The spatial connectivity of neural circuits and the various activity patterns they exert is what forms the brain function. How these patterns link to a certain perception or a behavior is a key question in neuroscience. Recording the activity of neural circuits while manipulating arbitrary neurons leads to answering this question. That is why acquiring a fast and reliable method of stimulation and imaging a population of neurons at a single cell resolution is of great importance. Owing to the recent advancements in calcium imaging and optogenetics, tens to hundreds of neurons in a living system can be imaged and manipulated optically. We describe the adaptation of a multi-point optical method that can be used to address the specific challenges faced in the in-vivo study of neuronal networks in the cerebral cortex. One specific challenge in the cerebral cortex is that the information flows perpendicular to the surface. Therefore, addressing multiple points in a three dimensional space simultaneously is of great interest. Using a liquid crystal spatial light modulator, the wavefront of the input laser beam is modified to produce multiple focal points at different depths of the sample for true multipoint two-photon excitation.
The challenge to wide application of optical tweezers in biological micromanipulation is the photodamage caused by high-intensity laser exposure to the manipulated living systems. While direct exposure to infrared lasers is less likely to kill cells, it can affect cell behavior and signaling. Pushing cells with optically trapped objects has been introduced as a less invasive alternative, but the technique includes some exposure of the biological object to parts of the optical tweezer beam. To keep the cells farther away from the laser, we introduce an indirect pushing-based technique for noninvasive manipulation of sensitive cells. We compare how cells respond to three manipulation approaches: direct manipulation, pushing, and indirect pushing. We find that indirect manipulation techniques lessen the impact of manipulation on cell behavior. Cell survival increases, as does the ability of cells to maintain shape and wiggle. Our experiments also demonstrate that indirect pushing allows cell–cell contacts to be formed in a controllable way, while retaining the ability of cells to change shape and move.
Optical tweezers have emerged as a promising technique for manipulating biological objects. Instead of direct laser exposure, more often than not, optically-trapped beads are attached to the ends or boundaries of the objects for translation, rotation, and stretching. This is referred to as indirect optical manipulation. In this paper, we utilize the concept of robotic gripping to explain the different experimental setups which are commonly used for indirect manipulation of cells, nucleic acids, and motor proteins. We also give an overview of the kind of biological insights provided by this technique. We conclude by highlighting the trends across the experimental studies, and discuss challenges and promising directions in this domain of active current research.
The shapes of unilamellar lipid vesicles are driven out of equilibrium by direct forcing with holographic optical tweezers. Vesicles have been studied extensively due to their relevance as a model for the membrane of cells as well as their potential practical uses e.g. for drug delivery or chemical confinement. We use multipoint laser tweezers formed by a spatial light modulator (holographic optical tweezers) to apply forces to such vesicles in several points simultaneously. To apply forces we utilize an index of refraction difference between the fluid inside the vesicle and the external fluid. Since this higher index of refraction material is fluid, the vesicle shape can changes in response to the optical forces. This shape change reveals the mechanical properties of vesicles subject to multiple stresses. We find that the surface forces on the membrane are localized near the points of forcing. Restoring forces from lipid tethers are used to estimate the total applied optical forces, which are below the pN level. The relaxation of deformations can be decomposed into its Fourier modes. The relaxation of all observable modes can be described well by a third order Landau equation. Ellipsoidal deformations relax more slowly than higher order deformation modes.
The aim of our work is to develop new optical tools to quantify parameters that may enter into models of cell motion in response to chemical gradients (chemotaxis). Dictyostelium discoidium is a well-known model organism for studying chemotaxis. We have developed a technique for manipulating Dictyostelium cells directly using a holographic laser tweezer array. Using this technique we have perturbed crawling Dictyostelium cells by changing their direction of motion. After tens of seconds, the cells generate protrusions perpendicular to the rotated polarization as they reorient in the direction of the local cAMP gradient. Here we describe how such micromanipulation may be used to test proposed biochemical pathways and their connection to mechanical deformations.
Vesicles are phospholipid bilayers that form a surface enclosing a volume of water or solution. They are of importance as model systems to study cells, as well as having practical applications such as containers for performing nanochemistry and facilitating drug delivery. Their properties have been studied for decades. Using a holographic laser tweezer array (LTA), which converts a single laser beam into many laser tweezer points, we stretch the vesicles in controlled ways from several points at once, measuring each force applied. Here, we present data on shape deformations of simple, spherical vesicles and on membrane fracture.