Cell Viability and DNA Denaturation Measurements by Two-Photon Fluorescence Excitation in CW Al:GaAs Diode Laser Optical Traps.

Cell viability and DNA denaturation are measured through two-photon fluorescence excitation using a single diode laser beam as the trapping and exciting source simultaneously. Two-photon fluorescence emission spectra are presented for CHO cells and T lymphocytes loaded with various fluorescent probes. This single beam method is demonstrated to be a safe tool to monitor the viability of optically trapped cells, even under intense 809 nm diode laser illumination (∼106 W/cm2). The dynamics of cellular necrosis is monitored by adding ethanol to the cell suspension during trapping. Thermal damage to heat-treated samples is assessed by recording shifts in the emission spectra from trapped cells loaded with the nucleic acid probe, acridine orange. © 1999 Society of Photo-Optical Instrumentation Engineers.

Effect of pentoxifylline on the intrinsic swimming forces of human sperm assessed by optical tweezers.

It is still controversial whether in vitro exposure of sperm to pentoxifylline increases sperm motility and force, which is defined as the product of velocity by beat frequency of the tail. Laser optical tweezers have been successfully used in the past to evaluate sperm force in basal conditions. The aim of this prospective study was to determine whether exposure of human sperm to pentoxifylline has any effect on sperm intrinsic forces. Twelve healthy subjects undergoing routine semen analysis were enrolled in the study. Ten exhibited normal semen parameters, 2 exhibited asthenozoospermia. Each semen specimen was washed and, after swim-up, resuspended in human tubal fluid (HTF) and divided into 2 aliquots. One aliquot was incubated with pentoxifylline for 30 minutes (final concentration = 3.6 mM); the second aliquot, without pentoxifylline, served as a control. After 30 minutes the pentoxifylline-treated aliquot was divided into 2 portions, 1 of which was washed to remove the pentoxifylline, the other was left in prolonged coincubation with the chemical. The main outcome was the measurement of sperm intrinsic force in milliwatts (mW), which was assessed by means of a noninvasive infrared laser optical trap created by a continuous wave, 1064-nm Nd:YAG laser beam directed in an inverted microscope. Exposure of sperm to pentoxifylline consistently increased sperm relative escape force from the laser optical trap. The increase ranged from 33% to 154% over baseline force compared with controls. The average absolute increase in sperm force rose from 37 mW to 79 mW (P < .05). Specimens with sperm having an initial low relative escape force gained the highest relative increase. The effect of pentoxifylline on sperm force, already apparent after 5 minutes, reached a peak at 30 minutes and persisted for up to 3 hours in sperm that were left in coincubation and in those on which the pentoxifylline had been washed off. In conclusion, pentoxifylline significantly increases sperm intrinsic relative force in normozoospermic and asthenozoospermic samples. This experiment confirms that optical tweezers can provide an accurate determination of sperm force in in vitro conditions. Clinical data must now establish whether a documented increase in sperm force is an important parameter for assessing sperm fertilizing capacity.

The use of exogenous fluorescent probes for temperature measurements in single living cells.

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 6-dodecanoyl-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 degrees C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1 degree 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.

Autofluorescence spectroscopy of optically trapped cells.

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 beta-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.

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.

Low power response of all-optical crossbar networks in quantum well heterostructures.

A new device configuration has been examined for its potential as a compact, all-optical modulator, operating at low input powers (~ 3 mW). This device utilizes the large optical nonlinearities of Al(x)Ga(1-x)As/GaAs multiple quantum well heterostructures to modulate a low power-guided wave test beam with an orthogonally propagating control beam. This configuration has the advantage of maximizing the packing density of nonlinear modulators having interaction lengths of only ~ 5 microm. Several modes of device operation are possible, including thermal modulation, optical limiting, bistable switching, and multiple-input logic. We present experimental results for the throughput of single and multiple waveguide beams, as well as for the operation of this device as a waveguide modulator array. A theoretical model is compared with the experimental results for input pulse lengths ranging from ~ 300 ns to 1 s and input powers up to ~ 3 mW. Several optical nonlinearities are experimentally observed, including an electronic nonlinearity and two thermal effects, which are classified as either local or global in origin, with time constants of ~ 30 ns, ~ 4 micros, and ~ 40 ms, respectively. For an optimized waveguide geometry, the intrinsic optical device described herein should be suitable for use in systems requiring fast, high-density waveguide arrays for optical computing and serial/parallel data-processing applications.

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.

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.

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.

Wavelength-division multiplexing and demultiplexing on locally sensitized single-mode polymer microstructure waveguides.

A four-channel wavelength-division-(de)multiplexing [WD(D)M] device, operating over optical wavelengths of 543.0 to 632.8 nm, has been successfully fabricated on newly developed locally sensitized polymer (photo-lime gelatin) microstructure waveguides (PMSW's). The WD(D)M device exhibits a cross talk of less than -40 dB between adjacent channels and a diffraction efficiency of better than 50%. The angular and spectral bandwidths for the device are ~0.2-0.4 degrees and ~4-10 nm, respectively. Such sensitivities can significantly increase the WD(D)M channel density for optical interconnect architectures. Since the PMSW device can be constructed on a variety of substrates, including insulators, semiconductors, conductors, and ceramics, the demultiplexing technique that we report is suitable for use in a variety of optical-computing, signal-processing, and communication applications.

Multiphoton fluorescence excitation in continuous-wave infrared optical traps.

The multiphoton fluorescence excitation observed in acontinuous-wave (cw) single-beam gradient force optical trap(optical tweezers) is reported for latex beads labeled with ayellow-green fluorescent dye (BODIPY). The fluorescenceemission spectra of the yellow-green beads trapped and excited by thesame 1064-nm laser light are identical to the spectra excited by the365-nm UV light. The influence of the numerical aperture of theobjective on the slope of the log-log power-dependence has beendemonstrated for BODIPY-Oil solution under cw and pulsed-laserconditions. The possibility that three-photon excitation processoccurs is discussed within the context of a dog-bone saturationmodel. Other possibilities for the observed fluorescence excitationhave been discussed.

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.

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.

Directed movement of chromosome arms and fragments in mitotic newt lung cells using optical scissors and optical tweezers.

A pulsed-laser microbeam at 532 nm wavelength (optical scissors) and a laser-induced optical trap at 1064 nm wavelength (optical tweezers) have been successively combined to dissect and manipulate chromosomes in live newt lung epithelial cells. These preliminary experimental results demonstrated that chromosome fragments dissected by laser microbeam surgery, regardless of their size, could be easily pulled or rotated by optical forces when positioned at the periphery of the mitotic spindle. In addition, chromosome arms which were not subjected to laser microsurgery also could be moved with the optical tweezers at the spindle periphery. In our previous study on rat kangaroo kidney cells (PTK2), this degree of facilty in manipulating chromosome movement was not possible, most likely due to the close proximity of the intermediate filament "cage" to the spindle. It is concluded herein that optical scissors and tweezers can be used in combination to study the interaction of chromosomes with the mitotic spindle in cells where the peripheral regions of the spindle are unobstructed by intermediate filaments. This can be performed on newt cells, where the diameter of the cage can be substantially larger than the diameter of the spindle.