Two-photon polymerization (2PP) has provided the field of cell biology with the opportunity to fabricate precisely designed microscaffolds for a wide range of studies, from mechanobiology to in vitro disease modelling. However, a multitude of commercial and in-house developed photosensitive materials employed in 2PP suffers from high auto-fluorescence in multiple regions of the spectrum. In the context of in vitro cell biological studies, this is a major problem since one of the main methods of characterization is fluorescence microscopy of immuno-stained cells. This undesired auto-fluorescence of microscaffolds affects the efficiency of such an analysis as it often overlaps with fluorescent signals of stained cells rendering them indistinguishable from the scaffolds. Here, we propose two effective solutions to suppress this auto-fluorescence and compare them to determine the superiority of one over the other: photo-bleaching with a powerful UV point source and auto-fluorescence quenching via Sudan Black B (SBB). The materials used in this study were all commercially available, namely IP-L, IP-Dip, IP-S, and IP-PDMS. Bleaching was shown to be 61.7–92.5% effective in reducing auto-fluorescence depending on the material. On the other hand, SBB was shown to be 33–95.4% effective. The worst result in presence of SBB (33%) was in combination with IP-PDMS since the adsorption of the material on IP-PDMS was not sufficient to fully quench the auto-fluorescence. However, auto-fluorescence reduction was significantly enhanced when activating the IP-PDMS structures with oxygen plasma for 30 s. Moreover, we performed a cell culture assay using a human neuroblastoma cell line (SH-SY5Y) to prove the effectiveness of both methods in immunofluorescence characterization. SBB presented a lower performance in the study especially in presence of 2PP-fabricated microchannels and microcages, within which the differentiated SH-SY5Y cells migrated and extended their axon-like processes, since the SBB obstructed the fluorescence of the stained cells. Therefore, we concluded that photo-bleaching is the optimal way of auto-fluorescence suppression. In summary, this study provides a systematic comparison to answer one of the most pressing issues in the field of 2PP applied to cell biology and paves the way to a more efficient immunofluorescence characterization of cells cultured within engineered in vitro microenvironments.

Atherosclerotic plaque rupture in carotid arteries is a major cause of cerebrovascular events. Plaque rupture is the mechanical failure of the heterogeneous fibrous plaque tissue. Local characterization of the tissue's failure properties and the collagen architecture are of great importance to have insights in plaque rupture for clinical event prevention. Previous studies were limited to average rupture properties and global structural characterization, and did not provide the necessary local information. In this study, we assessed the local collagen architecture and failure properties of fibrous plaque tissue, by analyzing 30 tissue strips from 18 carotid plaques. Our study framework entailed second harmonic generation imaging for local collagen orientation and dispersion, and uniaxial tensile testing and digital image correlation for local tissue mechanics. The results showed that 87% of the imaged locations had collagen orientation close to the circumferential direction (0°) of the artery, and substantial dispersion locally. All regions combined, median [Q1:Q3] of the predominant angle measurements was -2° [-16°:16°]. The stretch ratio measurements clearly demonstrated a nonuniform stretch ratio distribution in the tissue under uniaxial loading. The rupture initiation regions had significantly higher stretch ratios (1.26 [1.15-1.40]) than the tissue average stretch ratio (1.11 [1.10-1.16]). No significant difference in collagen direction and dispersion was identified between the rupture regions and the rest of the tissue. The presented study forms an initial step towards gaining better insights into the characterization of local structural and mechanical fingerprints of fibrous plaque tissue in order to aid improved assessment of plaque rupture risk. Statement of significance: Plaque rupture risk assessment, critical to prevent cardiovascular events, requires knowledge on local failure properties and structure of collagenous plaque tissue. Our current knowledge is unfortunately limited to tissue's overall ultimate failure properties with scarce information on collagen architecture. In this study, local failure properties and collagen architecture of fibrous plaque tissue were obtained. We found predominant circumferential alignment of collagen fibers with substantial local dispersion. The tissue showed nonuniform stretch distribution under uniaxial tensile loading, with high stretches at rupture spots. This study highlights the significance of local mechanical and structural assessment for better insights into plaque rupture and the potential use of local stretches as risk marker for plaque rupture for patient-specific clinical applications.

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture. The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.

A functional vascular system is a prerequisite for bone repair as disturbed angiogenesis often causes non-union. Paracrine factors released from human bone marrow derived mesenchymal stromal cells (BMSCs) have angiogenic effects on endothelial cells. However, whether these paracrine factors participate in blood flow dynamics within bone capillaries remains poorly understood. Here, we used two different microfluidic designs to investigate critical steps during angiogenesis and found pronounced effects of endothelial cell proliferation as well as chemotactic and mechanotactic migration induced by BMSC conditioned medium (CM). The application of BMSC-CM in dynamic cultures demonstrates that bioactive factors in combination with fluidic flow-induced biomechanical signals significantly enhanced endothelial cell migration. Transcriptional analyses of endothelial cells demonstrate the induction of a unique gene expression profile related to tricarboxylic acid cycle and energy metabolism by the combination of BMSC-CM factors and shear stress, which opens an interesting avenue to explore during fracture healing. Our results stress the importance of in vivo - like microenvironments simultaneously including biochemical, biomechanical and oxygen levels when investigating key events during vessel repair. Statement of significance: Our results demonstrate the importance of recapitulating in vivo - like microenvironments when investigating key events during vessel repair. Endothelial cells exhibit enhanced angiogenesis characteristics when simultaneous exposing them to hMSC-CM, mechanical forces and biochemical signals simultaneously. The improved angiogenesis may not only result from the direct effect of growth factors, but also by reprogramming of endothelial cell metabolism. Moreover, with this model we demonstrated a synergistic impact of mechanical forces and biochemical factors on endothelial cell behavior and the expression of genes involved in the TCA cycle and energy metabolism, which opens an interesting new avenue to stimulate angiogenesis during fracture healing.

Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high-contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry. Journal compilation