1.1.

Theoretical Basis for Single-Beam Optical Trapping

Optical manipulation is a term we use to encompass the application of optical forces to move, guide, and trap (in 2-D and 3-D) mesoscopic objects. Optical tweezers—single-beam 3-D trapping—is the method we describe here in great detail, given its importance for the field of biophotonics. Optical tweezers rely on a highly inhomogeneous spatial field distribution of the beam. This causes a small but significant optical force (approximately a piconewton) to act upon a dielectric or biological particle (of higher refractive index than its surroundings) near the focus and is the case we deal with here. This attracts the particle toward the highest intensity part of the field through what is commonly termed the gradient (dipole) force. A stable equilibrium is attained where any opposing force originating from light scattering becomes equal in magnitude. Consequently, such a particle can be trapped in a stable equilibrium when it is very close to the beam focus. For further details of the theoretical basis of optical trapping, we refer the reader elsewhere.¹⁹

Notably, any thermal forces can be detrimental compared to the optical forces. Such thermal forces typically originate from temperature gradients in the medium surrounding the particles caused by the absorption of radiation, creating an effect known as photophoresis.²³ Thus, optical trapping is typically restricted to the use of relatively transparent media and particles where any thermal effects are negligible—indeed, in the context of biophotonics, any residual heating can adversely affect cell viability and should be avoided. We shall discuss this later in the context of applications of trapping to cell biology.

The ratio between the dimension of the trapped particles (of radius $r$ ) and the wavelength of the trapping laser source (denoted by $\lambda$ ) determines how we mathematically treat the light-particle interaction. First, if we consider a dielectric particle whose radius is much smaller than the optical wavelength, the particle may be considered as an oscillating dipole, and the appropriate mathematical formulation to be used is the Rayleigh limit.^{13, 24, 25} Second, for cases where the particle size significantly exceeds the trapping laser wavelength, the geometric optics or ray optics regime suffices to describe the phenomena of optical forces that result from refraction and reflection.^{26, 27} We will look at both the Rayleigh and ray optics limits in a little more detail.

1.2.

Rayleigh Regime

To implement an optical trap entails the use of a highly intense and strongly focused beam. The trap occurs from a balance between the gradient force pulling the particle of a higher refractive index than its surroundings to the most intense region of the optical field (i.e., the beam focus), and the repulsive scattering force, which pushes the particle away from the beam focus. If we consider the dipole approximation, then the force on a particle is given by the Lorentz force

The total Lorentz force, experienced by a particle in a medium of refractive index $n_m$ , naturally includes both a gradient and scattering force. If we consider a linear response to the (plane-wave) electric field and that $\mathbf{p}=\alpha \mathbf{E}$ , where $\alpha$ is the polarizability, then the total optical force may be written as^{13, 24, 25}

Optical trapping in the Rayleigh regime has been demonstrated in numerous experiments with dielectric and metal microparticles with sizes ranging from a few tens of nanometers upward.^{28, 29, 30, 31} It is interesting to comment on the exact role in optical trapping of the polarizability $\alpha$ and refractive index because these are issues of particular interest when considering metallic nanoparticles. In any material, the polarizability depends on the electronic properties and the mutability of the charge distribution under the influence of the electromagnetic field. Typically, the index of refraction is an effective medium concept based on a homogenization of the microscopic polarizability over the particle in question. This assumes that the constituent molecules or atoms of the material experience the equivalent environment that indeed is the same as the bulk counterpart.

For dielectric nanoparticles, this approach is valid because the local polarizability originates from bound electrons. Generally, the gradient force is proportional to the particle volume and, for this reason, it is very difficult to trap inert dielectric nanometer-sized objects. Metallic particles, though, differ in several respects, as per unit volume they possess a much larger inherent polarizability. To model the optical forces exerted on metal nanoparticles accurately, dispersion needs to be considered. Furthermore, the behavior of the conduction electrons alters when these electrons are in close proximity to the metal surface. If the particle dimensions are smaller than the corresponding skin depth, then the incident field interacts with the whole particle volume, resulting in a linear dependence between force and volume, resulting in the particle being essentially transparent. However, for particle sizes greater than the skin depth, the field interacts only with a thin outer layer and the force varies as the square of the diameter.^{31, 32, 33, 34, 35} Such considerations are of importance for the biomedical community because metallic nanoparticles have come to prominence in a number of studies for their ability to enhance of Raman signals, as photothermal agents in the selective destruction of cancer cells, as highly sensitive molecular diagnostic indicators,³⁶ as well their ability to act as handles using optical forces.²⁹

1.3.

Ray Optics Regime

Optical tweezers are also able to trap larger particles, on the micron scale. At this scale, the particle is sufficiently large for the propagation of light through the trapped sphere to be determined through the principles of simple ray optics. Calculations based on the reflection and refraction of light on a model sphere may be used to determine the optical forces. Figure 1 shows a qualitative representation of a dielectric particle trapped by a single optical beam.²⁶ We may decompose the entire beam into individual rays, each with an appropriate intensity, direction, and polarization, propagating in straight lines within a medium of refractive index $n$ . The individual rays can change direction when each reflects or refracts, and may change polarization at dielectric interfaces in a manner determined by the Fresnel equations. The refraction or reflection of a light ray at the particle-medium interface results in a transfer of momentum from the trapping laser to the particle.

Fig. 1

Qualitative geometrical view of trapping, applicable to large spherical particles. A selected pair of rays demonstrate that the refraction from the trapping beam results in force attracting the particle toward the beam focus. For simplicity, we ignore the reflections because they do not play an important role in the final force balance. For a more detailed explanation, the reader is referred to Ref. 13, 26. (a) Shows the lateral restoring force upon the sphere towards the beam focus. (b) Shows the axial restoring force acting upon a sphere that is axially displaced downstream from the focal plane. Were the bead to be located upstream of the focal plane, it can be simply shown that the restoring force would point downward. (c) Outside the scope of geometrical optics, for Rayleigh particles it is the particle’s polarizability, and not its transparency, that leads to trapping. The particle is aligned to the center of the trap because the electric field component of the light generates an induced dipole in the sphere, which will align in the highest region of the field gradient (in the static field approximation).

From this description, we can calculate the forces due to individual rays of power $P$ and the ray incident angle on the surface of the sphere surface. The quantities $R$ and $T$ denote the Fresnel reflection and transmission coefficients of the surface, and we find that the total force exerted on a particle is the sum of the contributions due to the reflected rays of power $PR$ , and an infinite number of emergent refracted rays, of successively decreasing power, $P{T}^2{R}^m$ , where $m=0,1,2,\dots$ , leading to the overall force as²⁷

1.4.

Mie and Lorenz–Mie Regime

The Rayleigh and Mie (ray optics) regimes are important, but the majority of experiments employ trapped particles whose sizes are actually comparable to the trapping laser wavelength. In this case, classical electrodynamics is required and the net optical force exerted on an arbitrary object may be calculated by the Maxwell stress tensor $\stackrel{\mathrm{⃡}}{\mathbf{T}}(\mathbf{r},t)$ approach³⁷

Detailed calculations of this approach can be found in work by Barton³⁸ and Barton ³⁹ The stress tensor can be calculated for a sphere with the help of Mie theory, considering all fields inside and outside of the sphere to be continuous at the surface. An alternative approach to the Maxwell stress tensor has been described by Rohrbach⁴⁰ and Rohrbach and Stelzer.⁴¹ One advantage of this approach is that the particles can take on an arbitrary shape and the interaction can be determined for arbitrary modes of the electromagnetic field.

Having an appreciation now of the theoretical platforms used to explain the optical forces and confinement of mesoscopic particles, we briefly comment on how such optical gradients are created, in practice, to produce single-beam optical tweezers. In the majority of systems, high numerical aperture microscope objectives with high magnification are used. Typically, a numerical aperture of $>1.2$ is used and, in turn, this produces the necessary strong lateral and axial gradients of the light field, yielding strong gradient forces essential for 3-D optical trapping. The choice of laser is also important and will be discussed later in this paper. For full flexibility in maneuvering the trapped object, we need to effectively steer the beam across the sample plane without it ever drifting away from the back “entrance” aperture of our objective lens, and this is achieved using optical conjugates. A more detailed description of the assembly and implementation of single-beam optical tweezers can be found elsewhere.^{42, 43} It is also possible to develop a homebuilt system by adapting commercially available microscopes.⁴⁴ We will now discuss why optical traps have been so important and how they have been used in biophotonics.

3.1.

Molecular Motors

Our understanding of the workings of both linear motors, such as kinesin, myosin, and dynein, and rotary motors, such as bacterial flagella, have without a doubt been improved by the use of optical traps. They have provided the means for the analysis of molecular motion, step sizes, forces, and crucially how these mechanical movements are related to the underlying biochemistry. Some of the first investigations into mechanical enzymes and molecular motors with optical tweezers were performed by various laboratories in the late 1980s and early 1990s. Prior to this time, molecular motor analysis was limited to bulk biochemical data and the passive tracking of beads by video microscopy.

Molecular motors can either be processive or nonprocessive in nature. Examples of the former include DNA and RNA polymerase, kinesin, and myosin V, and examples of the latter include myosin II.⁷⁷ The key difference is the number of steps along the bound polymer the motor takes; processive motors bind to a polymer chain and take many steps, and nonprocessive motors bind and take only approximately one step before dissociating.

One of the most prominent examples of studies in this era is that of kinesin,⁴⁵ which are microtubule associated mechanical enzymes responsible for anterograde vesicle transportation⁷⁸ and meiotic and mitotic spindle organization.^{79, 80} Kinesin is a homodimeric molecule with a coiled stalk linked to two head domains. The hydrolysis of adenosine triphosphate (ATP) drives the movement of the two head domains along a microtubule (toward its plus end) in an asymmetric “hand-over-hand” motion.⁸¹ It is processive, moving $\sim 100$ steps before detaching.⁸²

For processive motors such as kinesin, the single-bead optical tweezers assay is appropriate. Such an assay was demonstrated by Svoboda,⁸³ where a microsphere coated with kinesin was placed onto microtubule tracks that were bound to a coverslip. The authors then integrated a dual-beam interferometer into an optical tweezers setup to analyze the beads movements. Prior to entering the objective, a Wollaston prism was used to split the laser light into two orthogonally polarized beams, which were then focused onto the specimen as diffraction-limited overlaid probe regions. The microsphere-bound kinesin, when entering or moving through the probe region, introduced a relative phase shift between the two beams which, after recombination via another Wollaston prism, manifested itself as ellipticity. This ellipticity was then measured by passing the beam through a quarter waveplate (to circularly polarize the light), a polarizing beamsplitter, and finally two photodetectors and a normalizing differential amplifier. The net result was a detection sensitivity of $<1\mathrm{nm}$ , and after calibration, force measurements of the molecular motor could be made.

A number of key points relating biophysical movements to biochemical activity have been gleaned from the above study and subsequent work. Kinesin moves with $\sim 8\mathrm{nm}$ step sizes,^{83, 84} which corresponds to the molecular spacing of tubulin dimers. Intriguingly, in reduced ATP conditions, the molecular motion after each such step stops, awaiting the addition of new energy in the form of ATP molecules from the surrounding buffer. Low concentrations of ATP cause single steps that are still $\sim 8\mathrm{nm}$ ; therefore, the hydrolysis of one ATP molecule is directly related to a single step.^{81, 83, 85, 86} Analysis of the timing of the kinesin movements implied that it remains completely bound during its activity, hence, confirming the “hand-over-hand” hypothesis⁸³ (which was later shown to be asymmetric in nature⁸¹). Speeds⁸⁷ of up to $800\mathrm{nm}∕\mathrm{s}$ and forces of $5–7\mathrm{pN}$ for single kinesin molecules were measured.^{84, 87, 88} There are still many unanswered questions about the exact mechanism and dynamics of kinesin, and we refer elsewhere for a more in-depth discussion of many of the current issues.⁸¹

Myosin is another molecular motor, which consists of a long tail and two heads. Enzymatic digestion of myosin can divide it into smaller units, heavy meromyosin (HMM), which has a truncated tail but a fully functioning double head, and subfragment 1 (S1), which has a truncated tail and a single head. Its collective action along actin monomers drives muscle contraction, and in a sense, it can be considered as a nanoactuator. To explore the dynamic actin-myosin system, a different experimental geometry was required from the previous kinesin studies. This is because nonprocessive motors such as myosin only bind briefly to their actin substrate before detaching. To measure this molecular motion, Finer ⁴⁷ developed a novel assay in which an actin molecule was held between two beads and a third bead functionalized with myosin was brought into contact with the actin. Forces on the beads were then measured using a calibrated optical tweezers system. When the actin-myosin made contact, ATP in the buffer medium was hydrolyzed, resulting in the motion (a single “kick”) of actin. One of the two trapped particles was imaged onto a quadrant photodiode to allow monitoring of these “kicks” over extended periods of time. Step sizes of $11\mathrm{nm}$ and forces of $3–6\mathrm{pN}$ were measured.^{47, 89}

Significant insight into the mechanics of myosin movement along actin monomers was gained by the subsequent experiments of Molloy ^{90, 91} The authors demonstrated that single-myosin S1 heads acted as independent force generators when moving along actin filaments. Conceptually, unlike kinesin, which moves forward in an asymmetric “hand-over-hand” motion, a both double-headed HMM and a single-headed S1 myosin fragment use a single “hand” or head to move along actin by repeatedly binding and releasing. Like the Finer study before it, the authors held an actin molecule suspended between two trapped $1.1\mu \mathrm{m}$ diameter beads, and the filament was then placed near either an HMM (doubled-headed) or S1 (single-headed) functionalized bead that was attached to a coverslip. The position of one of the beads was accurately measured using a quadrant photodiode. Another observation by the same study is highlighted in Fig. 2 . Here, the authors moved the two actin beads laterally in a sinusoidal motion and plotted length and force against time [Fig. 2] and force against position [Fig. 2]. This showed that the myosin S1 head attachment occurred at regular positions (arrows) $\sim 40\mathrm{nm}$ in distance from each other, corresponding to the periodic $38.5\mathrm{nm}$ twist in the actin structure.

Fig. 2

The movement of a single S1 myosin head along an actin filament. See text for details. (This image was published in Ref. 91. © Elsevier.)

Optical tweezers have also helped elucidate the biophysical characteristics of the related molecular motor, dynein. Dynein is a microtubule-associated molecular motor that drives the motion of the axoneme, the core of eukaryotic flagella or cilia.⁹² Like kinesin, dynein “walks” along microtubules and, in so doing, transports a variety of cytoplasmic vesicular cargo, but a key difference is that kinesin walks toward the plus end of microtubules and dynein walks toward the minus end. It is also involved with spindle assembly, retrograde vesicle transportation,⁴⁶ and chromosome segregation. Only a handful of articles have been written on the subject dynein force measurement by optical tweezers. Shingyoji optically tweezed singlet microtubules via bead handles and overlaid them perpendicular to doublet microtubules; the authors subsequently measured the forces acting on the singlet arm by the connecting dynein molecules.⁹³ To activate the dynein, they used UV flash photolysis to uncage ATP and, from these studies, they established that dynein exhibited a peak force of $\sim 6\mathrm{pN}$ . Hirakawa ⁹⁴ later showed that processive movements of dynein are dependent on the ATP concentration; only low concentrations of ATP allow processive movement to occur. More recent studies demonstrated $8\mathrm{nm}$ step sizes for dynein, but these step sizes can vary between 4 and $24\mathrm{nm}$ , depending on the load it is carrying.⁹⁵

Other molecular motors have also been characterized with optical tweezers. During infection of bacteria, the viral bacteriophage $\varphi 29$ packages DNA into precursor capsids via a molecular motor called a porter motor. Rickgauer ⁴⁸ were able to measure the force and rate with which the motor assembly packaged dsDNA into the viral prohead (Fig. 3 ). Their experiment consisted of two tweezers, one of which was controlled by an acousto-optic deflector, and the other was static, and using this setup, the deflection forces were measured on the static trap using a position sensing detector. Essentially, this is the tug-of-war configuration discussed in Sec. 3 (Table 1). Using an improved protocol from the original 2001 study,⁵⁹ the Rickgauer group attached a portal motor complex to one sphere, DNA to another, tweezed them together to initiate packaging, and monitored the resulting forces. The authors then showed that this motor has an average power of $\sim 700\mathrm{zW}$ with a $30\mathrm{pN}$ load and a $65\text{bp}∕\mathrm{s}$ velocity. From these studies, there is now a growing interest in the use of genetically engineered variants of the $\varphi 29$ motor, adapted to be lipid membrane bound, for a variety of biomedical and nanotechnological applications.⁹⁶ It also furthers our understanding of bacteriophage, which has been proposed one day to be used therapeutically against multidrug resistant bacteria.⁹⁷

3.2.

Studies on Nucleic Acids

3.2.3.

Measuring the transporting of DNA through a nanopore

It is possible to measure directly the force acted on a single DNA molecule as it is threaded through a nanopore due to electrophoresis. In an experiment described by Sischka,¹⁰⁰ a single linear dsDNA molecule was tagged with a bead handle on one end and placed near the entrance of an $80\mathrm{nm}$ diameter pore previously milled on $20\mathrm{nm}$ thick ${\mathrm{Si}}_3{\mathrm{N}}_4$ membrane (Fig. 5 ). On the application of a $100\mathrm{mV}$ potential difference across the membrane, the charged dsDNA molecule threaded itself through the membrane pore and, as it did so, the force acted on the bead handle was measured to be $16\pm 2.5\mathrm{pN}$ . With this technique, the position of the DNA molecule within the pore-to-nanometer resolution is possible.¹⁰¹ The charge of individual DNA molecules can also be measured, which previously had to be taken from ensemble studies using electrophoresis.¹⁰² Finally, there is excellent potential with this experimentation to perform single-molecule DNA sequencing, haplotyping, and to investigate DNA-protein interactions.¹⁰³

Fig. 5

(a) An $80\mathrm{nm}$ diameter nanopore was milled into a $20\mathrm{nm}$ thick ${\mathrm{Si}}_3{\mathrm{N}}_4$ membrane, around which a custom fluid chamber was built. A microsphere functionalized with $\lambda \text{-}\mathrm{DNA}$ (48,202 base pairs, or $16.4\mu \mathrm{m}$ in length) was placed in the bottom chamber, and an electrical potential difference of $\sim 25\mathrm{mV}$ was applied across the membrane, resulting an almost instantaneous threading of the DNA through the pore. (b) Force measurements on the DNA during the threading were taken using optical tweezers. Each additional DNA molecule entering the pore generates an individual force step. (c) Active pulling of the DNA molecule out of the nanopore. (Reprinted with permission from Ref. 100. © 2008, American Institute of Physics.)

3.2.4.

Measuring the translation of RNA into protein

Recent studies with optical traps have also observed, in real time, the first steps of a ribosome during translation. Wen ⁵⁰ followed the movement of a single ribosome along an mRNA molecule as the ribosome translated the mRNA into protein along the length of a hairpin loop (Fig. 6 ). This type of single-molecule analysis is of particular value because it is impossible to synchronize ribosomal activity, rendering ensemble studies difficult. To achieve this analysis, the authors optically held a single mRNA molecule using bead handles at the terminal ends held under a constant tension force of $20\mathrm{pN}$ . As the ribosome translated along the length of the mRNA molecule through the hairpin loop, it unwound the loop. This manifested itself in the mRNA molecule extending. The extension as a function of time revealed, in detail, the amount of time a ribosome spends translating each codon $(<0.1\mathrm{s})$ , and additionally demonstrated that each translocation step was equivalent to the distance of three base pairs, or one codon.

Fig. 6

Configuration used to observe the step size and timing of a single ribosome during translation. An RNA sequence encoding for a hairpin loop was linked to two beads via complementary DNA linker sequences and other chemistries. Ribosome activity was stalled by the omission of aminoacyl-tRNA until the experiment was ready to commence. The RNA sequence was held under a constant tension of 20 pN between a micropipette and an optical tweezers, which was used to measure the forces acted up on the RNA as the hairpin loop unwound due to the ribosomal movement. (Reprinted by permission from Macmillan Publishers Ltd., Nature, Ref. 50, © 2008.)

3.3.

Protein Folding

Correct protein folding is of fundamental importance to the viability of a cell, and incorrect folding can results in a variety of disease states. The amino acid sequence of a protein determines its configuration. For example, the hiding of hydrophobic groups minimizes the free energy from the protein-water interaction and is generally the dominant mechanism that drives protein folding. Electrostatic interactions during the folding process, such as salt bridges, hydrogen bonds, or disulfide bonds, limit the number of folding choices as folding ensues.^{104, 105} AFM studies have been used to study protein folding, but their much higher spring constants make it difficult to directly observe folding. However, optical tweezers have now been used successfully to study directly this folding process. In 2005, Cecconi ⁵¹ analyzed the unfolding and refolding of a small 155 residue protein called RNase H (Fig. 7 ). This protein was tethered to two bead handles using two $500\mathrm{bp}$ dsDNA linkers. Force measurements on one of the beads indicated an intermediate folding state between the native and the unfolded state. As such, this technique can be used to further our understanding of protein-folding dynamics.

Fig. 7

Single molecule investigation of protein denaturation and refolding. A protein molecule, RNAase H, is attached via cysteine residues to DNA linkers, each of which were attached to two microspheres. Note that the blue region of the protein is the central, stable core region. One of the microspheres was held by capillary suction, and the other by optical tweezers, which were used to generate the force-extension curve on the right. This curve shows the transition of the protein between native (N) to unfolded (U) state during stretching (red line) and relaxation (blue line), as well as a transition between an unfolded (U) and intermediate (I) state at lower forces. Inset: multiple stretching/relaxation curves. The yellow line is a control curve for the DNA linkers. (From Ref. 51. Reprinted with permission from AAAS.)

4.1.

Viability of Optically Manipulated Cells

It is of note that for any optical trapping experiment with cells, ensuring their viability and continued biological function is a crucial facet to ensure meaningful results. Cell survival during optical manipulation depends on the wavelength, power, the duration of manipulation, the presence of oxygen, and whether the cell has been prestressed.

With regard to eukaryotic cells, one of the most reliable indicators of viability, cloning efficiency, was explored by Liang ¹¹⁰ to investigate laser related injury (Fig. 8 ). Using Chinese hamster ovary (CHO) cells, laser-trapping wavelengths ranging from $700\text{to}1064\mathrm{nm}$ , and doses ranging from $1\min$ at $88\mathrm{mW}\text{to}3\min$ at $176\mathrm{mW}$ , they found that $990\mathrm{nm}$ was the optimum wavelength for tweezing. In contrast, tweezing at $760\mathrm{nm}$ resulted in a 100% loss of viability after as little as $1\min$ of exposure to the trapping beam. A separate study using electron microscopy, investigated the response of cells to tweezing for many minutes at a $980\mathrm{nm}$ wavelength and low tweezing powers. Primary Ambystoma tigrinum Tiger Salamander neurones showed normal mitochondria, cytoskeleton, cytoplasm, and nucleoplasm.¹¹¹

Fig. 8

The viability of CHO cells in single beam gradient optical tweezers at $176\mathrm{mW}$ , assessed by colony formation, as a function of the time and wavelength they are held within the beam. Note that these are quite high powers; a lower $88\mathrm{mW}$ exposure at $990\mathrm{nm}$ for $5\min$ could result in 100% viability. (Reprinted from Ref. 110. © 1996, with permission from Elsevier.)

Prokaryotic cells show a remarkably similar response in viability to different tweezing wavelengths. A study by Neuman explored the viability of Escherichia coli, and found that a wavelength of $970\mathrm{nm}$ provided optimum viability.¹¹² At this wavelength, cells could be tweezed for $5\min$ at $88\mathrm{mW}$ without loss of viability. The authors also demonstrated that the loss of viability is exacerbated greatly by the presence of oxygen. Cells tweezed in anaerobic conditions or in the presence of oxygen-scavenging molecules had greatly increased viabilities. They propose that the damage is a one-photon process that may involve singlet oxygen. In another bacterial study using much lower tweezing powers, Rasmussen ¹¹³ used the maintenance of intracellular pH as an indicator of cell survival during optical tweezing using a $1064\mathrm{nm}$ emitting laser and compared four different bacterial species, E. coli, L. monocytogenes, L. inocua, and B. subtilis. At $6\mathrm{mW}$ , under aerobic conditions, all these species with the exception of B. subtilis remained viable for $1\mathrm{h}$ or more; under anaerobic conditions, the B. inocua strain only survived for $10\min$ . Three main points were evident from this study: (i) different bacterial species demonstrate different tolerances to optical tweezers, (ii) intraspecies variations also exist, with some subpopulations of cells within a species being more naturally resistant to tweezing than others, and (iii) aerobic conditions can actually prevent subsequent loss of viability induced by tweezing (especially in L. inocua). Although this latter point seems to be in contradiction to the Neuman study, the experimental conditions were very different (including an eightfold difference in tweezing power and the species of bacteria used to draw this conclusion). It was proposed by the authors that oxidatively stressing cells can potentially confer protection from the subsequent stress of tweezing,¹¹³ although it could also be the case that hypoxia-induced stress made the cells more sensitive to tweezing.

The idea of prestressing a cell in order to prevent loss of viability during tweezing was also highlighted in a recent study with human breast adenocarcinoma (MCF-7) cells. When cells were preirradiated with a $632.8\mathrm{nm}$ source, a significantly greater number of cells survived subsequent optical tweezing at $1064\mathrm{nm}$ .¹¹⁴ It has been proposed that the mechanism for preirradiation protection may involve activation of the cytochrome c.¹¹⁵ Again in mammalian cells, light at optical tweezing wavelengths, has also been shown to induce an expression of heat shock proteins,¹¹⁶ which are chaperone molecules that might aid the cell to repair light-denatured proteins.

In spite of these potential limitations, optical tweezers have been used successfully to micromanipulate biological material often in ways that would otherwise be impossible to achieve. Minimizing the time the cell is in the trap, choosing the appropriate wavelength, and using novel tweezers configurations such as the Laguerre–Gaussian (LG) beam¹¹⁷ can all serve to minimize photodamage to the biological specimen. A LG mode may offer an annular profile allowing trapping with a lower power than a Gaussian beam counterpart and exposing less of the intracellular volume of a cell to high fluences. Overall, these studies highlight the importance of choice of laser wavelength and beam shape in trapping studies of cells. As a summary, although the “therapeutic window” of tweezing wavelengths has classically been considered to be between 700 and $1064\mathrm{nm}$ , the viability of tweezed cells changes dramatically with wavelength. For mammalian cells tweezed within this wavelength range, maximum viability lies between 950 and $990\mathrm{nm}$ (with $800–850\mathrm{nm}$ also demonstrating a smaller maximum) and minimum viability is between 740 and $760\mathrm{nm}$ .¹¹⁰ For prokaryotic cells tweezed within the range $790–1064\mathrm{nm}$ , maximum viability is observed at $970\mathrm{nm}$ (with a smaller maximum at $830\mathrm{nm}$ ) and minima are observed at 870 and $930\mathrm{nm}$ .¹¹² Tweezing with the popular $1064\mathrm{nm}$ Nd:YAG source is not necessarily the most appropriate wavelength choice if subsequent subculturing is desired.

4.2.

Optical Stretching

Emergent studies at the single-cell level have included the development of optical stretching of cells. The dual counterpropagating trap arrangement leads, in fact, to a bulging of a trapped deformable object of higher refractive index than its surroundings. This is a consequence of the change in photon momentum during passage from a medium of lower to higher refractive index—that is, as the light passes from the medium into the trapped object and reemerges on the other side. The direction of the force at the cell-medium interface can be readily derived using the Minkowski formulation.⁶ An in-depth discussion of the deformation of soft materials by optical tweezers was presented by Møller and Oddershede.¹¹⁸ In the case of a cell, the bulging and elliptical shape can be used to infer the state of the cytoskeleton. This is an internal polymer structure that can resist external optical forces. The optical stretcher allows the recording of the cell’s ellipticity and thus informs on when it changes (Fig. 9 ).⁶ The original cell-stretching work looked at the elasticity of erythrocyte (red blood cell) and fibroblast BALB 3T3 cells.⁶ Subsequent work has employed optical stretching as a biological marker for cancer. Essentially, neoplastic and diseased cells have weaker cytoskeletons and thus show on average larger deformations/ellipticity than normal cells. Thus, recording the distribution of a cell’s ellipticity can be indicative of the onset of disease. In their 2005 study, Guck compared the elasticity of a variety of healthy and cancerous cells lines, including models of breast cancer, and found that the cancerous cells were significantly more deformable than their noncancerous counterparts.¹¹⁹ Furthermore, cancer cells treated to either increase or decrease their metastatic competence had a corresponding increase or decrease in deformability. Subsequent work revealed a significant increase in the deformability of acute promyelocytic leukemia¹²⁰ and oral squamous cell carcinoma¹²¹ cell types when compared to their noncancerous counterparts, further confirming the diagnostic potential of the technique.

Fig. 9

An erythrocyte being optically stretched in a dual fiber trap. As power increases, the cell becomes more elliptical. Theoretical peak stresses, ${\sigma }_{\mathrm{o}}$ , are also shown. (Reprinted from Ref. 6. © 2001, with permission from Elsevier.)

As well as the dual counterpropagating trap, optical stretching can also be performed with single-beam optical tweezers. Erythrocytes and yeast cells are among common cell types studied with optical tweezers.^{22, 76, 107} Liao ¹²² describe an optical stretcher based on an acousto-optic modulator, which was used to scan the focal point of the tweezers between two ends of an erythrocyte at a rate of $100\mathrm{Hz}$ . As a result of the viscous drag imparted by the solution on the cell, instead of hopping between the two traps, the cell remains stationary and becomes stretched up to $0.5\mu \mathrm{m}$ in extra length.¹²² Erythrocytes have also been optically stretched using microspheres acting as “handles” on opposite ends of the cell. This was first described by Bronkorst,¹²³ and shortly thereafter used by Sleep ¹²⁴ to study the elasticity of erythrocyte membranes after chemical disruption of spectrin, ankyrin, or actin (Fig. 10 ). More recently, Suresh ¹²⁵ used optical tweezers to measure the elastic modulus of erythrocytes during various stages of infection by the malarial parasite P. falciparum. As an infection progresses, cells become less deformable and more adhesive. The advantage of this optical technique is that during the final (schizont) stage of infection, this morphological change makes it difficult to measure the elasticity of cells using classical micropipette aspiration or laminar shear flow methods.¹²⁵

Fig. 10

A red cell ghost being before and during optically stretching. The cell was treated with $N$ -ethylmaleimide prior to stretching, which dissociated spectrin tetramers to dimers. (Reprinted from Ref. 125. © 1999, with permission from Elsevier.)

Whole cell mechanics aside, the mechanical properties of small intracellular regions can also be analyzed. Wei performed a study of viscoelasticity of alveolar epithelial cells using a piezoelectric-driven oscillating optical tweezers and compared two methods: tweezing extracellularly bound (via an antigen-antibody interaction) $1.5\mu \mathrm{m}$ spheres, and tweezing endogenous intracellular granules.¹²⁶ In this work, the amplitude of the particle displacement and its phase shift, both of which are frequency dependent, were used to deduce the spring constant of the trap, and from there the magnitude of the complex modulus $\left|{\mathrm{G}}^{*}\right|$ was calculated. The frequency-dependent complex modulus ${\mathrm{G}}^{*}$ is a combination of the materials elastic [ ${\mathrm{G}}^{\prime }\left(\omega \right)$ —the shear storage modulus] and viscous [ ${\mathrm{G}}^{″}\left(\omega \right)$ —the loss storage modulus] response, with ${\mathrm{G}}^{*}\left(\omega \right)={\mathrm{G}}^{\prime }\left(\omega \right)+i{\mathrm{G}}^{″}\left(\omega \right)$ . The authors demonstrated that significantly lower internal storage and shear moduli were measured using the intracellular granules than the extracellular beads.

4.3.

Trapping for Cell Biology and Microfluidic Environments

Gene technology has advanced to such an extent that we can alter a single cell’s function. The ability to record the response of single cells to a range of gene manipulation opens up a new avenue for establishing more knowledge about individual cellular functions. Microfluidic systems, typically a series of channels with diameters of up to $100\mu \mathrm{m}$ , can assist in such studies and can mimic in vivo conditions by allowing cells to be exposed to different biochemical media. The physics of such narrow channels allows laminar flow (low Reynolds number regime) with little mixing. Optical forces can play a pivotal role in such geometries because they can controllably transport a cell between different chemical environments. This ability has been demonstrated by a number of groups, and this type of in situ assay allows the real-time analysis of fluorescent, Raman, or morphological changes in response to a changing chemical environment. For example, as described below, cells can be tweezed from different chambers or between laminar flow streams in order to dose them with different reagents, all during microscopic examination. Alternatively, microdroplets containing femtoliter–attoliter volumes can be tweezed to deliver reagents to a specified cell, or to extract, transport, or analyze subcellular vesicles or lysates.

In the study by Eriksson, flow velocities had to be high enough that the reagents did not appreciably mix, but low enough that the cells were not displaced from the trap by shear.¹²⁷ The studies revealed real-time changes in gene expression in response to the tweezing between different reagent streams. In one example, the nuclear localization of Yap1p, a transcription factor involved with the induction of defense against oxidative stress, was observed in response to the tweezing of a BY4741 wild-type yeast cell from normal cell culture medium to the oxidatively stressing tert-butyl hydroperoxide containing medium.

Other cellular experiments have been demonstrated without microfluidics but still exposing the cells to different media. Capitalizing on the slow diffusion rate of solutes out of $4\mathrm{mm}$ long, $50\mu \mathrm{m}$ id. capillary tubes, Brown demonstrated the optical tweezing of single HL60 cells between three different reagents: fixative, permeabilization buffer, and Trypan Blue.¹²⁸ Indeed, recent work has used optical forces in a new way, employing Airy light fields and moving on parabolic trajectories to transfer cells from one medium to another in the absence of any microfluidic flow.¹²⁹

Liposomes and other micro- and nanoparticles commonly used for the transfection of cells are also tweezable vehicles for targeted reagent delivery. Once tweezed in close proximity, contents can be either be released or injected into the cell.¹³⁰ In one chemical release experiment, optical tweezed particles containing carbachol (a ligand for the muscarinic acetylcholine receptor) were lysed in close proximity of a cell expressing the receptor, using a pulsed UV laser (Fig. 11 ). Activation of the receptor was assessed fluorescently.^{131, 132} The controlled release of a compound in this manner is highly desirable as localized concentration gradients of diffusible chemotactic agents that are ubiquitous control signals for cell migration, differentiation, developmental biology, and the immune system. This type of experiment is traditionally performed using a finely extruded glass capillary tube,¹³³ but the advantage with using optical tweezers is that it would provide far greater spatial and temporal control of the released reagent. Another excellent example was recently demonstrated by Kress, who used arrays of holographic optical tweezers to release compounds from optically manipulated microparticles.¹³⁴ By releasing chemoattractants, they demonstrated the polarization and directed migration of a neutrophil and the generation of new lamellipodia in HL60 cells. By releasing inhibitors of actin polymerization, they demonstrated the highly localized retraction of lamellipodia and the redirection of migration in the same cell type. In effect, this robust technique allows the full optical control of cell migration.

Fig. 11

A polystyrene particle containing carbachol (white dot, II) was optically tweezed to a cell that was expressing the muscarinic acetylcholine receptor (of which carbachol is a ligand) and that was pre-loaded with fluo-3 (which fluoresces proportionally to intracellular calcium release). Upon photolysis of the carbachol containing particle by UV pulse, the highly localized drug caused an increase in intracellular calcium (III). (Reprinted with permission from Ref. 133. © 2004 American Chemical Society.)

Finally, the activation dynamics of membrane receptors using microbeads that are coated with ligands can be studied using optical tweezers. Monneret used holographic optical traps to maneuver various microparticles to specific regions of individually targeted COS-7 cells suspended within a microfluidic environment.¹³⁵

4.4.

Imaging and Spectroscopy with Optical Manipulation

A theme that has emerged in the last decade has been the combination of an optical trapping with other optical methods, namely, imaging and spectroscopic analysis. This ability is essential if we wish to monitor and observe the cellular behavior over long time courses, and indeed, such combinations of techniques are emerging as forerunners for redefining modern microscopes: a future “biophotonics workstation” may be capable of a plurality of studies on one platform.

In terms of imaging, optical tweezers have been combined with imaging by epi-fluorescence¹²⁷ confocal laser-scanning microscopy (CLSM),¹³⁶ and multiphoton microscopy.¹³⁷ Fluorescent systems can either be homebuilt, or commercially available fluorescent microscopy platforms can be modified to incorporate tweezing beams.⁴⁴ The Hoffmann study was one of the first of its kind to address the technical challenge of implementing CLSM and optical tweezers via the same microscope objective.¹³⁶ During CLSM, the microscope stage is moved axially (i.e., in $Z$ ) in order to obtain scanned $XY$ 2-D image slices, which are later reconstructed into a 3-D image. The image plane remains a fixed axial distance from the objective. When a sample object is being optically tweezed, however, it also remains at a fixed distance relative to the objective. The two relative axial positions of the CLSM and the tweezers therefore had to be decoupled. To achieve this, the authors placed a translation stage onto the source of the trapping beam, changing the relative distance between it and the first lens in the system. This resulted in the ability to axially shift the optical tweezers focal plane relative to the CLSM focal plane by $15\mu \mathrm{m}$ $(\pm 0.25\mu \mathrm{m})$ . The simultaneous tweezing and confocal imaging of chloroplasts and cancer cells was demonstrated. Using a slightly different configuration, Goksör demonstrated the simultaneous multiphoton¹³⁷ imaging and tweezing of yeast cells. The group further demonstrated the versatility of its multiphoton imaging and trapping configuration by holding a single spermatozoon with a continuous wave 1064 beam, optically injecting a membrane impermeable dye into the spermatozoon cell using a femtosecond-pulsed $800\mathrm{nm}$ source, and imaging the entry of the dye over time using epi-fluorescence.

In what other contexts can this combined fluorescent imaging and tweezing be useful? Oddos demonstrated the imaging of a synapse formed between an immune cell and an antigen presenting cell.¹³⁸ This synapse was difficult to image with high resolution under normal microscopy conditions because the two cells naturally resided in the horizontal position. The authors capitalized on the fact that a cell couplet when optically tweezed assumed a vertical position. This vertical positioning allowed the rapid 2-D imaging of the immune synapse at the exact plane of the two contacting cells. Without the use of tweezers to position the couplet vertically, CLSM would otherwise require multiple Z slices to reconstruct the synapse, not only reducing the image resolution but also significantly increasing the acquisition time.

In another interesting biological application, Hayakawa ¹³⁹ used standard optical tweezers and CLSM to investigate the activation of mechano-sensitive channels. In an elegant experiment, they microinjected $400\mathrm{nm}$ phalloidin-conjugated beads into live HUVEC cells. The beads selectively bound to the actin cytoskeleton. By tweezing the beads with single beam gradient tweezers, they were able to demonstrate by patch clamping, that the mechanical force acting upon the actin results in mechano-sensitive channel activation. In contrast, the tweezing of microinjected control beads that were not tagged with phalloidin did not result mechanosensitive channel activation (Fig. 12 ).¹³⁹

Fig. 12

Mechanosensitive channel activation by the mechanical stimulation of actin fibres via optical tweezers. (a) Phalloidin laden $40\mathrm{nm}$ green fluorescent beads were microinjected into HUVEC cells, (b) which bound to actin stress fibers (red) observable under CLSM. (c) a: a transient inward current occurred (arrows) when beads were moved using optical tweezers, b: but no current was present in moved control beads that were not actin bound. (d) This mechanical stimulation via optical tweezers also caused an increase in intracellular calcium $(*)$ in the HUVEC cell (outlined with dotted lines) visualized by CLSM. [Reproduced with permission from the Journal of Cell Science http://dx.doi.org/10.1242/jcs.022053 (Ref. 140)].

Combining fluorescent imaging with holographic optical tweezing has also been demonstrated. Eriksson showed the ability to manipulate dozens of individual yeast cells such that the nuclei of each cell was in the correctly focussed imaging plane.¹⁴⁰ In other words, instead of imaging 3-D stacks of a large number of cells resting on a glass coverslip, the cells were manipulated above the coverslip into free solution and automatically repositioned axially such that all nuclei could be imaged concurrently. In the future, this type of configuration could allow the real-time in situ analysis of dozens of trapped cells in response to a drug challenge, for example.

In terms of spectroscopy, Raman spectroscopy has been successfully combined with optical traps for a variety of studies. Raman scattering is an inelastic process and generates a biochemical signature of the molecules. However, the Raman signal is very weak (for example, compared to fluorescence) and can be hampered by the immediate environment of the cell. In this regard, combining this method with trapping thus assists in isolating the cell from any local surface (e.g., a coverslip). Additionally, the cell can be exposed to different buffer media and the Raman signal recorded in situ, or the cell can be rotated and oriented as a Raman signal is recorded from different subcellular regions. Most molecules are termed Raman active and the process requires no external labelling or fluorophores making it attractive. Raman studies with optical trapping¹⁴¹ originated in 1994 and have found major applications for biomedical problems. In addition, innovation in the Raman trapping apparatus has also been pursued. Geometries have been explored both using the same beam for trapping and Raman, and decoupling these beams. Additionally, both holographically generated multipoint traps and counterpropagating dual-beam traps have been used. This latter trap was used to immobilize large cells for Raman analysis. Jess ⁸ used this system and showed Raman signatures from the cytoplasm, nucleus, and membrane, and the ability to discriminate between them.

In 2002, a single-beam gradient optical tweezers holding individual cells in place while simultaneously taking Raman spectra was demonstrated. The system operated at $785\mathrm{nm}$ using the same laser for trapping and Raman analysis. It was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different Raman spectra were observed for the different cells.¹⁴² The same team extended its work to look at sorting, using Raman identification.¹⁴³ In separate work by Creely and colleagues,¹⁴⁴ T-lymphocytes in suspension were held in place using holographic optical tweezers while being probed with a second Raman beam.

Raman may also be extended to look at cell disorders, disease, and even the onset of cancer. Thus early work by Zheng ¹⁴⁵ describe the potential of Raman tweezers in cancer diagnosis. They used Raman tweezers to analyze 200 normal and 200 cancerous colorectal cells and perform multivariate analysis for discrimination. Promising results for specificity and sensitivity were obtained in this work. Raman spectra from optically trapped cancerous and normal colorectal epithelial cells were also obtained by Chen and colleagues who aimed to investigate intact cells.¹⁴⁶ This study had clinical relevance because patients suffering from colorectal adenocarcinomas supplied the cell suspensions for this work. The Raman spectra indicated higher amounts of nucleic acids and proteins in the cancerous cells, which is consistent with other studies. Recent studies by Harvey looked at Raman tweezers as a tool to explore urological cell lines, with the aim of distinguishing malignant cells with principal component analysis.¹⁴⁷

Biological studies of erythrocytes and lymphocytes have been a prominent topic combining Raman and trapping. Such cells have major biological relevance for diseases such as leukemia¹⁴⁸ as well as being typically smaller than epithelial cells, thus making them more amenable to trapping. Another advantage using hematopoietic cells in this type of experimentation is that they do not readily adhere to surfaces, simplifying their preparation for tweezing. Finally, the porphyrin ring contained in hemoglobin strongly absorbs visible light, enhancing resonance and allowing the oxygenation and spin state of erythrocytes to be easily monitored.¹⁴⁹ Ramser explored the oxygenation state of an erythrocyte.¹⁵⁰ Using a microfluidics platform, different buffers passed through the channels using electro-osmotic flow. At the same time, the resonance Raman response of an optically trapped erythrocyte was recorded. This enabled them to monitor the oxygenation cycle of the cell in real time and to study the photoinduced chemistry caused by the chamber illumination, but naturally this could be extended to other biological end points. Chan showed highly reproducible Raman spectra could be obtained by trapping live cells obtained from leukemia patients; multivariate analysis could be used for distinguishing normal and abnormal cells.¹⁵¹ Other studies explored the oxygenation capability of diseased erythrocytes: Rusciano explored the characterization of an erythrocyte from $\beta$ -thalassemic patients obtained by using an optical tweezers system integrated with a Raman probe.¹⁵² They compared the Raman spectra of single thalassemic erythrocytes to those obtained from healthy subjects, relating the observed differences to the analyzed disease. The photoinduced effects caused by the Raman and trapping laser radiation were also investigated. The oxygenation capability of hemoglobin was shown to be reduced in $\beta$ -thalassemic erythrocytes. In other studies, Raman spectroscopy monitored changes in the oxygenation state of human erythrocytes placed under mechanical stress by optical forces leading to cell stretching. The optical force in this instance mimicked the stretching and compression that flowing cells undergo as they flow along smaller capillaries. Rao ¹⁵³ showed spectroscopic evidence of a transition between the oxygenation and deoxygenation states, which is induced by stretching the cell with optical tweezers. They attributed the transition to enhanced hemoglobin-membrane and hemoglobin neighbor-neighbor interactions.¹⁵³ This has just given a flavor of the area of Raman spectroscopy and optical trapping. For the interested reader, there are more in-depth reviews of this field.^{154, 155, 156}

4.5.

Tweezers and Nanosurgery

The application of manipulation to cellular material is achieved with minimal damage from the trapping beam. However, it can be advantageous to combine optical tweezers with other light fields that are designed to perform very precise cutting or controlled transient perforation with submicron resolution. Berns explained the use and history of optical scissors in Ref. 157.

UV laser fields, pulsed Nd:YAG lasers, or ultrashort pulsed fs beams may typically be employed to function as “optical scissors.” Such scissors were employed to study the intracellular organelles, including chromosomes, and the mitotic spindle. In 1991, Steubing ¹⁵⁸ brought two cells into contact using an optical trap. The application of a UV laser microbeam then cut the common wall of the two cells allowing them to fuse.¹⁵⁸ This enabled the creation of hybrid cells that retained cell function. In other work, Seeger ¹⁵⁹ used a laser microbeam to microdissect chromosomes collected by an optical trap and subsequently collected the chromosome segments using a glass capillary.¹⁵⁹ Similar methods can be used for the dissection of cellular organelles, cytoskeletal filaments,¹⁶⁰ or chromosomes.^{161, 162, 163, 164} More recently, such systems can be used for the separation of individual cells from cell clusters.¹⁶⁵ Finally, the cell membrane may also be transiently permeabilized by applying CW or pulsed light, leading to cell transfection.¹⁶⁶

Large liposomes (up to $50\mu \mathrm{m}$ diam) may be used to perform chemical reactions in closed nanovials using only picoliters of reagent. Kulin ¹⁶⁷ used optical tweezers to trap two individual liposomes, which were drawn then into contact. A “nanosurgery” beam, in this case a single pulse of ultraviolet laser light induces the fusion between liposomes.¹⁶⁷ This allowed reagents held within the liposomes to mix and react with one another. Such studies may enable quantitative studies of mixing of chemicals and combinatorial chemistry with picolitres of reagents.

Such nanosurgery may also facilitate the analysis of subcellular components. For example, vesicles can be extracted from cells and optically tweezed to a destination for proteomic^{168, 169, 170} or genomic¹⁶⁵ analysis. As a parallel example, lipid-coated oil droplets can be tweezed to the plasma membrane of a cell and used to extract membrane components.¹⁷¹ Once extracted, subcellular material can be further processed using tweezable microdroplet reactors.^{167, 172, 173} It is key to note that the handling of single subcellular components, such as mitochondria, endoplasmic reticulum, endosomes, etc., is technically challenging using other methods such as by an extruded capillary tube. Therefore, optical tweezers will no doubt continue to play a valuable role in the transport and analysis of these precious samples.

4.6.

Cell Patterning and Directed Growth

Since Ashkin and Dziedzic first demonstrated the tweezing of a virion and a bacterium,¹⁷⁴ optical manipulation has found a variety of applications in tissue engineering and neuroscience where, in particular, patterning and organization of biological material is essential. 2-D and 3-D patterns of nerves have been demonstrated,¹⁷⁵ sometimes with the goal of generating artificial neural networks.¹¹¹ In 1998, Townes-Anderson tweezed a rod cell, cone cell, and multipolar neuron (cells all from the retina) adjacent to each other. Subsequently, new processes interconnecting these cells were subsequently formed.¹¹¹ Manipulation by optical tweezers can also be used to deliver single cells to microfabricated sensors^{176, 177} or microwells.¹⁷⁸ In 2009, Pine and Chow demonstrated the delivery of rat hippocampal neurones to parlene plastic cages containing electrodes for stimulation and recording,¹⁷⁷ opening up a new type of electrophysiological experimentation (Fig. 13 ). Other studies have investigated heterotypic cell-cell patterning on a larger scale for tissue engineering. In 2006, Akselrod ¹⁷⁹ used a combination of a SLM and an AOD to generate arrays of live cells onto a photopolymerizable polyethylene glycol diacrylate hydrogel. 2-D ( $21\times 21$ matrix) or 3-D ( $3\times 3\times 3$ matrix) arrays of P. aeruginosa bacteria were produced, which could be subsequently fixed into position using the photopolymerizable gel. Heterotypic arrays of Swiss 3T3 cells and bacteria were also generated.

Fig. 13

Parlene neurocages, designed to hold, electrically stimulate, and record the activity of individual neurones,¹⁷⁷ are seeded one cell at a time using optical tweezers. [Image reproduced from Ref. 178 with permission (© 2009 IEEE)].

The real-time optical control of cell growth has also been demonstrated. Both the axons of a growing neuron and the hyphal tip of fungi have been optically controlled using optical tweezers. Burnham ¹⁸⁰ demonstrated the controlled growth of the fungal hyphae using engineered light fields to constrict and induce branching in a highly reproducible manner.¹⁸⁰ Light fields have also been used to guide the axons of mammalian PC-12 and NG-108 cells.^{181, 182, 183, 184} These experiments are surprising, and the underlying mechanisms are not fully understood.

This precise cellular patterning and cell delivery using lasers may have future consequences in tissue engineering. It is well established that the microenvironment of a cell greatly influences its state of differentiation, and one of the great challenges of tissue engineering has been the precise control of cell-cell contacts.¹⁸⁵ One classic example is the primary hepatocyte, which rapidly loses a number of hepatocyte-specific markers, such as cytochrome P-450¹⁸⁶, albumin, and urea¹⁸⁷ once removed from its natural environment. This loss of function can be ameliorated by coculture with nonparenchymal cells.¹⁸⁸ Using a weakly focussed $830\mathrm{nm}$ wavelength beam, Nahmias explored tissue engineering possibilities using laser cell patterning.¹⁸⁹ By carefully depositing either one cell type (homotypic) or two different cell types (heterotypic) in linear arrays, cells spontaneously aggregated into tissuelike structures. In the former instance, laser-deposited human umbilical vein endothelial cells (HUVEC) self-assembled into vascular (blood-vessel–like) structures. In the latter case, a combination of primary rat hepatocytes heterotypically laser patterned with HUVEC cells resulted in tubular structures reminiscent of the hepatic sinusoid, a highly specialized liver blood vessel. Therefore, the ability to pattern cocultured cells using optical tweezers may help elucidate the mechanisms of phenotype maintenance in all cell types such as hepatocytes, neurons, and potentially stem cells.

Arneborg used cell patterning to observe synergistic yeast ecosystem dynamics.¹⁹⁰ The authors organized two species of yeast cells to study their growth characteristics using the generalized phase contrast technique as their multiple-trap generation method. In this study, Hanseniaspora uvarum cells were surrounded by Saccharomyces cerevisiae cells. The S. cerevisiae cells indeed influenced the growth of H. uvarum. The generation time of surrounded H. uvarum cells was 15% greater than that of nonsurrounded cells. The confinement imposed by viable S. cerevisiae cells on H. uvarum led to a significant proliferation of the latter cell. The conclusion was that such confinement determined growth in a microbial ecosystem.

4.7.

Cell Sorting

Since its invention in 1965,¹⁹¹ fluorescence-activated cell sorting has been a powerful technique in the separation of fluorescently tagged cells. In this technique, cells are hydrodynamically focused so they pass through an orthogonally directed laser beam surrounded by detectors. A combination of forward scatter, side scatter, and fluorescence is measured. The flow stream is then split into droplets (with approximately one cell per droplet), and an electrostatic charge proportional to the measured fluorescence is placed on the cell containing droplet. The charged droplets may then be electrostatically separated.

There is a continuing trend to miniaturize this device to an on-chip system. The sorting of cell types, particularly in a microfluidics environment, is another major application of optical forces at the single-cell scale. Here tweezers can “actively” drag biologically tagged cells from a flow stream, creating a microscopic fluorescence activated cell sorter¹⁹² or in fact aim to sort cells on a potential energy landscape by relying on differing physical attributes (e.g., size) of one cell type compared to another.¹⁹³ One example can be found in the work of Perroud who used a microfluidic system in combination with optical tweezers in order to separate fluorescently green labeled macrophages from a mixed cell population.¹⁹⁴ High purities (93%) were reported with reasonable recoveries (39–60%). More recent studies have explored techniques of passive optical sorting where the very shape or size difference between cells can be used to sort cells. Arrays of optical traps create what are termed optical potential energy landscapes.^{195, 196} Optical potential energy landscapes are in a key position to aid with the trend in miniaturization particularly in the field of optical sorting and separation. Motion of biological material across such a landscape depends crucially on the interplay between the physical attributes of the particle and the forces exerted by the landscape, which depend not only on laser power but on its optical form (e.g., periodicity). One example of an optical landscape that can be used for cell sorting is the Bessel beam. A Bessel beam offers the profile of a central “nondiffracting” maximum of light surrounded by a series of concentric rings. It is typically produced by sending a Gaussian beam through a diffractive optical element termed a conical axicon lens or may be generated with a spatial light modulator. Paterson ¹⁹⁶ demonstrated the passive optical sorting of erythrocytes and lymphocytes. In the Bessel beam, the erythrocytes become trapped in the concentric rings, whereas the lymphocytes migrate to the central core of the beam and are subsequently guided along its axis.¹⁹⁶ Mononuclear and sphere-tagged T-lymphocytes have also been sorted in this manner. ¹⁹⁷

A further separation technology of note is that of optical chromatography, which separates particles or cells based on a competing drag force from a fluid flow and radiation pressure from a laser.¹⁹⁸ As particles flow in solution toward a weakly focused beam, they are trapped by the beam. Depending on their size and refractive index, they will be trapped in different positions along the length of the light field, allowing extremely sensitive separation to occur. In this technique, stokes drag is balanced by radiation pressure; particles with a higher refractive index and a larger size will be affected more strongly by the radiation pressure than the stokes drag. This configuration has recently realized the separation of B. anthracis from a genetically related spore, B. thuringiensis.¹⁹⁹ A mixed spore population of the two species were flowed along a microfluidic channel against the optical pressure of a loosely focussed $850\mathrm{nm}$ laser. The B. anthracis strain was affected more strongly by the radiation pressure of the laser and was therefore separated many hundreds of microns upstream from the B. thuringiensis spore. Optical chromatography has also been used for highly sensitive immunodetection. Antibody-coated spheres, when challenged with antigen, bound to each other as couplets. These couplets were then able to be separated by optical chromatography from unbound spheres;²⁰⁰ the ratio of the separated singlet and couplet spheres is proportional to the amount of antigen present.²⁰¹