Ninad Ingle

Trapping and two-photon fluorescence excitation of microscopic objects using ultrafast single-fiber optical tweezers

Analysis of trapped microscopic objects using fluorescence and Raman spectroscopy is gaining considerable interest. We report on the development of single fiber ultrafast optical tweezers and its use in simultaneous two-photon fluorescence (TPF) excitation of trapped fluorescent microscopic objects. Using this method, trapping depth of a few centimeters was achieved inside a colloidal sample with TPF from the trapped particle being visible to the naked eye. Owing to the propagation distance of the Bessel-like beam emerging from the axicon-fiber tip, a relatively longer streak of fluorescence was observed along the microsphere length. The cone angle of the axicon was engineered so as to provide better trapping stability and high axial confinement of TPF. Trapping of the floating objects led to stable fluorescence emission intensity over a long period of time, suitable for spectroscopic measurements. Furthermore, the stability of the fiber optic trapping was confirmed by holding and maneuvering the fiber by hand so as to move the trapped fluorescent particle in three dimensions. Apart from miniaturization capability into lab-on-a-chip microfluidic devices, the proposed noninvasive microaxicon tipped optical fiber can be used in multifunctional mode for in-depth trapping, rotation, sorting, and ablation, as well as for two-photon fluorescence excitation of a motile sample.

Detachment and reorientation of cells using near-infrared laser microbeam

Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.

Live atomic force microscopy imaging of laser microbeam assisted cellular microsurgery

Ultrafast laser microbeam is finding growing usage in causing highly localized damage to cellular structures. This has specifically enhanced efficiency of optoporation-injection of exogenous impermeable substances into the cell by transient pore formation. However, kinetics of laser microbeam induced pore formation and sealing of membrane has not been visualized at nanoscale resolution. Here, we report realization of live atomic force microscopy (AFM) imaging of ultrafast tunable Ti: Sapphire laser microbeam assisted cellular microsurgery. AFM imaging was carried out using Nanonics Multiview system in parallel to exposure of the laser beam. Red blood cells (RBCs) were chosen as cellular model for micro-surgery due to their smooth surface topography. The transparent nature of the Nanonics fiber-optic AFM cantilever allowed simultaneous bright field/phase contrast imaging of the RBC. Measurement of pore size by AFM revealed true pore size as a function of laser exposure duration in contrast to phase contrast imaging. Further, AFM imaging of live cells showed fine topography of sealed pores that could not be comprehended from conventional microscopy.

Development of a digital holographic microscopy system integrated with atomic force microscope

Atomic Force Microscope (AFM) imaging, due to the scanning method of recording, requires significant recording time for examination of wide sample area. In contrast, digital holographic microscopy (DHM), owing to the wide-field method, allows recording of the hologram in very fast rate which could be numerically analyzed to reveal surface of the sample with axial resolution at the nanometer scale. However, DHM yields quantitative phase properties of the sample, and therefore sensitive to changes in refractive index along with physical thickness. Therefore, to accurately determine the refractive index map, it is imperative to estimate the physical thickness map of the sample. This was achieved by AFM imaging. Further, since the transverse resolution of DHM is limited by diffraction limit, co-registration of AFM image provided higher transverse resolution at nanometer scale. The interference of the AFM probe was observed to be minimal during simultaneous AFM and DHM recording due to the transparent nature and bent configuration of the optical fiber based AFM cantilever. Integration of DHM and AFM led to realization of a powerful platform for nanoscale imaging. The integrated AFM-DHM system was built on an inverted fluorescence microscope to enable fluorescence imaging of the sample. The integrated system was employed to analyze fluorescent polystyrene microspheres, two-photon polymerized microstructures and red blood cells.

Development of a two-photon polymerization and optical tweezers microscope for fabrication and manipulation of microstructures

We report development of a two-photon polymerization (TPP) microscope, for micro-fabrication of microstructures, which is capable of optical manipulation by use of optical tweezers. The system is based on an inverted Nikon microscope with a tunable Ti: Sapphire femto-second (fs) laser coupled to the upper back port. While in modelocked condition, nanoparticles and wires were fabricated in photo-polymerizable synthetic materials using TPP. By axial positioning of the focused TPP laser beam, 1D-structures (for use as wave guide) were fabricated at desired height above the surface of the substrate. In the mode lock-OFF condition the same tunable laser microbeam was employed as optical tweezers to the hold the nanostructures and manipulate them even in highly viscous medium before immobilizing. Size of the TPP induced structure was found to depend on the fs laser intensity and exposure. Further, by shaping the fs laser beam to line pattern, linear 1D structures could be fabricated without scanning the beam or stage, which remain aligned along the line intensity profile due to anisotropic trapping force of the line tweezers in X and Y-directions. Use of optical tweezers with two-photon polymerization not only allowed in-situ corrective positioning of the polymerized structures, but also the integration of fluorescent microspheres (resonator/detector) with polymerized waveguide.

Femtosecond single optical fiber tweezers enabled two-photon fluorescence excitation of trapped microscopic objects

Analysis of trapped microscopic objects using fluorescence and Raman spectroscopy is gaining considerable interest. We report on the development of single fiber femto second optical tweezers and its use in two-photon fluorescence (TPF) excitation of trapped fluorescent particles. Trapping of the floating objects led to stable fluorescence emission intensity over a long period of time, suitable for spectroscopic measurements. Trapping depth of few cm was achieved inside colloidal sample with TPF from the trapped particle being visible to the naked eye. Furthermore, the fiber optic trapping was so stable that the trapped particle could be moved in 3D even by holding the fiber in hand and slow maneuvering of the same. Owing to the propagation distance of the Bessel-like beam emerging from the axicon-fiber tip, a relatively longer streak of fluorescence was observed along the microsphere length. The cone angle of axicon was engineered so as to provide better trapping stability and high axial confinement of TPF. The theoretical simulation of fiber optical microbeam profiles emerging from the axicon tip and trapping force estimations was found to be in good agreement with the experimentally observed stiffness and TPF patterns. Apart from miniaturization capability into lab-on- a-chip micro-fluidic devices, the proposed non-invasive micro axicon tipped optical fiber can be used in multifunctional mode for in-depth trapping, rotation, sorting and ablation as well as for two-photon fluorescence excitation of motile sample which will revolutionize biophysics and research in material science.

Laser induced detachment and re-orientation of cells

Re-orientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The cell gets detached after irradiation for few seconds, followed by vertical orientation. The detached cell was transported along axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by Gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. The cell could be brought back to the substrate by reducing the laser beam power using a polarizer or blocking the laser beam. Viability of the detached and manipulated cell was found not to be compromised as confirmed by PI fluorescence exclusion assay. The re-oriented cell was allowed to re-attach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for non-contact modification of cellular orientations that will enable study of inter-cellular interactions and design of engineered tissue.

Photothermal mediated transformation of carbon nanoparticles

Irradiating carbon nanoparticles (CNPs) with near-infrared laser beam leads to generation of heat, therefore it has potential to be used in many applications including the destruction of cancer cells. Though pulsed laser beams have been used earlier to transform shapes of metallic and semiconductor nanoparticles, changing shape of CNPs required intense electron beam irradiation. In this paper, we report significant size reduction of CNPs under continuous-wave (cw) near-infrared (NIR) laser beam micro-irradiation which was attributed to melting and vaporization or fragmentation of the carbon nanoparticles. Further, we show that the spherical shape of the CNPs can be transformed into ellipsoidal, by exposure to cw NIR laser microbeam irradiation for a few seconds. In-situ measurements using atomic force microscopy (AFM) reveal the shape and size changes of the CNPs upon laser micro-irradiation. Most importantly, cw NIR laser microbeam irradiation led to ultra-structural phase transformation of CNPs as detected via Raman spectroscopic imaging. While the graphitic CNPs could be changed to diamond-like carbon (DLC), no phase change in DLC nanoparticles was observed. These transformations did not require presence of any special chemical (catalyst, functionalization) or physical (pressure, temperature) arrangement. In-situ control of CNP-size, shape and ultra-structural properties opens new possibilities in multiple nanotechnology adventures.

In-Situ formation of microstructures near live cells using spatially structured near-infrared laser microbeam

Here, we report in situ formation of microstructures from the regular constituents of culture media near live cells using spatially-structured near infrared (NIR) laser beam. Irradiation with the continuous wave (cw) NIR laser microbeam for few seconds onto the regular cell culture media containing fetal bovine serum resulted in accumulation of dense material inside the media as evidenced by phase contrast microscopy. The time to form the phase dense material was found to depend on the laser beam power. Switching off the laser beam led to diffusion of phase dark material. However, the proteins could be stitched together by use of carbon nanoparticles and continuous wave (cw) Ti: Sapphire laser beam. Further, by use of spatially-structured beam profiles different structures near live cells could be formed. The microfabricated structure could be held by the Gravito-optical trap and repositioned by movement of the sample stage. Orientation of these microstructures was achieved by rotating the elliptical laser beam profile. Thus, multiple microstructures were formed and organized near live cells. This method would enable study of response of cells/axons to the immediate physical hindrance provided by such structure formation and also eliminate the biocompatibility requirement posed on artificial microstructure materials.

In-depth fiber optic two-photon polymerization and its applications in micromanipulation

Two photon polymerization (TPP) has enabled three-dimensional microfabrication with sub-diffraction limited spatial resolution. However, depth at which TPP could be achieved, has been limited due to the high numerical aperture microscope objective, used to focus the ultrafast laser beam. Here, we report fiber-optic two photon polymerization (FTP) for in-depth fabrication of microstructures from a photopolymerizable resin. A cleaved single mode optical fiber coupled with tunable femtosecond laser could achieve TPP, forming extended waveguide on the fiber itself. The length of the FTP tip was found to depend on the laser power and exposure duration. Microfabricated fiber tip using FTP was employed to deliver continuous wave laser beam on to polystyrene microspheres in order to transport and manipulate selected particles by scattering force and 2D trapping. Such microstructures formed by TPP on tip of the fiber will also enable puncture and micro-surgery of cellular structures. With use of a cleaved fiber or axicon tip, FTP structures were fabricated on curved surfaces at large depth. The required Power for FTP and the polymerization rate was faster while using an axicon tip optical fiber. This enabled fabrication of complex octopus-like microstructures.