During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we used a laser dissector system based on a sub-nanosecond pulsed UVA laser to inflict a partial damage to the axon of mouse hippocampal neurons. The use of such laser gives the possibility to deliver very low average power to the sample to be ablated. Therefore, the collateral damage due to temperature rise was reduced and for the first time, we were able to manipulate the neurite of cultured mouse neurons during the first days in vitro with sub-cellular precision. Force spectroscopy measurements were performed in parallel during and after the partial ablation of the neurite, by the use of a bead attached to the neurite membrane and held in an optical trap. The sub-piconewton and millisecond resolution of the force spectroscopy measurements allowed to quantify the damage inflicted to the process and to monitor the viscoelastic properties of the axonal membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term (24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.
Articles tagged with: Holography
Cultured neural networks represent a useful model in between single neurons and brain tissue for studying neural development, coding, pathology and regeneration. Neurons functionally reshape their interconnectivity not only in response to incoming activity from other cells, but also to external stimuli and changes of environmental conditions (Muotri and Gage. 2006). Thus, studying cell physiology, cell motility and interconnectivity by interacting with cells or their organelles and controlling the intercellular organization and enviroment.
Complementary optical technologies such as optical tweezers (OTs) and laser microdissectors (LMDs) are among the most versatile tools for performing such manipulation experiments with high spatio-temporal resolution under sterile conditions.
In contrast to most commercial OTs and LMDs, which are installed on inverted microscopes, the setup is mounted on an upright microscope. Nevertheless, both inverted and non-inverted optical pathways are optically accessible. This configuration allows the placing of specimen on transparent and non-transparent substrates alike without affecting the overall performance of the optical setup. The system is configured to work in combination with experimental techniques such as multichannel microelectrode array recording, patch clamp electrophysiology, and fluorescence imaging.
During morphogenesis, neuronal precursor cells migrate from the zone where they are born to their final destination, which, in some cases, is at a distance of several millimeters. After reaching their destination, neurons must establish appropriate synaptic connections by sending out from their soma projections called neurites. The motion of neurites is guided by growth cones located at their tips. Growth cones contain a variety of chemical and
mechanical receptors and sophisticated biochemical machinery that couples these receptors to the cytoskeleton. Extruding from the tip of the growth cone are highly motile structures called filopodia and lamellipodia that are used to explore and probe the environment. All these complex events, which are at the basis of neuronal development and differentiation, involve cell motility requiring a precise control of cellular and molecular motors.
We study, by optical tweezers and force spectroscopy, the dynamic of cytoskeletal elements in the growth cone, and how the growth cone navigate in a controlled mechano-chemical micro-enviroment.
Currently the two most commonly configurations of optical microscopy used in neuroscience laboratories are wide field illumination and laser scanning imaging. In wide field microscopy, the whole field of view is simultaneously illuminated, allowing fast image acquisition or fast repetitive stimulation, but preventing the application of complex spatial light patterns (fig. 1A). Differently, in laser scanning microscopy a diffraction limited laser spot is sequentially deflected in the field of view, allowing the selective illumination of portions of the sample that depend on the scanning trajectory. This configuration leads to an increase of the spatial but to a significant loss in the time resolution of the optical system. Both approaches thus have intrinsic limitations with respect to the degree of complexity with which spatio-temporal patterns of light can be projected onto the biological sample. Illumination with structured light represents an innovative alternative to overcome these limitations. In this experimental approach, the laser wavefront is engineered (or shaped) to simultaneously and selectively illuminate only specific regions of interest in a given field of view. This technique offers flexibility in the pattern of illumination that cannot be achieved with more traditional optical approaches and gives the opportunity of imaging/stimulating simultaneously multiple portions of a given cell or different cells within a neuronal network, or the possibility to manipulate simultaneously different object to controll the micro-enviroment around a cell.