Mouse Fibroblast Cell Adhesion Studied by Neutron Reflectometry

Abstract

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be approximately 180 A. The membrane region ( approximately 80 A thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

Analysis of biosurfaces by neutron reflectometry: From simple to complex interfaces

“…47 Neutrons are the perfect probe for the investigation of living tissue because they offer unparalleled resolution on the 1-1000 Å level where even the best microscopy techniques fail. Moreover, NR provides global information about the cellular monolayer averaged over the footprint size.…”

“…Cell monolayers were perfused with culture medium at controlled conditions to reach a laminar shear stress level of 1.5 Pa or left static, as previously described. 47 In all experimental data presented below, D 2 O based buffers were used as the subphase. From our experience, we know that the cells can survive up to 6 h in such conditions without visible changes in the cells' viability.…”

“…NR can be applied in the study of living tissue to better understand biological mechanisms such as cellular adhesion within the vascular system and the differences that may be specifically associated with brain tumor invasion. [45][46][47] …”

Neutron reflectometry in biological applications

“…We recently employed NR to examine live mouse fi broblast cells, which represent a signifi cant advancement towards employing NR to measure biological samples [7]. The fi broblast cells were adherent to a quartz substrate in a deuterated phosphate-buffered saline enOpen Access Neutron News authors can now choose to publish their articles open access with Taylor & Francis Open Select.…”

Tuning endothelial monolayer adhesion: a neutron reflectivity study

“…Neutrons probe matter in a nonperturbative manner with minimal energy transfer; furthermore, because neutrons interact with nuclei and not electrons, chemical bonding is minimally affected (7,20,34). Using neutron reflectivity, we developed a novel tool that probes confluent endothelial monolayer adhesion under dynamic conditions and with nanometer resolution for the first time.…”