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.

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.

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.

Optical tweezers are an important tool for use as non-contact micromanipulators of biological cells and organisms. The ability to apply optical forces in the pN regime means that optical tweezers can also be used as transducers of the fundamental forces responsible for cell locomotion, cell adhesion, and intracellular transport. Herein, we describe our recent results in modeling and measuring the optical trapping forces that are applied to dielectric microsphere and biological sperm cell samples

Using a tunable titanium-sapphire laser, we have compared different wavelengths (from 700 to 840 nm) for their utility in optical trapping of chromosomes in mitotic rat kangaroo Potorous tridactylus (PtK2) cells.It was found that irradiation with a near-infrared light induces the sticking together of chromosome shoulders.The attached chromatids failed to separate, or separated with significant delay and formed a chromosome bridge during anaphase.Using this bridge (and induced c-mitosis) as a reference, we compared the action of different wavelengths (from 700 to 840 nm).Chromosomes were irradiated at metaphase and the cells were observed until the end of cytokinesis.Chromosomes were irradiated for different periods of time, using 130 mW of power at the objective focal plane.The biological responses observed after optical trapping were: (1 ) normal cell division, (2) formation of a temporary chromosome bridge, (3) formation of a permanent chromosome bridge, (4) complete blockage of chromosome separation (c-mitosis).The chromosomes were found to have a maximal sensitivity to 760-765 nm light and minimal sensitivity to 700 and 800-820 nm light.Cells with chromosomes irradiated for a long time, using wavelength 760-765 nm, generally were incapable of going through anaphase and remained in c-mitosis.We conclude that the optimal wavelengths for optical trapping are 700 and 800-820 nm.

An optical scalpel and optical tweezer have been combined to perform intracellular microsurgery and micromanipulation in vivo. When only laser microsurgery was performed on metaphase chromosomes, the dissected sister chromatid fragments drifted off to either the side of the spindle or completely off the spindle. At anaphase the fragments separated and the two arms generally moved to their respective daughter cells. When the chromosome arm was cut during anaphase A and B, the distal chromosome fragment separated from the rest of the chromosome and moved toward the pole, following the proximal chromosome fragment. Distal chromosome fragments laser-dissected during metaphase were held together throughout anaphase using the optical trap. Optical trapping of dissected chromosome fragments during anaphase A and B inhibited movement of the chromosome fragment to its pole. As a result, the trapped chromosome fragments were (1) incorporated into the opposite daughter cell, (2) lost in the cleavage furrow during cytokinesis, or (3) eventually incorporated into the correct daughter cell. These results indicate that optical traps are effective in holding laser-dissected chromosome fragments throughout mitosis. This new tool should be useful for studies on chromosome movement and cell genetics.

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wave-length of 1.06 μm. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.

We describe the use of the optical trapping technique to the study of micron-sized particles and cell samples, from which scattering, particle size, and optical parametric data can be derived. Its application to the study of phytoplanktonic cells and other biogenic particles is also presented. In comparison to conventional flow cytometric or volume scattering measurement systems, an optical trap utilizes the radiation forces, derived from a highly focused laser beam, to confine the particle under study to an optical potential well. The optical trap, therefore, functions simultaneously as both a non-contact micromanipulator and microforce transducer. In addition, forward angle light scattering measurements can be made while the cell sample is held by the focused laser beam. Forward light scattering measurements and calculations for optically trapped spherical test particles and mammalian cells, under low power (< 10 mW), are presented for the cases when the laser beam spot size (omega) o is approximately r, (lambda) /2n < (omega) o < r, and (omega) o approximately (lambda) /2n, respectively, where r is the cell radius and n is the refractive index of the surrounding water medium. Scattering data over the range from approximately 0 degree(s) to 45 degree(s) is shown to be a sensitive function of beam radius, particle size, and relative refractive index. The optical trapping technique should prove to be a powerful tool in the study of the optical properties of marine cells and organisms, and their dependence on external optical stimuli.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A method for quantitative characterization of port wine stain (PWS) is presented. Pulsed photothermal radiometry (PPTR) uses a non-invasive infrared radiometry system to measure changes in surface temperature induced by pulsed radiation. When a pulsed laser is used to irradiate a PWS, an initial temperature jump (T-jump) is seen due to the heating of the epidermis as a result of melanin absorption. Subsequently, heat generated in the subsurface blood vessels due to hemoglobin absorption is detected by PPTR as a delayed thermal wave as the heat diffuses toward the skin surface. The time delay and magnitude of the delayed PPTR signal indicate the depth and thickness of the PWS. In this report, we present an initial clinical study of PPTR measurements on PWS patients. Computer simulations of various classes of PWS illustrate how the PPTR signal depends on the concentration of epidermal melanin, and depth and thickness of the PWS. The goal of this research is to provide a means of characterizing PWS before initiating therapy, guiding laser dosimetry, and advising the patient as to the time course and efficacy of the planned protocol.

Various microsurgical procedures at the cellular and subcellular levels using laser non-touch techniques are presented and reviewed. In these procedures, the beams of light, varying in their wavelength (range: 14 ns to continuous wave), were directed via microscopes towards the target area. Micromanipulation of human spermatozoa with a laser-generated optical trap enabled the assessment of possible effects on sperm motility and measurements of the relative force generated by each single spermatozoon. Furthermore, the optical trap also provides a new approach to the measurements of intracellular forces without physically touching the cell or its organelles, and inducing chromosome movement during cell division is also possible. Laser beams in a specific configuration are able to induce minimal superficial damage to the zona pellucida of oocytes from various species. This manipulation is aimed at increasing the fertilization rate following insemination with low-quality spermatozoa. Another intracellular application of the laser beams is the destruction of extra pronuclei in polyspermic fertilized human oocytes. These procedures require special equipment which is not commonly available. However, simpler devices may be developed if the advantages of this novel technology are demonstrated.

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.

The precise removal of stratum corneum from cadaveric swine skin by a mid-infrared erbium:yttrium scandium gallium garnet laser (lambda = 2.79 microns; 250 microseconds pulse width) was assessed by electrical resistance measurements and documented by histology. The effects of stratum corneum removal by laser ablation and by adhesive tape-stripping on the in vitro penetration of 3H-hydrocortisone and 125I-gamma-interferon were determined. Excised swine skin was irradiated with laser (1 J/cm2; 31 mJ/pulse; 1 Hz; 2 mm spot diameter). For skin penetration studies, laser pulses were delivered to discrete 2-mm areas to ablate up to 12.6% of the total 3-cm2 stratum corneum diffusional area. Franz in vitro skin penetration chambers were used to measure the cumulative 48-h penetration of 3H-hydrocortisone and 125I-gamma-interferon in laser-treated and tape-stripped skin. Electrical resistance measurements and histologic studies demonstrated that 10-14 laser pulses at the above energy density were required to abolish skin resistance and selectively ablate stratum corneum without damage to adjacent dermal structures. Laser ablation of 12.6% of the surface area of stratum corneum produced a 2.8 and 2.1-times increase in permeability constant (kp) for 3H-hydrocortisone and 125I-gamma-interferon, respectively. These studies demonstrate that a pulsed mid-infrared laser can reliably and precisely remove the stratum corneum, facilitating penetration of large molecules such as 125I-gamma-interferon that cannot penetrate intact skin. This new technique may be useful for basic and clinical investigation of skin barrier properties.

To study the potential use of optical forces to manipulate chromosome movement, we have used a Nd:YAG laser at a wavelength of 1.06 microns focused into a phase contrast microscope. Metaphase and anaphase chromosomes were exposed while being monitored by video microscopy. The results indicated that when optical forces were applied to late-moving metaphase chromosomes on the side closest to the nearest spindle pole, the trapped chromosomes initiated movement to the metaphase plate. The chromosome velocities were two to eight times the normal rate depending on the chromosome size, geometry, and trapping site. At the initiation of anaphase, a pair of chromatids could be held by the optical trap and kept motionless throughout anaphase while the other pairs of chromatids separated and moved to opposite spindle poles. As a result, the trapped chromosome either was incorporated into one of the daughter cells or was lost in the cleavage furrow, or the two chromatids eventually separated and moved to their respective daughter cells. If the trap was removed at the beginning of anaphase B, the chromosome moved back to the poles. Our experiments demonstrate that the laser-induced optical force trap is a potential new technique to study noninvasively the mitotic spindle of living cells.

The development of the single beam gradient force optical trap has made it possible to manipulate cells solely by laser light. A continuous wave Nd:YAG (1.06 microns) laser beam was directed into a conventional microscope and focused onto the viewing plane by the objective lens. The laser beam power at which human sperm were released from the trap was measured and correlated to the sperm's linear velocity before trapping. The mean trapping power readings for slow, medium, and fast motile sperm were 57, 73, and 84 mW, respectively. The analysis of measurements over the total population demonstrated that zig-zag motile sperm had significantly higher mean power readings when compared with straight motile sperm with similar mean linear velocities. In two cases, specimens required significantly less trapping power when the measurements were repeated 24 hours later.

Optical traps offer the promise of being used as noninvasive micromanipulators for biological objects. We have developed an analytical model 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 an effective trapping range. A minimum trapping power of approximately 5 mW and an effective trapping range of 2.4-mu-m have been measured for 10-mu-m diameter dielectric microspheres, and are in reasonable agreement with expected results. In cell biology, we have used the optical trap 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. Chromosome velocities as high as 48-mu-m/min were observed, and are 24 times faster than normal during cell division. In sperm trapping experiments, mean sperm velocities were unchanged following short (< 30 s) exposures in the trap, while longer exposures resulted in a decrease in mean sperm velocities. Given its noninvasive nature, optical traps should prove to be useful tools in the study of biological processes.

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam In order to perform laser Induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 jim, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogenpumped dye laser (366 rim), was collinear with the trapping beam. The maximum transverse force exerted on a moving cell (NS- 1) by the optical trap was determined by measuring the velocity at which the cell fell out of the trap. For a laser power of 55 mW and a cell diameter from 9 to 20 rim, the drag force was calculated using Stokes' law to be in the range from 1.42 0.22 x 106 to 1.13 0.25 x 10-6 dynes. For the fusions, the transverse force needed to capture two NS- 1 cells and to bring them into close contact, was > 200 mW. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of 1 &J/pulse In the object plane. This method of laser Induced cell fusion should provide Increased selectivity and efficiency in generating viable hybrid cells.

The force generated by the radiation pressure of a low power laser beam induces an optical trap which may be used to manipulate sperm. We studied the effect of the optical trap on sperm motility. A Nd:YAG laser beam was coupled to a conventional microscope and focused into the viewing plane by the objective lens. Sperm were caught in the trap and manipulated by a joy stick controlled motorized stage. After different exposure periods, the velocity and patterns were analysed by a computerized image processor. There were minor changes in sperm velocity when exposed to the trap for 30 seconds or less. A gradual decrease in the mean linear velocity was observed after 45 seconds of exposure. This optical micromanipulator may also be useful for studying the force generated by a single spermatozoa and evaluating the influence of drugs on motility.

A laser-induced optical force trap was used to alter the movement of chromosomes in mitotic cells in vitro. The trap was produced by using a 1.06-microns neodymium YAG (yttrium/aluminum garnet) laser focused through a phase-contrast microscope. The trap was applied to one side of centrophilic chromosomes off the mitotic spindle and to late-moving chromosomes on the mitotic spindle. In both situations, chromosome movement was initiated in the direction opposite to that of the applied force. When the force was applied, chromosomes moved at velocities 10-20 times normal. These studies verify and extend the feasibility of using this new technique to study factors that influence organelle motility.