid tumours, we analysed data of cancer of the uterine cervix to demonstrate that local tumour spread is not isotropic but follows predictable growth patterns.However, increasing evidence points to fundamental flaws in the wide excision strategy. Even when adequate wide excision margins are obtained surgically, local relapse rates remain high14, we hypothesized that these anisotropic growth patterns would fit to an ontogenetic distance map, with tissues exhibiting a lesser ontogenetic distance to the uterine cervix having a higher probability of being infiltrated by the tumour than those of greater ontogenetic distance. The corresponding null-hypothesis would predict that the observed tumour infiltration patterns could be explained by physical distance alone, which is a function of metric distance and physical tissue type (i.e. adipose tissue or muscle). In this investigation, we use data derived from detailed pathology reports of patients who underwent surgical treatment for cervical cancer at our institution. Based on the results from pathological examination of all tissues removed during surgery, we determine in each case which pelvic structures are infiltrated by cancer. We then use area-proportional Euler diagrams and computational tumour growth simulations to determine whether ontogenetic tissue distance (relative to the uterine cervix) is a better parameter for the prediction of cancer infiltration than microenvironmental  distance. The assumption of predictable and ordered rather than stochastic tumour growth patterns could form the basis of a roadmap for local tumour growth which might help surgeons or radiotherapists identify tissues at high risk for tumour infiltration.Based on clinical experience during the last two decadesTo determine the clinical relevance of our findings and to facilitate their clinical application, we characterized our patient sample according to standard criteria; general patient- and tumour characteristics are compiled in Table ). A detailed description of the ontogenetic anatomy that exhibits some differences to conventional anatomy is available in the methods section.Detailed two-dimensional anatomical maps in the sagittal and transversal plane of the pelvis were drawn and tumour infiltration probabilities for different tissues and tissue compartments adjacent to the uterine cervix were mapped to these drawings Fig.  based on1Dρ = 0.599). In contrast, we observed only a weak correlation between the two parameters when a higher number, n ≥ 3, of structures were infiltrated (ρ = 0.340). A very similar picture was seen when grouping the tumour data in two sets, one set with tumour diameter ≤4 cm in greatest dimension (FIGO staging IB1/IIA1) and one with >4 cm (FIGO staging IB2/IIA2). The correlation between infiltrated structures and tumour size for small tumours below 4 cm was again quite reasonable (Spearman’s rank order correlation of ρ = 0.584), whereas the same correlation was weaker for larger tumours (ρ = 0.389).We compared the gross tumour diameter with the number of tumour-infiltrated tissue compartments categorised in Fig. In Fig. in silico simulations, each incorporating one of the two models. Results from the tumour growth simulations are shown in Fig. We ran two different In the ontogenetic model Fig. , tissue 17 of the histopathological data of tumour infiltration were drawn in Fig. Tumour infiltration data was gathered from the pathology reports containing the findings of the examination of the surgically resected tissues. Area-proportional Euler diagramsIn this work, we demonstrate that local tumour growth potential of cervical cancer is not isotropic and that the infiltration probability differs greatly among different anatomical structures and compartments in close proximity to the uterine cervix.As a first step to investigate whether tumour growth displays isotropic patterns, we compared the gross tumour diameter with the number of tumour-infiltrated tissue compartments Fig. . If all 20.Consequentially, we analysed whether the average tumour shape aspect ratio in our cohort was close to spherical as would have been predicted by our null-hypothesis assuming similar infiltration likelihoods of all surrounding tissues. Contrarily to an idealised, isotropically growing tumour – which is assumed in a wide excision strategy and would have an aspect ratio of very close to 1 – we found that the majority of aspect ratios significantly diverge from 1 with an average aspect ratio of approx. 0.4. While the localisation and shape of the initially formed tumour may influence the shape of the growing tumour, the surgically resected and pathologically analysed tumours presented in Figs in silico simulations for a physical microenvironmental and an ontogenetic model. The microenvironmental model is agnostic to ontogenetic compartment boundaries. The infiltration probabilities of different compartments are vastly overlapping, thus the expected number of cases with, for instance, mesorectum infiltration without mesobladder infiltration should be far beyond a singular case as observed in our cohort. Therefore, the microenvironmental model does not adequately reflect tumour infiltration patterns from a probabilistic point of view and physical distance is not a useful parameter in predicting local tumour growth. In the ontogenetic model, however, invasion of spatially close compartments happens stepwise, which is in accordance with the histopathological data. In detail, the tumour infiltration probabilities of tissues with growing ontogenetic distance from the uterine cervix increase suddenly with distinct onsets, in contrast to the overlapping infiltration probabilities of the microenvironmental model. Our tumour growth modelling data shows that introducing ontogenetic distance of tissues relative to the uterine cervix leads to a significant improvement in predicting the clinically observed tumour growth patterns as compared to models assuming isotropic tumour growth in the context of microenvironmental factors alone . However, some points in each model deserve further discussion:To further investigate whether ontogenetic distance is a better parameter than physical  distance to predict local tumour growth patterns, we ran 22 (which would translate into an increased cancer cell proliferation rate rij). However, tumour cells would be expected to exhibit comparable growth and proliferation behaviour in similar tissue types. The fibro-fatty connective tissues surrounding the uterine cervix ranging to the urinary bladder, the rectum, and the ureter are morphologically very similar and therefore cancer should show similar propensities to extend in either direction rendering tumour growth isotropic on average. Furthermore, the supporting (meso-) structures of bladder, ureter and rectum are all composed of fatty tissue, yet are infiltrated at significantly different rates13, see Fig. First, one should consider how differences in the tumour microenvironment might lead to preferential infiltration of specific abutting tissues. Fatty, muscular and fibrous connective tissues all constitute different microenvironments in which tumour cells might behave differently. For example, fatty tissue has been shown to promote of tumour growth24. To date, no measurements of stiffness of the tissues surrounding the uterine cervix have been performed; therefore, we could not include tissue stiffness as a parameter in our model. Nonetheless, it seems unlikely that morphologically similar tissues such as the fatty tissue of the bladder mesentery or the rectal mesentery exhibit significantly different degrees of rigidity or rigidity gradients. In fact, morphologically similar cells share important mechanical and regulatory characteristics and respond and adapt to the rigidity of their surroundings in similar ways28. The impact of the microenvironment is also reflected in the active regulation and fine-tuning of the cell’s cytoskeleton via crosslinker and filament concentrations35 and therefore the resulting mechanical properties of the cells39. While it cannot be ruled out that the tissue microenvironment may have an influence of the growth direction and anisotropy, it can be argued that the physical microenvironmental factors cannot be the dominant factor in the process.Second, during the past decade, there has been an increasing notion that rigidity of the tumour microenvironment plays an important role in directing tumour growth. Extracellular matrix (ECM) stiffness might in fact promote malignant transformation and direct tumour growth along gradients of ECM rigidity, a process termed durotaxiset al. have shown dense, parallel aligned collagen layers between abutting pelvic tissue complexes such as the uterine cervix and the rectum or urinary bladder40. These collagen layers may halt locally invading tumour cells. In fact, it has previously been shown that low-to-high concentrated collagen interfaces drastically limit cell migration43. The implementation of collagenous tissue boundaries in our model via uniform, passive resistive boundaries – that is, as a simple mechanical barrier – did not sufficiently lead to the clinically observed growth patterns and shape of cervical cancer. It should be noted, however, that the use of strict inter-compartmental barriers in our model predicts a step-wise tumour progression into neighbouring tissue compartments and thus captures one aspect of the clinically observed tumour permeation pattern. It fails, however, to adequately reflect the anatomical distribution of tumour spread. Nonetheless, dense collagen lamellae remain a crucial physical barrier in (cancer) cell migration. Especially the ontogenetic lineage boundary between the mesometrium and mesorectum, as well as the mesureter, is extraordinary resilient against tumour transmigration, whereas the collagen lamella between the mesometrium and bladder compartment seems to be a minor obstacle; an unexpected observation considering that all collagen lamellae are of comparable thickness and composition and thus should display the same migratory resistance. Given the high spatial density of segregated compartments and therefore proximity of compartment boundaries, the tumour growth is influenced and shaped by these compartment boundaries, described in previous findings14. However, when interpreting the cancer cell migration resistance across compartment boundaries due to initial ontogenetic tissue incompatibility – that is, cancer can only invade ontogenetically close tissues at first and acquires the trait to invade ontogenetically distant tissue later on – the reason for the migration resilience may become apparent. The bladder compartment is ontogenetically close, whereas the ureter and rectum compartment are ontogenetically far from the uterine cervix compartment. This translates then to a lower hurdle for cervical cancer to acquire the ability to invade the bladder compartment, but a profound step in tumour progression for the rectum and ureter compartment. The patterns of cervical cancer growth that we observed can be mapped to the tissue domains that are identified using the bifurcational developmental paradigm. Crucially, there is a clear correlation between the degree of ontogenetic proximity to the uterine cervix and the likelihood of infiltration by cervical cancer for any pelvic tissue. Indeed, when introducing ontogenetic distance on the tissue tree into our model of local tumour spread, the predicted step-wise tumour infiltration of the various pelvic tissue compartments is nearly identical to the ones observed in clinical practice, as seen in Fig. Third, it should be assessed whether fascial tissue structures, i.e. dense collagen layers between neighbouring tissues which usually coincide with developmental compartment boundaries, impede tumour growth and thus lead to anisotropic permeation patterns. Recently, using a novel slicing and staining technique, Steinke stepwise tumour progression which we observed in the ontogenetic tumour progression model. To confirm that ontogenetic tissue compartment infiltration occurs in a predictable and step-wise fashion, tumour infiltration data was gathered by pathological examination of the surgical resected tissues and area-proportional Euler diagrams of the histopathological data of tumour infiltration were drawn  was never infiltrated without simultaneous infiltration of the bladder mesentery which strongly suggests a stepwise tumour progression based on tissue compartments with specifically different risks of tumour infiltration. Interestingly, in the one patient with rectum but without bladder infiltration, the bladder was spared during the initial surgery. After six months, this patient developed a tumour recurrence in the bladder compartment, indicating probable occult involvement of the bladder at the time of the initial operation. The nearly perfect sub-set characteristic of Fig. 14. In the current analysis examining 518 cases of primary cervical cancer, these patterns were slightly different with the ureter and mesureter exhibiting a greater likelihood of being infiltrated than the rectum and mesorectum. This demonstrates that tumour growth is different in cases of recurrent cancer as previous treatment (e.g. pelvic surgery or irradiation) probably affects ontogenetic compartment boundaries leading to different tumour progression patterns.One significant strength of the present work is that it is based on the histopathological data of numerous patients with advanced disease who underwent surgery without any preoperative treatment. Because patients with locally advanced disease are usually submitted to primary chemo-radiotherapy in most institutions, there is generally no comprehensive pathological data available. In a previous series of 88 cases with locally advanced and/or recurrent cervical cancer, the bladder with its support structures showed tumour infiltration in 84%, the rectum in 34% and the ureter in 24% of all casesThere are some weaknesses of our study which should be addressed. First, we used 2-dimensional anatomical maps instead of 3-dimensional anatomic templates. This was due to computational limitations and limited knowledge of the exact 3-dimensional positional information of all pelvic fascia. However, given the fact that the adjacent pelvic planes  are nearly identical to the ones we used, 3-dimensional simulation should lead to similar results.44, they can be thought of as spatial extensions from that region providing a similarly fertile ground for tumour cell proliferation. We would speculate, that the knowledge about which tissue domain is connected to which lymph nodes could provide the basis for mapping the lymphatic landscape according to the display of antigens characteristic of specified ontogenetic compartments. Consequently, we would expect tumours to proliferate and spread preferentially to lymph nodes displaying antigens of close ontogenetic proximity to the peripheral tissue of tumour origin. Regarding hematogenic cancer spread causing metastasis in distant organs, we would again predict that organs which are ontogenetically closer related to the organ of tumour origin have a higher likelihood of being affected. However, the multitude of organs and anatomic structures sharing equal ontogenetic distance at this late stage of cancer progression makes it difficult to predict patterns of metastasis.Second, we disregarded other than continuous modes of tumour spread, i.e. lymphatic and haematogenous cancer dissemination. Although these routes of tumour propagation are clinically highly significant and have an important impact on a patient’s prognosis, we did not cover them, as our goal was to provide a roadmap for local tumour growth only. Furthermore, constructing precise anatomical templates of the lymphatic and circulatory system as a basis for modelling is currently not feasible. However, considering that lymph nodes receiving drainage from a specific anatomical region display antigens from that region45. Furthermore, the correlation of carcinogenesis and ontogenesis has been shown for various tumor entities, such as rectum46, pancreas47, and mid-facial skin cancers48, wherein the local tumor spread can be well-described with embryological structures such as compartments and fusion planes.Third, this analysis only includes cervical cancer cases and its applicability to other tumour entities is therefore limited. However, the same principles regarding ontogenetically determined tumour growth seem to apply to vulvar cancer as was demonstrated in a recent clinical trialOur findings provide a framework to conceptualize and predict local spread of cervical cancer. As ontogenetic compartment borders can clearly be identified intraoperatively, ontogenetic tissue mapping can probably serve as a roadmap to local cancer growth in other tumour entities. Knowing the embryological origin of any tissue in which cancer arises, one can derive a relative likelihood of different adjacent tissues to be infiltrated by tumour cells, depending on their ontogenetic proximity to the tissue of cancer origin, deduce an ontogenetic tumour stage, and tailor surgical resection exactly to the tissue at risk. In the future, this might help to decrease operative morbidity by sparing low-risk tissue while improving local tumour control by resecting all tissues of potential (occult) tumour infiltration.49. GRN’s enable cells to concertedly change protein expression patterns in response to environmental changes and have a tendency to stabilize themselves including pathological states which might foster tumour progression50. This would lead to a predictable evolution which is reflected by the acquisition of all hallmarks of cancer on a cellular level and by specific growth patterns on the tissue level. Interestingly, GRNs seem to play an important role in epithelial-mesenchymal transition (EMT) which is a key mechanism of cell and tissue differentiation, both during ontogenesis and during cancer progression53 including hybrid epithelial/mesenchymal states56. We hypothesize that the stepwise re-activation of GRNs, which are normally executed during ontogenetic development, leads to the observed growth and infiltration patterns.What could be the underlying mechanisms on a cellular and molecular level? One explanation might be the existence of transcriptional programs which sequentially activate and silence specific genes. Such programs are executed by gene regulatory networks (GRNs), which are defined as units of interacting transcriptional elements which exhibit specific regulatory relationships and therefore cause a predictable pattern of gene expression with a defined structural and functional outputIn conclusion, we have shown that the potential local tumour growth of cervical cancer is not isotropic. The data representation and our tumour growth simulation indicate that ontogenetic tissue mapping might profoundly help in predicting the probability of tumour invasion into a given tissue by a certain cancer. Future work needs to corroborate these findings, ideally in other cancer entities.In order to obtain histopathological information for further analysis, patient and tumour data was extracted from our institutional study databases. Figure Numerical and qualitative data used for preparation of the Euler diagrams and in-silico models were extracted from the original detailed pathology reports archived in our database. These pathology reports contained all relevant information regarding tumour entity (i.e. squamous or non-squamous histology), tumour size measured directly within the surgical specimen, and extent of infiltration into neighbouring pelvic tissues.The terms we use to describe pelvic anatomy diverge from the terminology commonly used by gynaecological surgeons. Generally, structures and landmarks in surgical anatomy consist of functional units while “non-functional” connective tissue is frequently not classified any further. In contrast, ontogenetic anatomy provides a means to group all tissues into distinct groups regardless of form and function. Therefore, some of the terms which are less commonly used in conventional surgical anatomy or which have different meanings are described here. Figure The Müllerian compartment is the sum of all adult tissues which are derived from the embryonic paramesonephric ducts. Cranio-caudally, this includes the Fallopian tubes, the uterine corpus, the uterine cervix, the proximal two thirds of the vagina and the Müllerian adventitia (see below). Each of these structures constitutes a sub-compartment of the Müllerian compartment.Fallopian tubes – parasalpinxUterine corpus – paracorpusUterine cervix – paracervixVagina – paracolposThe para tissues constitute together the Müllerian adventitia. This is a coat of fibrovascular connective tissue enveloping the entire Müllerian compartment. In a cranio-caudal fashion the Müllerian adventitia can be divided into the following segments according to the adjacent organs:Each para-tissue constitutes a sub-compartment of the Müllerian compartment.Note that the term “parametrium” in surgical anatomy conventionally denotes a poorly defined composite structure including more tissues of different ontogenetic origin . The para-tissues contain a tight network of anastomosing arteries and veins as well as lymph vessels and nerves.The term mesentery is here applied to the connective tissues which connect the pelvic organs to the pelvic and abdominal wall laterally and caudally . The mesenteries are composed primarily of fibrofatty tissue containing lymph- and blood vessels and therefore serve an important nutritional role. In addition, they contain nerves and interspersed condensations of fibrous tissue which serves a suspensory function. Ontogenetically, they are closely related to the organs which they support and are therefore prone for early tumor infiltration. The mesenteries develop and elongate during the embryonic and fetal period as the associated organs move within the abdominal and pelvic cavity as a consequence of differential growth.The mesometrium supporting the uterus (corpus and cervix)The mesureter supporting the ureterThe mesobladder supporting the urinary bladder.Within the pelvis, the urogenital tract is supported by the urogenital mesentery. This mesentery can be subdivided intoIn addition, the rectum is supported by the mesorectum.By conventional nomenclature, only the mesorectum is a proper mesentery . From an ontogenetic perspective, the term “mesentery” is broader to include the above-mentioned structures.13. During the first stage of tissue expansion in a given embryonic domain, cellular proliferation is homogenous and isotropic. At a certain point, presumably when a morphogen gradient is formed, and/or an unstable critical concentration is reached, cellular segregation and the emergence of two new cell populations with new phenotypes can be observed. Figure We have previously described embryogenesis in terms of bifurcational tissue differentiation based on morphologic observations in human embryosstatistically averaged tumour to show that the clinically assumed isotropic growth is not sufficient for explaining the clinically and pathologically observed tumour growth patterns. The descriptive model is based on the pathological and ontogenetic characterization presented Table As described in the beginning, singular tumours are usually not of spherical shape and do not necessarily grow isotropically. Nevertheless, permeation of cancer cells into surrounding tissues is assumed isotropic in order to define a safe surgical resection margin, hence they are assumed to have an isotropic growth potential. Here, we introduce a description of tumour growth dynamics for a i located in the tissue compartment j at a given time. The index k accounts for the cancer cell’s coarse-grained aggressiveness or staging, and i at the stage k in the compartment j. The local cancer net proliferation rate 62. Some or many of inhibitory factors may lose effect in the progress of tumour progression65, thus altering the cancer proliferation and spreading dynamics. In vivo, the local cancer proliferation rate is generally thought to depend on supply of oxygen and nutrients per cancer cell, which is an inverse measure of the local cancer cell density, among potential other physical and chemical microenvironmental factors70. The reaction term can be expanded to include features of nutrient supply, microenvironmental effects or growth factors to create a model closer resembling in vivo tumour physiology and allowing for enforced anisotropic tumour growth. Here, the basic tumour growth rate was modelled by a generalized Fisher-Kolmogorov term73 agnostic of microscopic interactions of single cancer cells,rijk (as compared to Rijk) and the proliferation inhibition coefficient Rijk initially increases with an increasing number of cancer cells in a given volume and slows down when it approaches the maximum cancer cell density probability i and j, and ρij the resistance for cancer cell migration across the interface. The resistive barrier simulates an initially low fraction of cancer cells with the acquired capacity of invasion into a neighbouring compartment. The finite-element model was simulated via COMSOL Multiphysics® software74. Further detailed descriptions and values of model parameters are given in the Supplement The growth dynamics for a statistically averaged tumour were described by the following set of reaction-diffusion equationsSupplement