Yagang Liu

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.

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.

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.

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.

AUTOFLUORESCENCE SPECTROSCOPY OF OPTICALLY TRAPPED CELLS

Abstract— Cellular autofluorescence spectra were monitored in a single‐beam gradient force optical trap (“optical tweezers”) in order to probe the physiological effects of near infrared and UVA (320–400 nm) microirradiation. Prior to trapping, Chinese hamster ovary cells exhibited weak UVA‐excited autofluorescence with maxima at 455 nm characteristic of β‐nicotinamide adenine dinucleotide (phosphate) emission. No strong effect of a 1064 nm NIR microbeam on fluorescence intensity and spectral characteristics was found during trapping, even for power densities up to 70 MW/cm2 and radiant exposures of 100 GJ/cm2. In contrast to the 1064 nm trap, a 760 nm trapping beam caused a two‐fold autofluorescence increase within 5 min (about 20 GJ/cm2). Exposure to 365 nm UVA (1 W/cm2) during 1064 nm trapping significantly altered cellular autofluorescence, causing, within 10 min, a five‐fold increase and a 6 nm red shift versus initial levels. We conclude that 1064 nm microbeams can be applied for an extended period without producing autofluorescence changes characteristic of alterations in the cellular redox state. However, 760 nm effects may occur via a two‐photon absorption mechanism, which, in a manner similar to UVA exposure, alters the redox balance and places the cell in a state of oxidative stress.

In situ microparticle analysis of marine phytoplankton cells with infrared laser-based optical tweezers

We describe the application of infrared optical tweezers to the in situ microparticle analysis of marine phytoplankton cells. A Nd:YAG laser (λ= 1064 nm) trap is used to confine and manipulate single Nannochloris and Synechococcus cells in an enriched seawater medium while spectral fluorescence and Lorenz-Mie backscatter signals are simultaneously acquired under a variety of excitation and trapping conditions. Variations in the measured fluorescence intensities of chlorophyll a (Chl a) and phycoerythrin pigments in phytoplankton cells are observed. These variations are related, in part, to basic intrasample variability, but they also indicate that increasing ultraviolet-exposure time and infrared trapping power may have short-term effects on cellular physiology that are related to Chl a photobleaching and laser-induced heating, respectively. The use of optical tweezers to study the factors that affect marine cell physiology and the processes of absorption, scattering, and attenuation by individual cells, organisms, and particulate matter that contribute to optical closure on a microscopic scale are also described.

Optical switch based on thermally-activated dye-doped biomolecular thin films

A new optical switch has been developed, based on laser-induced heating and the temperature-dependent fluorescence emission properties of biomolecular thin films comprised of the phospholipid 15OPC. For films doped with the Laurdan dye probe, a 200 mW CW Nd:YAG laser (1060 nm) was used to heat submicron thick films by /spl sim/5/spl deg/C, and induce switching between two phase states of the bilayer, at the peak emission wavelengths of 440 nm and 490 nm, with rise and fall times of 50 ms and 275 ms, respectively. The thermal switching times are shown to be dependent on the initial device setpoint temperature, and the thermal sensitivity of the film in the phase transition region.<<ETX>>

Beam magnification and the efficiency of optical trapping with 790-nm AlGaAs laser diodes

The efficiency of dielectric particle confinement with 790 nm AlGaAs laser diode optical traps is investigated as a function of beam magnification and polarization state. When an anamorphic prism pair is used to correct for diode beam ellipticity, trapping efficiencies of nearly 0.37 are achieved with a magnification factor of 3/spl times/, laser powers of 4-18 mW, and an overfilled microscope objective entrance aperture. Results are compared for diodes having small (8 /spl mu/m) and large (57 /spl mu/m) astigmatisms, but comparable far-field divergence angles.<<ETX>>

Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap (laser tweezers)

We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into an inverted confocal laser scanning microscope. Fluorescence of cells in the single- beam gradient force optical trap was excited with a 488 nm microbeam (laser scanning microscopy) or with 365 nm radiation from a high- pressure mercury lamp. Modifications to NADH-attributed autofluorescence and Rhodamine- and Propidium Iodide-attributed xenofluorescence indicate a significant cell-damaging effect of 760 nm trapping beams. 760 nm effects produce a biological response comparable to UVA-induced oxidative stress and appear to be a consequence to two-photon absorption.

Image processing and trapping of microscopic objects using a phase conjugate Michelson interferometer

We report the feasibility study of a versatile optical configuration consisting of a phase conjugate Michelson interferometer in conjunction with microscopic imaging optics for image processing and trapping of microscopic objects. Our test samples include phase gratings, amplitude gratings (i.e., Ronchi rulings), polystyrene microspheres, and biological samples such as liposomes and yeast cells. We have experimentally demonstrated (1) the novelty filtering feature which enhances the image of moving phase objects by suppressing the stationary background, (2) contrast reversal which is useful for the imaging of light absorbing (or scattering) particles, (3) the aberration correction capability of the system to enhance the image quality of microscopic objects embedded in or otherwise distorted by aberrators, and (4) optical trapping of polystyrene microspheres. The potential of using this technique for the manipulation and diagnosis of biological cells and tissues is discussed.