Khyati Mohanty

Dynamics of Interaction of RBC with optical tweezers.

It has recently been shown that a red blood cell (RBC) can be used as optically driven motor. The mechanism for rotation is however not fully understood. While the dependence on osmolarity of the buffer led us to conclude that the osmolarity dependent changes in shape of the cell are responsible for the observed rotation, role of ion gradients and folding of RBC to a rod shape has been invoked by Dharmadhikari et al to explain their observations. In this paper we report results of studies undertaken to understand the dynamics of a RBC when it is optically tweezed. The results obtained support our earlier conjecture that osmolarity dependent changes in shape of the cell are responsible for the observed rotation.

Manipulation of mammalian cells using a single-fiber optical microbeam

The short working distance of microscope objectives has severely restricted the application of optical micromanipulation techniques at larger depths. We show the first use of fiber-optic tweezers toward controlled guidance of neuronal growth cones and stretching of neurons. Further, by mode locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes and thus allowing study of the subsequent response of neurons to localized injury. At high average powers, lysis of a three-dimensionally trapped cell was accomplished.

Organization of microscale objects using a microfabricated optical fiber

We demonstrate the use of a single fiber-optic axicon device for organization of microscopic objects using longitudinal optical binding. Further, by manipulating the shape of the fiber tip, part of the emanating light was made to undergo total internal reflection in the conical tip region, enabling near-field trapping. Near-field trapping resulted in trapping and self-organization of long chains of particles along azimuthal directions (in contrast to the axial direction, observed in the case of large tip cone angle far-field trapping).

In depth fiber optic trapping of low-index microscopic objects

We demonstrate that a focused beam through a microaxicon built on the tip of a single mode optical fiber can trap low-index objects at much larger depths as compared to the vortex beam tweezers generated using high numerical aperture microscope objectives. The measured transverse trapping force for low-index objects in Mie regime was found to depend on particle size and on distance of trapped objects from the fiber tip. While axial movement of trapped low-index objects was achieved by variation in trap beam power, transportation in three dimensions was achieved by maneuvering the fiber.

Fibre based cellular transfection

Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

Single fiber optical tweezers for manipulation of microscopic objects

Trapping of microscopic objects using fiber optical traps is gaining considerable interest since it has the potential to manipulate objects inside turbid medium such as tissue, thus removing the limitation of short working distance of the conventional optical tweezers based on high numerical aperture microscope objective. Here, we show that scattering force of an output beam from a single fiber can be reduced as compared to the axial gradient force when an axicon is built on the tip of the fiber, thus enabling single beam fiber-optic tweezers. Trapping of wide range of objects in size range of few hundreds of nanometers to tens of micrometers could thus be achieved. This fiber optic tweezers could be easily maneuvered in all three directions by moving the mechanical manipulator holding the axicon tip fiber. Further, chain of upto 40 particles could be trapped along the axial direction, which can be attributed as longitudinal optical binding where each trapped object acts as lens to trap subsequent object near its focal point. Apart from miniaturization capability, axicon tipped optical fiber can be used in multi-functional mode for cellular manipulation, as well as for two-photon fluorescence excitation for biomedical diagnosis.

RBCs under optical tweezers as cellular motors and rockers: microfluidic applications

Recently, we have reported self-rotation of normal red blood cells (RBC), suspended in hypertonic buffer, and trapped in unpolarized laser tweezers. Here, we report use of such an optically driven RBC-motor for microfluidic applications such as pumping/centrifugation of fluids. Since the speed of rotation of the RBC-motor was found to vary with the power of the trapping beam, the flow rate could be controlled by controlling the laser power. In polarized optical tweezers, preferential alignment of trapped RBC was observed. The aligned RBC (simulating a disk) in isotonic buffer, could be rotated in a controlled manner for use as a microfluidic valve by rotation of the plane of polarization of the trapping beam. The thickness of the discotic RBC could be changed by changing the osmolarity of the solution and thus the alignment torque on the RBC due to the polarization of the trapping beam could be varied. Further, in polarized tweezers, the RBCs in hypertonic buffer showed rocking motion while being in rotation. Here, the RBC rotated over a finite angular range, stopped for some time at a particular angle, and then started rotating till it was back to the aligned position and this cycle was found repetitive. This can be attributed to the fact that though the RBCs were found to experience an alignment torque to align with plane of polarization of the tweezers due to its form birefringence, it was smaller in magnitude as compared to the rotational torque due to its structural asymmetry in hypertonic solution. Changes in the laser power caused a transition from/to rocking to/from motor behavior of the RBC in a linearly polarized tweezers. By changing the direction of polarization caused by rotation of an external half wave plate, the stopping angle of rocking could be changed. Further, RBCs suspended in intermediate hypertonic buffer and trapped with polarized tweezers showed fluttering about the vertical plane.

Fiber optic trapping of low-refractive-index particles

Since the low index particles are repelled away from the highest intensity point, trapping them optically requires either a rotating Gaussian beam or optical vortex beams focused by a high numerical microscope objective. However, the short working distance of these microscope objectives puts a limit on the depth at which these particles can be manipulated. Here, we show that axicon like structure built on tip of a single mode optical fiber produces a focused beam that is able to trap low index particles. In fact, in addition to transverse trapping inside the dark conical region surrounded by high intensity ring, axial trapping is possible by the balance of scattering force against the buoyancy of the particles. The low-index particle system consisted of an emulsion of water droplets in acetophenone. When the fiber was kept horizontal, the low index spheres moved away along the beam and thus could be transported by influence of the scattering force. However in the vertical position (or at an angle) of the fiber, the particles could be trapped stably both in transverse and axial directions. Chain of such particles could also be trapped and transported together by translation of the fiber. Using escape force technique, transverse trapping force and thus efficiency for particle in Mie regime was measured. Details of these measurements and theory showed that trapping of Raleigh particle is possible with such axicon-tip fibers. This ability to manipulate low-index spheres inside complex condensed environments using such traps will throw new insights in the understanding of bubble-bubble and bubble-wall interactions, thus probing the physics behind sonoluminescence and exploring new applications in biology and medicine.

Single-fiber optical tweezers for cellular micro-manipulation

Optical Tweezers: First discovered in 1970, optical tweezers can now be generated and controlled.

Rotational behavior of erythrocytes in optical trap: revisited by confocal fluorescence microscopy

There has been considerable current interest in rotational behavior of red blood cells (RBC) in optical tweezers. However, the mechanism of rotation in polarized tweezers is still not well understood and there exists conflicts in the understanding of this phenomenon. Therefore, we re-examined the underlying phenomenon by use of confocal fluorescence microscopy. Under different osmolarities of the buffer, the three dimensionally reconstructed images showed that the trapped RBC maintains its discotic shape and is oriented in vertical direction. Using dual optical tweezers, the RBC could also be oriented three-dimensionally in a controlled manner. Since, no folding of the RBC was observed under optical trapping beam, the rotational mechanism based on optical birefringence caused by folding of RBC can be ruled out. The alignment of RBC with polarization of the tweezers beam can be attributed to its formbirefringence. We also present the mechanism for possible rotational behavior of RBC in circularly polarized beam.