Anatomy of the fetal membranes: insights from spinning disk confocal microscopy.

PURPOSE: The fetal membranes are essential for the maintenance of pregnancy, and their integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. METHODS: In this proof of principle study, we collected fetal membranes from women undergoing vaginal delivery or cesarean section. Small membrane sections were then fixed, stained for nucleic acids, actin, and collagen using fluorescent probes, and subsequently imaged in three dimensions using a spinning disk confocal microscope. RESULTS: Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro) fractures could be identified in several samples. CONCLUSION: The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro) fractures and cellular jamming correlate with the timing of membrane rupture and the mode of delivery. In future measurements, this method could be combined with mechanical probing techniques to compare optical and mechanical sample information.

Collagen networks determine viscoelastic properties of connective tissues yet do not hinder diffusion of the aqueous solvent.

Collagen accounts for the major extracellular matrix (ECM) component in many tissues and provides mechanical support for cells. Magnetic Resonance (MR) Imaging, MR based diffusion measurements and MR Elastography (MRE) are considered sensitive to the microstructure of tissues including collagen networks of the ECM. However, little is known whether water diffusion interacts with viscoelastic properties of tissues. This study combines highfield MR based diffusion measurements, novel compact tabletop MRE and confocal microscopy in collagen networks of different cross-linking states (untreated collagen gels versus additional treatment with glutaraldehyde). The consistency of bulk rheology and MRE within a wide dynamic range is demonstrated in heparin gels, a viscoelastic standard for MRE. Additional crosslinking of collagen led to an 8-fold increased storage modulus, a 4-fold increased loss modulus and a significantly decreased power law exponent, describing multi-relaxational behavior, corresponding to a pronounced transition from viscous-soft to elastic-rigid properties. Collagen network changes were not detectable by MR based diffusion measurements and microscopy which are sensitive to the micrometer scale. The MRE-measured shear modulus is sensitive to collagen fiber interactions which take place on the intrafiber level such as fiber stiffness. The insensitivity of MR based diffusion measurements to collagen hydrogels of different cross-linking states alludes that congeneric collagen structures in connective tissues do not hinder extracellular diffusive water transport. Furthermore, the glutaraldehyde induced rigorous changes in viscoelastic properties indicate that intrafibrillar dissipation is the dominant mode of viscous dissipation in collagen-dominated connective tissue.

The two faces of enhanced stroma: Stroma acts as a tumor promoter and a steric obstacle.

In addition to genetic, morphological and biochemical alterations in cells, a key feature of the malignant progression of cancer is the stroma, including cancer cell motility as well as the emergence of metastases. Our current knowledge with regard to the biophysically driven experimental approaches of cancer progression indicates that mechanical aberrations are major contributors to the malignant progression of cancer. In particular, the mechanical probing of the stroma is of great interest. However, the impact of the tumor stroma on cellular motility, and hence the metastatic cascade leading to the malignant progression of cancer, is controversial as there are two different and opposing effects within the stroma. On the one hand, the stroma can promote and enhance the proliferation, survival and migration of cancer cells through mechanotransduction processes evoked by fiber alignment as a result of increased stroma rigidity. This enables all types of cancer to overcome restrictive biological capabilities. On the other hand, as a result of its structural constraints, the stroma acts as a steric obstacle for cancer cell motility in dense three-dimensional extracellular matrices, when the pore size is smaller than the cell's nucleus. The mechanical properties of the stroma, such as the tissue matrix stiffness and the entire architectural network of the stroma, are the major players in providing the optimal environment for cancer cell migration. Thus, biophysical methods determining the mechanical properties of the stroma, such as magnetic resonance elastography, are critical for the diagnosis and prediction of early cancer stages. Fibrogenesis and cancer are tightly connected, as there is an elevated risk of cancer on cystic fibrosis or, subsequently, cirrhosis. This also applies to the subsequent metastatic process.

Simultaneous manipulation and detection of living cell membrane dynamics.

We report a novel optical-tweezers-based method to study the membrane motion at the leading edge of biological cells with nanometer spatial and microsecond temporal resolution. A diffraction-limited laser spot was positioned at the leading edge of a cell, and the forward scattered light was imaged on a quadrant photodiode that served as a position sensitive device. The universality of this technique is demonstrated with different cell types. We investigated the membrane motion at the leading edge of red blood cells in detail and showed that this technique can achieve simultaneous manipulation and detection of cellular edge dynamics with unprecedented precision.