PHOTONIC-NEUROSURGERY LAB

Difato Francesco

Neuroscience research has recently taken advantage of optical approaches to record, modulate and manipulate the physiological activity of neurons. Studies on the nature of light-matter interactions have paved the way for emerging fields in biophysics and neuroscience, while advances in optical systems have provided minimally invasive approaches for studying the structure and function of living cells. Precise engineering of light-matter interactions allows contact-free manipulation of biological samples, such as the use of optical tweezers and laser dissector for precise and reproducible “optical surgery”. At the same time, molecular engineering has provided a new generation of optical probes to detect and modulate the activity of living cells.

My work has focused on the development of optical systems for the precise and controlled spatio-temporal manipulation of biological samples, and on the integration of optical setups with electrophysiological recording devices to study the central nervous system at various levels of complexity.

UOPTYoungaward  Taylor

  The Koh Young Best Paper Award 2012

      

Integration of Optical Manipulation and Electrophysiological Tools to Modulate and Record Activity in Neural Networks.  F. Difato, L. Schibalsky, F. Benfenati, and A. Blau. International Journal of Optomechatronics, 2011, 5(3), 191-216.

Corresponding Author: Difato F.

Photonic-neurosurgery lab @ JOVE

click on the image below to watch the published video article

JoVE

Articles tagged with: optical tweezers

Electrophysiology

on Sunday, 18 March 2012. Posted in Home

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.

Force spectroscopy

on Saturday, 17 March 2012. Posted in Home

Force spectroscopy

Complex biological phenomena, such as cell differentiation and locomotion, require long range tracking capabilities with nanometer resolution over an extended period, to resolve molecular processes on the cellular scale.
Living cells face mechanical forces which are converted into biochemical signals and integrated into the cellular responses (mechanotransduction). Therefore, the development of new techniques for exerting mechanical stresses on cells and observe their responses is crucial to clarify the molecule‐and cell‐level structures that may participate in mechanotransduction.
Forces can be exerted on a cell by a variety of experimental techniques. If the force is in the right range of magnitude it is capable of eliciting a biological response from the cell. The magnitude of force that can be produced by a single myosin molecule8, consistent with the theory that active cellular  contraction can induce cell signalling. Therefore, all this would suggest that the critical values of force exerted on a single molecule fall within the pN range. Interestingly to notice, this is also the range of forces exerted on the particle in an optical
trap by the radiation pressure of light. This relatively young technique provides the nonmechanical manipulation of the biological particles such as virus, living cells and subcellular organelles. Moreover, optical tweezers are recognized single-molecule technique to resolve forces and motion on the molecular scale.
Optical Tweezers are now being used in the investigation of an increasing number of biochemical and biophysical processes, from the mechanical properties of biological polymers to the multitude of molecular machines that drive the internal dynamics of the cell.
The combination of chemical and mechanical stimulation is useful and relevant in cell biology, since cells test the extracellular matrix rigidity during their differentiation.
Cells need to organize their internal space and interact with their environment. The network filaments, i.e., the cytoskeleton, represent connections to distribute tensile forces through the cytoplasm to nucleus as a signaling pathway. Transmembrane receptor, as integrins, function as mechanoreceptors and provide a preferred path to transfer mechanical forces across cell surface. During motion, cells organize integrins distribution in clusters at special complexes called focal adhesions. Forces transmitted to the cytoskeleton from these sites produce a stress in associated cytoskeleton molecules which can be converted in gene expression, protein recruitment at local sites and dynamic cytoskeleton architecture modulation. Thus cell shape and stability is the effect of a mechanical force balance between cytoskeleton traction forces and extracellular substrate local rigidity. Optical tweezers can be utilized to measure and analyze the force operated by cells in exploratory motion and therefore to  understand how cell operate mechanic transduction. With optical microscopy, it is possible to follow the motion of the cell, but force
measurements permit to quantify how many molecular motors are involved in a movement and therefore to quantify how much the molecular machinery of the cell is recruited in such action. This means that thanks to force measurement, it is possible to distinguish random motion from well organized shift of the cell.
It has been observed that filopodia, which explore the environment by rapidly moving in all directions, modulate their activity changing the duration of the collision with a bead, in response to different stiffness of the load. This could represent sensing of the obstacle force and is in agreement with measurements on single  microtubule catastrophic time modulation. Lamellipodia, which follow the pathway analyzed by filopodia, showed a more complex
behavior in response to the obstacle: sometimes, they entirely retracted, other times they moved around it to progress forward or they removed the obstacle by lifting it and giving it back. Lamellipodia have a more differentiated structure and are thought to exert a force with variable directions in space. Therefore, multi‐tweezers measurements have been applied to understand their overall organization and to measure complex forces exerted during growth cone motion.
This kind of measurements can be used to understand the  response of the cells to molecular cues or mechanic‐chemical stimuli localized in the trapping volume, or to understand the mechanisms and which molecules are involved in different steps of motor planning. Moreover,  when the system is working in force-clamp condition, can be exploited  to study the role of statically and dynamically applied forces in neuronal differentiation.

Growth cone

on Saturday, 17 March 2012. Posted in Home

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.

Holography

on Saturday, 17 March 2012. Posted in Home

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.

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 (fig. 1B). 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 (fig. 1C). 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.

Structured light

Force spectroscopy

Growth cone navigation

Electrophysiology

Laser Dissection

Axon Regeneration