Gregory J. Sonek

Evidence for localized cell heating induced by infrared optical tweezers

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.

Parametric study of the forces on microspheres held by optical tweezers

Optical-trapping forces exerted on polystyrene microspheres are predicted and measured as a function of sphere size, laser spot size, and laser beam polarization. Axial and transverse forces are in good and excellent agreement, respectively, with a ray-optics model when the sphere diameter is ≥ 10 µm. Results are compared with results from an electromagnetic model when the sphere size is ≤ 1 µm. Axial trapping performance is found to be optimum when the numerical aperture of the objective lens is as large as possible, and when the trapped sphere is located just below the chamber cover slip. Forces in the transverse direction are not as sensitive to parametric variations as are the axial forces. These results are important as a first-order approximation to the forces that can be applied either directly to biological objects or by means of microsphere handles attached to the biological specimen.

Laser trapping in cell biology

Optical traps offer the promise of being used as noninvasive micromanipulators for biological objects. An analytical model was developed that accurately describes the forces exerted on dielectric microspheres while in a single-beam gradient force optical trap. The model can be extended to the trapping of biological objects. The model predicts the existence of a stable trapping point and effective trapping range. A minimum trapping power of approximately 5 mW and an effective trapping range of 2.4 mu m were measured for 10- mu m-diameter dielectric microspheres and are in reasonable agreement with expected results. In cell biology, the optical trap was used to alter the movement of chromosomes within mitotic cells in vitro and to hold motile sperm cells. Results for the mitotic cells indicate that chromosome movement was initiated in the direction opposite to that of the applied force. >

Physiological monitoring of optically trapped cells: Assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source. An average temperature increase of < 0.1 +/- 0.30 degrees C/100 mW is measured for cells when held stationary with CW optical tweezers at powers of up to 400 mW. The same trapping conditions do not appear to alter DNA structure or cellular pH. In contrast, a pulsed 1064-nm laser trap (100-ns pulses at 40 microJ/pulse and average power of 40 mW) produced significant fluorescence spectral alterations in acridine orange, perhaps because of thermally induced DNA structural changes or laser-induced multiphoton processes. The techniques and results presented herein demonstrate the ability to perform in situ monitoring of cellular physiology during CW and pulsed laser trapping, and should prove useful in studying mechanisms by which optical tweezers and microbeams perturb metabolic function and cellular viability.

Radiation trapping forces on microspheres with optical tweezers

Axial trapping forces exerted on microspheres are predicted using a Gaussian beam electromagnetic field model and a ray‐optics model, and compared with experimental measurements. Ray‐optics predicts a maximum trapping efficiency Q= −0.14 for optically trapped polystyrene microspheres in water, compared to a measured value of −0.12 ± 0.014 for 10 μm diam microspheres. When the microspheres are composed of amorphous silica, the predicted ray‐optics Q decreases to −0.11, compared to a Q = −0.034 predicted by the electromagnetic field model, and a measured value of −0.012 ± 0.001 for 1 μm diam microspheres. These results indicate that the two models have applicability in two different size regimes, and thus, are complementary.

THE USE OF EXOGENOUS FLUORESCENT PROBES FOR TEMPERATURE MEASUREMENTS IN SINGLE LIVING CELLS

The fluorescent membrane probes 7‐nitrobenz‐2‐oxa‐1,3‐diazo1‐4‐y1 (NBD) and 6‐dodeca‐noy1‐2‐dimethylamino‐naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat‐induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane‐bound NBD‐conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature‐dependent Stokes shift as the membrane undergoes a gel‐to‐liquid‐crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady‐state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2°C) over a broad temperature range. Laurdans microenvironmental sensitivity permits better temperature resolution (0.1‐1°C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short‐pulse microirradiation on single cells.

Confinement and bistability in a tapered hemispherically lensed optical fiber trap

We describe the operation and performance of a dual fiber optical trap created using tapered lensed optical fibers pigtailed to 1300 nm laser diodes. Single‐mode fibers, having mode field diameters of ∼9.5 μm, and separated by up to 350 μm, are used to demonstrate dielectric particle confinement over two orders of magnitude in fiber trapping power ratio. Axial and transverse trap efficiencies, as well as the existence of bistable trapping positions, are predicted and experimentally confirmed. The use of fiber lenses results in the creation of an optical trap that provides strong transverse optical confinement and which is nearly insensitive to the fiber polarization state.

Large fanout optical interconnects using thick holographic gratings and substrate wave propagation

Substrate wave propagation and Bragg diffraction by multiplexed holographic gratings have been used to demonstrate a new 1-to-30 fanout optical interconnect having an overall diffraction efficiency of 87.8% at 514.5 nm and an individual channel efficiency of approximately 3.0 +/- 0.8%. The device configuration utilizes the large multiplexing capability of dichromated gelatin polymer films and substrate total internal reflection to realize large channel fanouts within the plane of a soda-lime glass substrate. A simplified theoretical formulation is presented to treat the corresponding three-dimensional holographic diffraction problem in the Bragg regime for slanted phase gratings. Results are compared with experimentally measured quantities for singly exposed phase gratings in different polarization conditions and incident angle orientations. The limitations of using multiplexed holograms in multiplanar substrate interconnection applications are also discussed.

Two-photon fluorescence excitation in continuous-wave infrared optical tweezers.

We report the observation of two-photon fluorescence excitation in a continuous-wave (cw) single-beam gradient force optical trap and demonstrate its use as an in situ probe to study the physiological state of an optically confined sample. In particular, a cw Nd:YAG (1064-nm) laser is used simultaneously to confine, and excite visible fluorescence from submicrometer regions of, cell specimens. Two-photon fluorescence emission spectra are presented for motile human sperm cells and immotile Chinese hamster ovary cells that have been labeled with nucleic acid (Propidium Iodide) and pH-sensitive (Snarf) fluorescent probes. The resulting spectra are correlated to light-induced changes in the physiological state experienced by the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers.

Five-channel polymer waveguide wavelength division demultiplexer for the near infrared

A five-channel wavelength division demultiplexer (WDDM) fabricated in polymer gelatin waveguides and operating over a 100-nm bandwidth centered at 770 nm in the near infrared is discussed. The device has a maximum diffraction efficiency of 80% at 730 nm, has a spectral bandwidth of 17+or-3 nm per channel, and effectively utilizes a portion of the large optical transparency bandwidth ( approximately 2400 nm) of the photo-lime gelatin polymer material at laser diode wavelengths. High-channel-density WDDM devices at longer infrared wavelengths should be possible.<<ETX>>