Multiphoton Laser Scanning Microscopy Research Articles

Skin Cancer Image Classification Using Artificial Intelligence Strategies: A Systematic Review

The increasing incidence of and resulting deaths associated with malignant skin tumors are a public health problem that can be minimized if detection strategies are improved. Currently, diagnosis is heavily based on physicians’ judgment and experience, which can occasionally lead to the worsening of the lesion or needless biopsies. Several non-invasive imaging modalities, e.g., confocal scanning laser microscopy or multiphoton laser scanning microscopy, have been explored for skin cancer assessment, which have been aligned with different artificial intelligence (AI) strategies to assist in the diagnostic task, based on several image features, thus making the process more reliable and faster. This systematic review concerns the implementation of AI methods for skin tumor classification with different imaging modalities, following the PRISMA guidelines. In total, 206 records were retrieved and qualitatively analyzed. Diagnostic potential was found for several techniques, particularly for dermoscopy images, with strategies yielding classification results close to perfection. Learning approaches based on support vector machines and artificial neural networks seem to be preferred, with a recent focus on convolutional neural networks. Still, detailed descriptions of training/testing conditions are lacking in some reports, hampering reproduction. The use of AI methods in skin cancer diagnosis is an expanding field, with future work aiming to construct optimal learning approaches and strategies. Ultimately, early detection could be optimized, improving patient outcomes, even in areas where healthcare is scarce.

Dynamic Pathology of Enteric Neural Network using Curcumin-assisted Multiphoton Laser Imaging in Hirschsprung Disease.

In living tissue, it has been difficult to make microscopic-level observations without damaging the tissue. We have invented a novel intravital fluorescent observation method (IFOM) for real-time tissue observation, combining multi-photon laser scanning microscopy (MPLSM) with curcumin vital staining (CVS-IFOM). The aim of this study was to use CVS-IFOM to analyze the enteric nervous system (ENS) in mice and human patients with hypoganglionosis and Hirschsprung disease. In an initial viability study, we compared live ENS images from non-fluorescent C57BL6 mice stained with curcumin (n=5) and GFP mice (n=5) using MPLSM. We then explored CVS-IFOM for the live examination of resected colon tissues from one hypoganglionosis and three Hirschsprung disease patients. In the viability study, detailed ENS histological features were only observed in the curcumin-stained mice. In the hypoganglionosis patient, CVS-IFOM provided ENS details that were not visualized under H&E staining or calretinin immunohistochemistry, allowing the analysis of ENS size, neural bundle number, and neural cell number per plexus. In Hirschsprung disease patients, CVS-IFOM showed a gradual hypoplastic change in the ENS from the oral wedge to the anal wedge, detecting disproportionate changes in the ENS within the same intestinal level, supporting a circumferentially uneven distribution of the intestinal ENS. CVS-IFOM may be supportive for intraoperative pathological diagnosis during surgeries in Hirschsprung disease.

Three Photon Excited Image Scanning Microscopy for in-Depth Super-Resolution Studies of Biological Samples

Multiphoton laser scanning microscopy is a powerful tool for deep imaging of thick biological samples. Image scanning microscopy (ISM) has demonstrated significant improvements in the signal-to-noise ratio in confocal laser scanning microscopy, while at the same time improving upon the effectively attainable resolution. Two-photon excitation (2PE), combined with ISM, has been shown to allow for deep tissue imaging with enhanced resolution compared to 2PE microscopy. Three-photon excitation (3PE) has enabled record imaging depth and contrast for multiphoton imaging, due to the superior suppression of out-of-focus signal generation. In this paper, we demonstrate super-resolution 3PE ISM. This is achieved using a single-photon avalanche detector array, and 1040-nm pulses for 3PE of blue fluorescence. This method enables subdiffraction limited resolution imaging of biological samples stained with blue fluorescent markers, such as mouse myocardial and spinal cord tissues stained with 4′,6-diamidino-2-phenylindole. Deconvolution improves the resolving power further and allows for imaging with better than λ/8 resolution with respect to the 3PE wavelength λ. With the ISM pixel reassignment procedure, we demonstrate a resolution enhancement of ∼1.6 laterally, compared to the resolution attained using a photomultiplier tube in a non-descanned detection arrangement, and a factor of ∼1.8 enhancement in axial resolution. The experimentally measured three-dimensional point spread function volume is shrunk ∼4.4-fold, which is close to the theoretically expected enhancement. Published by the American Physical Society 2024

Trauma in Neonatal Acute Brain Slices Alters Calcium and Network Dynamics and Causes Calpain-Mediated Cell Death.

Preparing acute brain slices produces trauma that mimics severe penetrating brain injury. In neonatal acute brain slices, the spatiotemporal characteristics of trauma-induced calcium dynamics in neurons and its effect on network activity are relatively unknown. Using multiphoton laser scanning microscopy of the somatosensory neocortex in acute neonatal mouse brain slices (P8-12), we simultaneously imaged neuronal Ca2+ dynamics (GCaMP6s) and cytotoxicity (propidium iodide or PI) to determine the relationship between cytotoxic Ca2+ loaded neurons (GCaMP-filled) and cell viability at different depths and incubation times. PI+ cells and GCaMP-filled neurons were abundant at the surface of the slices, with an exponential decrease with depth. Regions with high PI+ cells correlated with elevated neuronal and neuropil Ca2+ The number of PI+ cells and GCaMP-filled neurons increased with prolonged incubation. GCaMP-filled neurons did not participate in stimulus-evoked or seizure-evoked network activity. Significantly, the superficial tissue, with a higher degree of trauma-induced injury, showed attenuated seizure-related neuronal Ca2+ responses. Calpain inhibition prevented the increase in PI+ cells and GCaMP-filled neurons in the deep tissue and during prolonged incubation times. Isoform-specific pharmacological inhibition implicated calpain-2 as a significant contributor to trauma-induced injury in acute slices. Our results show a calpain-mediated spatiotemporal relationship between cell death and aberrant neuronal Ca2+ load in acute neonatal brain slices. Also, we demonstrate that neurons in acute brain slices exhibit altered physiology depending on the degree of trauma-induced injury. Blocking calpains may be a therapeutic option to prevent acute neuronal death during traumatic brain injury in the young brain.

Dynamic pathology in various disease-model mice using multiphoton laser scanning microscopy

Live imaging of experimental animals is now possible thanks to recent technological advances that overcome the limitations of conventional histological analysis. In contrast to conventional histological microscopy techniques, this intravital approach can reveal previously unknown morphogenetic and functional processes in live tissues. In addition, this approach can capture real-time information on these processes, compared with conventional histological microscopy and other techniques that only provide snapshots in time. We used multi-photon laser-scanning microscopy (MPLSM) for in vivo real-time imaging of intra-abdominal organs, and investigated the intravital microscopic changes in various disease-model mice, referred to as ‘dynamic pathology’. For example, we used this technology to examine bacterial translocation in dextran sodium sulfate (DSS)-induced colitis, thrombus formation in laser-induced endothelial injury, neutrophil extracellular traps, the dynamics of circulating free DNA in a model of DSS-induced colitis, and to obtain a comprehensive understanding of the development and blood flow dynamics of the small intestinal microcirculation in a mouse model of necrotizing enterocolitis. This mini review summarizes the in vivo observation methods that we have developed to observe the dynamic pathology in various disease-model mice using MPLSM.

Abstract C020: Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest forms of cancer with an extremely low survival rate. Despite the success of therapeutic T cells in hematologic cancers, many solid tumors, including PDA, remain unresponsive with such therapies, partly due to its mechanically complex, dense, and immunosuppressive stroma. Therefore, understanding the dynamic interplay between therapeutic T cells and the tumor microenvironment (TME) is crucial for revealing engineering targets for next-generation immunotherapies. We approach this problem with genetically engineered KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-1-Cre) mouse models that recapitulates PDA disease progression and stromal dynamics. Within live tumor slices from primary tumors, we are defining how stromal components such as dense collagen fiber networks and tumor-associated macrophages (TAMs) orchestrate an immunosuppressive landscape. We employ multiphoton laser scanning microscopy (MPLSM) to generate multiphoton excitation of fluorescence (cell dyes and immunofluorescence) and second harmonic generation (SHG) for collagen fibers to acquire multidimensional data (x, y, z, c, t) of T cell migration and interaction within the TME. We subsequently reveal T cell migration and interaction as a function of its physical and biological ECM components using advanced computer vision and machine learning algorithms based on the Clustered Random Convolutional Kernel Transform (cROCKET), revealing a hidden landscape in the feature space. Perturbations to the TME in KPC mice with CCR2 inhibitor CCX598 (in vivo) and clodronate liposomes (ex vivo) deplete CD11b+ myeloid cells, resulting in altered behaviors of the infiltrating T cells. Anti-PD-1 monoclonal antibody neutralizes the PD-1/PD-L1 axis in T cells, resulting in enhanced migration and infiltration in tumor slices. Citation Format: Guhan Qian, Hongrong Zhang, Aine Knott, Jon Zettervall, Ingunn Stromnes, Paolo Provenzano. Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C020.

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

#3197 A NOVEL INVESTIGATION OF AVF WITH A SECOND HARMONIC GENERATION MICROSCOPY IN PATIENTS WITH END STAGE RENAL DISEASE

Abstract Background and Aims The population of patients with end-stage renal disease is rapidly growing and hemodialysis (HD) remains the most common treatment option. In clinical practice, the formation of arteriovenous fistulas (AVFs) is considered a priority in order to ensure optimal vascular access for HD patients. One of the complications in the formation of AVF is the hyperplasia of the neointimal layer of the vein, which leads to the failure of the fistula. The impact of the arrangement of collagen fibers in the veins of AVFs is little studied and opens wide opportunities for investigations. The structural tissue can be imaged with polarimetric nonlinear microscopy. Collagen fibers have a non-centrosymmetric structure, so they can be effectively imaged by second harmonic generation (SHG) microscopy [1]. The polarimetric nonlinear microscopy, exploiting the multidimentional tensor nature of light-matter interactions, is emerging as a valuable research tool in biomedical investigations and applications to digital pathology, and is only now becoming technically feasible for potential clinical translation [2,3]. This proposal represents a novel application of multimodal polarimetric nonlinear microscopy in histopathology, and biomedical imaging. The aim of this study is to determine the influence of the arrangement of collagen fibers in veins on the formation efficiency of AVFs by using the innovative method of nonlinear SHG microscopy. Method In this prospective study, we first used a novel SHG microscopy method to investigate AVFs venous intima hyperplasia. V. Cephalica segment is being obtained during arteriovenous fistula formation surgery from patients with end stage renal disease and after the removal of aneurysmatic or fibrotic AVF. Cross-sections of venous specimens are being fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E). Further, sample investigation is being performed in Laser Research Centre, Vilnius University using a home-built multiphoton laser-scanning microscope. The investigation is being performed with the permission of Lithuanian Biomedical Research Ethics Committee. Results We determined the arrangement of collagen fibers in veins before AVF formation and after the removal of aneurysmatic AVFs. It was observed that tunica intima of V. Cephalica is dominated both by endothelial cells and connective tissue made up of high-density collagen fibers. Whereas tunica adventitia consists mainly of collagen fibers. Collagen in tunica intima and adventitia are found to be highly noncentrosymmetric. Thus a strong SHG signal, resulting in high-contrast structural visualization of the V. Cephalica segment is generated. Conclusion Our initial investigations of AVF show clearly visible collagen fibers. The future work will focus on the structural changes of collagen fibers in the vein intimal hyperplasia. Further investigations need to be done to create artificial intelligence-based identification of new predictive collagen markers.

The effect of Er:YAG laser treatment on biofilm formation on titanium and zirconia disc surfaces.

Lasers represent a promising method for implant decontamination, but evidence on implant surface changes and subsequent biofilm formation is limited. This study aimed to assess the effect of erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment on zirconia and titanium discs, and the differences in biofilm formation as a result of surface alterations. A two-stage (in vitro and in vivo) experiment utilizing Er:YAG laser on titanium and zirconia discs was performed. In vitro, surface alterations, roughness, and elemental-material weight differences following laser treatment were assessed using scanning electron microscopy and atomic force microscopy. In vivo, four participants wore custom-made intra-oral stents, embedded with laser-treated and untreated titanium and zirconia discs overnight. Biofilm-coated discs were stained using nucleic acid fluorescence dye and visualized using multiphoton confocal laser scanning microscopy. Biofilm 3D structure, biomass, thickness, and live-to-dead bacteria ratio were assessed. Both titanium and zirconia discs treated with Er:YAG laser resulted in visual surface alterations, but showed no significant change in average surface roughness (titanium P=0.53, zirconia P=0.34) or elemental-material-weight (titanium, P=0.98), (zirconia, P=0.96). No significant differences in biofilm biomass, average thickness, and live-to-dead bacteria ratio of laser-treated titanium and zirconia discs were identified compared to untreated groups (titanium P>0.05, zirconia P>0.05). Generally, zirconia discs presented with a lower live-to-dead bacteria ratio compared to titanium discs, regardless of laser treatment. Er:YAG laser treatment of titanium and zirconia implant surfaces does not significantly affect surface roughness, elemental material weight, or early biofilm formation in the oral cavity.

Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury

Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage invivo in an animal model. Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope. Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw. Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function. In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

Characterization of a decellularized rat larynx: comparison between microscopy techniques

BackgroundNo effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues. MethodsH&E, Masson Trichrome and DAPI staining as well as DNA quantification were used to evaluate decellularization efficiency. Van Gieson stain, Picrosirius Red stain (PRS) and multiphoton laser scanning microscopy (MPM) were carried out for collagen detection and quantitative assessment. Polarized PRS was used to investigate collagen network, and Weigert stain and MPM were used to detect and estimate elastin content. ResultsThe decellularization process removed the cellular components without affecting glycosaminoglycan, collagen and elastin content. Concerning collagen quantification, Van Gieson stain underestimated collagen content, while PRS, apparently less fading, did not reach reliable results when used as quantitative method. MPM effectively quantified collagen content. Collagen fibers were visualized much better under polarized light microscopy, allowing to underline that decellularization process affects the homogeneity of 3D collagen network. Concerning elastin detection, Weigert stain and MPM produced overlapping results. ConclusionsAn efficient protocol to decellularize the whole larynx was developed, allowing the removal of cells without affecting ECM integrity. The results supported the use of non-polarized PRS to highlight collagen, even the thin fibers, second harmonic generation for major fibrillar collagens and polarized PRS for 3D collagen network. Concerning elastin, Weigert stain and MPM showed similar results, thus the use of MPM, rather than that of the Weigert stain, may be suitable to avoid the additional time and costs of a histological staining.

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Lifetime imaging of the discrete nanophosphors in biological systems

The aim. Demonstrate a novel modality of laser-scanning multiphoton microscopy suitable for rapid acquisition of images of samples labelled with phosphorescent materials characterised by long emission lifetime measured in microseconds. The reported microscopy represents an advancement over the existing laser-scanning modalities, where the acquisition of images of phosphorescent materials takes unpractically long time.Materials and methods. The reported method is based on rapid scanning of the focussed excitation beam across a sample while continuously recording the photoluminescent (PL) signal. The resultant images of discrete phosphorescent nanoparticles appeared blurred. The diffraction-limited image was reconstructed by using a deconvolution algorithm, where the PL lifetime was the key input parameter. To test the method, two types of upconversion nanoparticles (UCNP) were synthesised, NaYF4:Yb3+:Er3+/NaYF4 (E-UCNP), β-NaYF4:Yb3+, Tm3+/NaYF4 (T-UCNP) and used to test a possibility of demultiplexing the two types of UCNPs ex vivo taken up in the mouse liver.Results. The resultant images of E-UCNP, T-UCNP on the background of the liver were fully reconstructed and exhibited the enhanced signal-to-noise ratio. Besides, the method allowed rapid (at the scale of seconds) acquisition of the UCNP PL lifetime and clear discrimination of the two types of UCNPs.Conclusion. We demonstrated a new approach for rapid PL image acquisition of samples containing PL materials, such as biological specimens labelled with discrete UCNPs. Blurred images were shown to be reconstructed at the post-processing stage by applying a deconvolution procedure. This enabled demonstration of multiplexing/demultiplexing using lifetime imaging mode, where the lifetime was engineered by the UCNP synthesis and reconstructed during multiphoton image acquisition using the deconvolution algorithm. The power of this method was demonstrated by the identification of two types of UCNPs accumulated in the liver of a laboratory animal. We believe that the demonstrated method can be useful for rapid lifetime imaging where several molecular specific labelling agents are required.

Multiphoton imaging of melanoma 3D models with plasmonic nanocapsules

We report the synthesis of plasmonic nanocapsules and the cellular responses they induce in 3D melanoma models for their perspective use as a photothermal therapeutic agent. The wall of the nanocapsules is composed of polyelectrolytes. The inner part is functionalized with discrete gold nanoislands. The cavity of the nanocapsules contains a fluorescent payload to show their ability for loading a cargo. The nanocapsules exhibit simultaneous two-photon luminescent, fluorescent properties and X-ray contrasting ability. The average fluorescence lifetime (τ) of the nanocapsules measured with FLIM (0.3 ns) is maintained regardless of the intracellular environment, thus proving their abilities for bioimaging of models such as 3D spheroids with a complex architecture. Their multimodal imaging properties are exploited for the first time to study tumorspheres cellular responses exposed to the nanocapsules. Specifically, we studied cellular uptake, toxicity, intracellular fate, generation of reactive oxygen species, and effect on the levels of hypoxia by using multi-photon and confocal laser scanning microscopy. Because of the high X-ray attenuation and atomic number of the gold nanostructure, we imaged the nanocapsule-cell interactions without processing the sample. We confirmed maintenance of the nanocapsules’ geometry in the intracellular milieu with no impairment of the cellular ultrastructure. Furthermore, we observed the lack of cellular toxicity and no alteration in oxygen or reactive oxygen species levels. These results in 3D melanoma models contribute to the development of these nanocapsules for their exploitation in future applications as agents for imaging-guided photothermal therapy. Statement of SignificanceThe novelty of the work is that our plasmonic nanocapsules are multimodal. They are responsive to X-ray and to multiphoton and single-photon excitation. This allowed us to study their interaction with 2D and 3D cellular structures and specifically to obtain information on tumor cell parameters such as hypoxia, reactive oxygen species, and toxicity. These nanocapsules will be further validated as imaging-guided photothermal probes.

Noise reduction in two-photon laser scanned microscopic images by singular value decomposition with copula threshold

Multi-photon laser scanning microscopy is an advantageous technique for layered imaging in thick tissue samples. It enables reduced phototoxicity as the region of fluorescence excitation is limited. But, the small number and stochastic nature of detected photons require the multifold acquisition of each image so the photodamage may accumulate. This paper aims to reduce the number of repeated image acquisitions, proposing a denoising technique based on singular value decomposition with a novel two-stage copula threshold, thus the name SVD-COPULA. The first stage defines the referent noise level based on copula analysis. Following the comparative analysis of classical techniques and different copula families, Frank's copula is selected to determine the regions for noise estimation. The second threshold stage analyzes the stationarity of the singular values and adjusts the number of the eigenimages necessary for reconstruction, an issue important for images without the distinctive background regions. A problem of the mixed discrete-continual nature of pixel values is noticed and resolved. The procedure is fully automatic, with the built-in set of parameters and without the necessity for prior image analysis. The proposed method, together with twelve state-of-the-art denoising methods, are tested using the microscopic images from the proprietary and open databases, and the test images with simulated mixed Poisson-Gaussian noise. It is shown that the performance of SVD-COPULA is consistently good for all investigated images.

Imaging the different timescales of germinal center selection.

Germinal centers (GCs) are the site of antibody affinity maturation, a fundamental immunological process that increases the potency of antibodies and thereby their ability to protect against infection. GC biology is highly dynamic in both time and space, making it ideally suited for intravital imaging. Using multiphoton laser scanning microscopy (MPLSM), the field has gained insight into the molecular, cellular, and structural changes and movements that coordinate affinity maturation in real time in their native environment. On the other hand, several limitations of MPLSM have had to be overcome to allow full appreciation of GC events taking place across different timescales. Here, we review the technical advances afforded by intravital imaging and their contributions to our understanding of GC biology.

Acid-induced gels from soy and whey protein thermally-induced mixed aggregates: Rheology and microstructure

In this study, we explored how substituting whey protein isolate (WPI) with soy protein isolate (SPI) affects the linear and non-linear rheological behavior of acid-induced gels, and their microstructures. Commercial SPI and WPI dispersions (pH 7.0, 3.0 mS/cm) were preheated (95 °C, 30 min) at different protein concentrations (2%, 4%, 6%, and 8% w/w) and SPI: WPI ratios (0: 4, 1: 3, 2: 2, 3: 1 and 4: 0). The resultant thermally-induced aggregates were characterized before gelation was induced by glucono-δ-lactone (GDL). Small and large amplitude oscillatory shear (SAOS and LAOS) tests showed that replacing WPI with SPI decreased the strength (lower G′) and stretchability (lower γc) of acid-induced gels in the linear viscoelastic (LVE) regime. Gels containing SPI behaved more similar to pure SPI gels in the non-linear viscoelastic (NLVE) regime: displaying a relatively elastic response at large strain and a gradual transition to plastic behavior. The changes in rheological properties were explained by the differences in the gel microstructures, via fractal scaling theory, multiphoton laser scanning microscopy (MLSM) and scanning electron microscopy (SEM). WPI gels formed denser and homogenous gel networks with very strong inter-floc links, while hybrid gels and pure SPI gels formed coarser and more porous networks with intermediate inter-floc links. The constituent flocs in the latter were larger, with rougher, more elongated and branched structures. The present results provide useful information for future attempts to replace WPI with SPI in food products based on acid-induced gelation.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN− vessels exhibiting higher flowrate and higher oxygen tension (pO2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN− vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering.

KRP-203 Is a Desirable Immunomodulator for Islet Allotransplantation.

Background.The current standard immunosuppressive regimens, calcineurin inhibitors, have diabetogenic and anti-vascularization effects on islet grafts. KRP-203, a sphingosine-1-phosphate functional antagonist, exerts its immunomodulatory function through lymphocyte sequestration. However, the effect of this antagonist on islets is unclear. We examined the effect of KRP-203 on the islet function and vascularization and sought a calcineurin-free regimen for islet allotransplantation.Methods.KRP-203 was administered for 14 d to mice, then diabetogenic effect was evaluated by blood glucose levels and a glucose tolerance test. Static glucose stimulation, the breathing index, and insulin/DNA were examined using isolated islets. Islet neovascularization was evaluated using a multiphoton laser scanning microscope. After islet allotransplantation with either KRP-203 alone, sirolimus alone, or both in combination, the graft survival was evaluated by blood glucose levels and immunohistochemical analyses. A mixed lymphocyte reaction was also performed to investigate the immunologic characteristics of KRP-203 and sirolimus.Results.No significant differences in the blood glucose levels or glucose tolerance were observed between the control and KRP-203 groups. Functional assays after islet isolation were also comparable. The multiphoton laser scanning microscope showed no inhibitory effect of KRP-203 on islet neovascularization. Although allogeneic rejection was effectively inhibited by KRP-203 monotherapy (44%), combination therapy prevented rejection in most transplanted mice (83%).Conclusions.KRP-203 is a desirable immunomodulator for islet transplantation because of the preservation of the endocrine function and lack of interference with islet neovascularization. The combination of KRP-203 with low-dose sirolimus may be promising as a calcineurin-free regimen for islet allotransplantation.