HK1092995B - Optical fractionation methods and apparatus - Google Patents

Description

Method and apparatus for optical fractionation

Technical Field

The present invention relates generally to embodiments of methods and systems for classifying minute particles. More specifically, the present invention is directed to the classification of minute objects such as macro-molecules, biomolecules, nanoclusters, colloidal particles, and biological cells using holographic optical tweezers technology.

Background

Optical tweezers use optical gradient forces to trap tiny, usually micron-scale, volumes of matter along two and three dimensions into a well. A holographic form of optical tweezers that can be created in large numbers from a single laser beam using computer generated diffractive optical elements. The tweezers can be arranged according to the practical requirements in any required structural form.

While some systems are known that move particles accurately and with relatively high confidence, typical systems require a separate projected hologram for each individual step of particle motion. Computing multiple holograms is time consuming and requires a significant computational effort. Furthermore, a computer-addressable projection system required to implement the above-described computer-generated optical tweezers, or other dynamic optical tweezers systems, such as scanning optical tweezers, would seem to be prohibitively expensive.

Disclosure of Invention

The operation of many technically and commercially important systems is produced by the classical migration of the morphology of the modulated potential energy. One method of utilizing these operations is optical fractionation. Optical fractionation is capable of continuously (over a given period of time) sorting populations of small objects into separate components based on their differing abilities to pass through an array of optical traps. In particular, an object driven by an external force, such as viscous drag in a flowing liquid, encounters an array of optical traps whose axes of symmetry are oriented at an angle relative to the driving force. Generally, these traps can be created using holographic optical tweezers technology. Those objects that are more strongly influenced by the potential energy wells created by these wells tend to jump from well to well, deviating from the direction of the driving force. Other objects that are more strongly influenced by the driving force or less strongly influenced by the light traps pass through the array without deflection. Depending on the configuration of the trap, the invention can be used to deflect different components by different amounts. In some cases, it may be desirable to use a clean separation of the two-component embodiment described above. However, it is within the scope of the present invention to select multiple components for collection. For example, in one embodiment, the non-uniform sample may be output in a fan shape over a range of directions of continuous distribution for an "optical chromatography" method. The deflected and undeflected components can be collected separately.

Generally, the non-uniform input sample and output components are dispersed in the liquid flowing through the channels. In a preferred embodiment, the channels take the form of a so-called H-shaped cross, where two outputs, one containing the input mixed sample and the other containing only background liquid, are brought together and run side by side for a set distance before being split into two output channels. If the channel is small enough, the reynolds number of the flowing liquid is also small enough that the two fluids do not mix but flow side by side in a laminar flow. As a result, objects in the input fluid generally do not cross the boundaries between the fluids and enter the buffer channels, except by chance or due to diffusion.

One aspect of the present invention relates to optical fractionation using an array of discrete optical traps to continuously sort micro objects based on their relative affinities for the optical traps and for competition from external forces. An undesired component is more diffusive or mobile than a desired component. However, another aspect of the invention relates to the "opposite" optical fractionation method. In the opposite optical fractionation method, the desired component is more diffusive or mobile than the undesired component.

Another aspect of the invention involves a modification to the so-called optical peristalsis technique, in which tiny objects are deterministically migrated by a projected sequence of optical trap patterns. The difference between optical peristalsis and the disclosed optical thermal ratchet technology characteristically confers new capabilities to the system and method, including without limitation bi-directional pumping through what is known as the flux reversal effect, and also with new possibilities for sorting non-uniform samples.

Drawings

FIG. 1 depicts an optical fractionation method in which a microfluidic H-shaped cross-over comprises a first fluid containing a heterogeneous sample to be sorted, and a second fluid consisting of a background or buffer liquid;

FIG. 2 depicts an inverse optical fractionation method in which the microfluidic H-intersection comprises two flowing liquids, one containing the heterogeneous sample to be fractionated and the other containing only buffer solution;

FIG. 3A shows a schematic side view of an optical fractionation process; FIG. 3B depicts a top view of the optical fractionation process of FIG. 3A;

FIG. 4 shows the optical separation of a large quartz sphere from a small quartz sphere; FIG. 4A depicts a representative trace of a 0.79 micron radius sphere measured every 1/60 seconds; FIG. 4B depicts the trace of a 0.5 micron radius sphere obtained at the same time; FIG. 4C is the time-averaged areal density of 0.79 micron radius spheres relative to their average areal density; and FIG. 4D is the time-averaged areal density of 0.50 micron radius spheres relative to their average areal density;

FIG. 5 shows the separated spatially resolved quality obtained for a single optical trap line;

FIG. 6A depicts a prior art optical peristalsis method in which an optical trap pattern positions an object; FIG. 6B depicts replacement of the light trap pattern by another light trap pattern displaced by a distance; FIG. 6C depicts yet another shifted pattern of optical traps; finally, FIG. 6D illustrates the completion of one cycle of optical peristalsis; and

FIG. 7 plots a numerical solution to the equation of motion for an optical thermal ratchet embodiment demonstrating flux reversal.

Detailed Description

The present invention relates to a method and apparatus for optical fractionation. One aspect relates to optical fractionation using an array of discrete optical traps to continuously sort small objects based on their relative affinities for the optical traps and for competition from externally applied forces. In another aspect, the invention relates to the "reverse" optical fractionation method. A third aspect of the invention involves the use of a "ratchet-type" optical separation technique.

To study the modulated migration, a model system has been developed in which individual colloidal spheres are driven through an array of regular potential wells created with discrete optical tweezers while their motion is analyzed with a digital video microscope. Experiments on this system show that as the array rotates relative to the driving force, the driven particles trace out a Devil ladder system of kinetically locked states. In each of these states, the trajectory of the particle follows a symmetrically selected direction through the lattice of wells, independent of the orientation of the array, and is thus deflected laterally away from the driving force. This deflection can be predicted to provide the basis for a continuous separation technique in which a selected population is deflected by the array of wells while the rest of the sample passes unimpeded. This method gives a practical demonstration of optical fractionation, and it has also been demonstrated that the resolution of optical fractionation can depend exponentially on the size of the particles. Thus, this method gives a sensitivity that is not comparable to any of the previously reported classification techniques.

One can demonstrate a conceptual form of optical fractionation that can continuously sort small objects based on their relative affinities for optical traps and for competing external forces by using an array of discrete optical traps. This demonstration utilized trajectories of two different sized colloidal quartz spheres dispersed in water passing through a linear array of optical tweezers arranged at an angle to the water flow. The colloidal dispersion of the flow was limited to 4mm x 0.7mm x 40 μm glass channels formed by bonding the edge of the cover plate to the microscope slide. The pressure differential applied across the channel produced a substantially constant Poisseuille flow of about 60 μm/sec over several minutes. Samples included a 0.79 μm radius sphere (Duke Scientific Corporation, 2463Faber Place Palo Alto, California 94303, Lot No.24169) and a 0.5 μm radius sphere (Duke Scientific Lot No.19057), both of which were tracked in a plane with 30nm accuracy every 1/60 seconds using conventional bright field microscopy and digital video analysis. In addition, the spheres can be reliably distinguished according to their appearance, thereby providing an ideal model system that can be monitored in real time for microscopic response to optical fractionation. Typical trajectories for the large and small spheres are shown in fig. 4A and 4B, respectively.

The quartz spheres are approximately twice as dense as water and therefore settle into a single layer just above the glass wall below the channel, the smaller spheres float higher because they are lighter. Given the Poisseuille flow line in the channel, the smaller ball has an average velocity of u_s17. + -.9 μm/s, u with larger spheres_bMove slightly faster than 13 ± 2 μm/sec. Viscous drag F of static ball₁γ u, characterized by a drag coefficient γ, which is related to the radius a of the sphere and the neighborhood of the interface with the sphere. The overall coefficient of resistance can be expressed by the Einstein-Smoluchowsky relationship D ═ k_bT/γ, estimated from their diffusivity, D, where K_BT is the proportion of heat energy at temperature T. The diffusivity, in turn, can be measured from the fluctuation of the lateral velocity in the traces shown, for example, in fig. 4A and 4B. More generally, the force F applied₁Can be provided by processes such as electrophoresis, electroosmosis, magnetophoresis, or gravity sedimentation.

The optical traps shown above were created using dynamic holographic optical tweezers technology. 12 discrete optical tweezers produced by converging 1.7 + -0.8 mW 532nm laser beam and arranged at 12.0 degree theta to the channel axisIn the line of ± 0.5 °, the center-to-center spacing b is 3.6 ± 0.1 μm. Each well can be roughly modeled by a Gauss potential well, the depth V of which_oAnd a width epsilon_TBoth of which are related to the radius a of the sphere.

If not for optical traps, then force F is applied₁The particles being driven through the viscous liquid will be at an average velocityAnd (4) moving. As long as the force F is applied₁Large enough that the optical trap deflects only one particle away from its trajectory. If the deflection is small, the particles will continue to move downstream, away from the line of traps, and so to speak have escaped from the line of traps. Instead, each well may be strong enough to deflect a particle into its neighboring domain of influence. In this case the particles will pass from one well to the other and be effectively trapped by the array. This is the mechanism of kinematically locked migration. The deflection angle θ is selected to be close to the maximum deflection of the lock-in migration. The relative deflection of the trajectory of the trapped particle to the trajectory of the escaped particle is the basis for classification by optical fractionation. The deflected and undeflected components may be collected separately and the process is schematically illustrated in figure 1.

Given the geometry of the trap, the laser power can be set between the empirically determined large ball and small ball escape thresholds. The traces of fig. 4A and 4B show that under these conditions, the larger spheres are systematically deflected by the trap array, while the smaller spheres are not. As a result, small spheres flow unimpeded into the shadow created in the distribution of large spheres where they can be collected. Instead, the deflected large spheres are concentrated into a small area at one end of the array of optical traps, where they can be collected separately. Because the purification of the beads and the concentration of the large beads result from the lateral deflection of the larger components, the optical fractionation process can be continued, thereby outperforming batch mode techniques such as gel electrophoresis.

This qualitative interpretation of only a few traces, by considering what was collected in FIGS. 4C and 4DThe statistics of thousands of tracks can make people convince. Here, we are working onArea of centerIn (1), the time-average areal density n of the drawing ballWherein the surface density n is averaged over time for each population_oAnd (6) normalizing. The relative affinity of the ball for the well can be estimated as follows: the probability of a large ball in a trap is roughly 18 times greater than in the entire fluid, while the probability of a small ball is only 3 times greater. Given the relative velocities of the balls, these ratios are consistent with the larger balls temporarily stopping in a local potential minimum, while the smaller balls are simply decelerated.

The resulting separation quality can be estimated by measuring the relative bulk concentration as a function of position in the fluid:

the figure of merit for the above equation, shown in figures 5A and 5B, reaches a maximum of one unit in the area containing only the large spheres and a negative one unit in the area containing only the small spheres. A section of the fluid along line a of figure 5A is traversed before the well array, revealing a fully mixed sample, q (y) 0, as shown by the small circles in figure 5B. A similar section along line B after the well array, drawn as a larger circle in fig. 5B, roughly demonstrates 40 percent purification on large and small spheres. Many of the backgrounds contribute to collisions in the trap array that can allow large spheres to escape. The escape caused by collisions is evident in the concentration curve of the large spheres downstream of the trap array of fig. 4C, with the probability of collisions and escapes gradually increasing as the large spheres saturate at the downstream end of the trap array. The most effective way to avoid such collisions is to project several parallel trap lines. Under the present experimental conditions, as few as three wires can provide a substantially perfect separation, in denser suspensions, more wires are required.

The data in fig. 4A, 4B and 5A, 5B show that a discrete array of optical traps can continuously separate the spheres according to their size. Consideration of the physical conditions that result in one type of particle escaping from the array of optical traps, while the other is captured, may provide the basis for optimizing the optical fractionation.

For simplicity, the analysis has only the effect of two discrete optical traps centered at x ═ b/2, with the particles near their midpoint x ═ 0. The total potential energy of the particles is

The point through which the particles escape is

Here, the y-component of the total force is equal to zero. The particles are likely to escape near x-0 because the force trapped in the trap is weakest, and at y- σ the force of separation is greatest. In this case, the maximum obtainable deflection of the captured trajectory is still allowed, given by

Here, relative trap strengthDepending on the particle material properties, including their size, but not on the configuration of the trap. Here V₁＝F₁σ characterizes the driving force. Similarly, the apparent expansion σ (a) of an optical trap depends not only on the width σ of the focused beam_oAlso depending on the size of the particles:

larger particles are affected to a greater extent by the optical traps than smaller particles. This dependence of σ on a in nature establishes conditions for exponential sensitive separations. We continue to use equation (5) for illustration.

For V currently obtained from big and small balls respectively_o/V₁Thermal fluctuation analysis was used to characterize the depth of the optical traps for data of 1.3 and 0.73. The same analysis shows that the apparent widths of the wells are 0.94 ± 0.07 μm and 0.74 ± 0.07 μm. These results suggest a critical angle for the large ball of 14 ° ± 1 ° and for the small ball of 3 ° ± 2 °, consistent with the observed phenomenon that a large ball is systematically trapped and a small ball escapes. For a barely trapped particle in an N well array, the total lateral deflection is (N-1) b sin θ. Thus, is composed of

Δ(a|b)＝bsinθ (6)

Each is establishedThe wells are laterally deflected, thereby characterizing the efficiency of the array. Selected at a value of 4/eV_o/F₁The inter-well spacing b above is 2 σ (a) to optimize the efficiency. This result is useful for the design of a practical optical fractionation system, but it is not necessary to optimize its sensitivity to particle size.

The sensitivity can be expressed by the formula

And can be optimized by

Thus obtaining

Here, the

Equation (9) establishes the inter-trap spacing b at which the array of optical traps is most sensitive to distinguishing between "large" particles that will be trapped and "small" particles that will escape at angle θ.

As a practical example, these results may be used to optimize optical fractionation in viscous fluids. For particles of a size comparable to the wavelength of the light or smaller, the depth of the potential well will be related to the particle volume V_o＝Aa³Proportional, at the same time, to the radius V of the particle₁Ba is proportional, so f (a) is proportional to a²Is in direct proportion. Substituting the optimized interval b into the criterion based on fluid separation in equation (4) yields:

equations (4) and (5) also show that the optical fractionation depends only linearly on the depth of the potential well. Thus, variations in the actual depth of the actual array of optical vortex bodies only linearly degrade the resolution of the separation and can generally be compensated for by a substantially greater dependence on particle size.

In summary, the foregoing examples have in fact demonstrated optical fractionation using a colloidal quartz sphere simulation system, and have also demonstrated that this technique has the potential to achieve exponential sensitivity for size-based separations. The foregoing considerations illustrate that the geometry of the optical separation system can be selected to optimize separation based on size, and that exponential sensitivity is standard. Separation based on other features can be optimized with the same arguments, but exponential sensitivity is generally not expected.

Equation (11) also provides a good understanding of the possibility of using optical fractionation for protein and nanocluster objects whose dimensions a are measured in tens of nanometers. In particular, equation (11) indicates that at a fixed angle θ, from a 1 micron scale object to a 10 nanometer scale object, the ratio A/B will be required to increase by several orders of magnitude. This can in principle be achieved by increasing the light intensity, reducing its wavelength, and selecting a wavelength at which the interaction with the particles is resonantly enhanced.

Optical fractionation is performed in this system, involving the creation of an array of traps spanning the input mixed fluid in a manner that enables the desired components of the particles to be deflected across the boundary into the buffer fluid. On the one hand, successful operation requires that the sample have a sufficiently low diffusivity or mobility that the unwanted components spontaneously cross the boundary at an acceptably low rate.

However, another aspect of the invention is directed to the opposite situation, where the desired component is more diffusive or mobile than the undesired component. Furthermore, this aspect also addresses the situation where the desired component interacts less strongly than the other component and is therefore not selected by conventional optical separation methods. The greatest benefit of the present invention will be realized in a system that can implement both conditions, although both conditions are not sufficient. Fig. 1 shows a microfluidic H-shaped intersection 100 with two liquid fluids. One fluid, the mixed input fluid 110, contains a heterogeneous sample to be separated. The other fluid, buffer fluid 120, is comprised of background or buffer liquid. Objects in the input fluid 110 encounter an optical tweezer array 130 arranged at an angle θ to the fluid, which optical tweezer array 130 deflects selected components of the sample into a buffer output fluid 140 for collection. The undeflected components of the sample remain in the original flow, output flow 150, where they are collected.

Instead of creating an array of optical traps that direct objects away from the mixed input fluid 110 and into the buffer fluid 120, the present invention can also use optical traps to direct objects back into the input mixed fluid as the objects attempt to cross the boundary, either by diffusion or by active swimming, as shown in fig. 2. As in the conventional method shown in fig. 1, the microfluidic H-shaped cross 200 contains two flowing liquid streams, one 210 of which contains the heterogeneous sample to be separated and the other 220 of which contains only buffer solution. Only those objects that attempt to cross the boundary between the two fluids in the mixed input fluid 210 will encounter the array of light traps 230, which is arranged to direct the objects back into the mixed input fluid 210. Objects that cross the boundary by the array of optical traps 230 are collected in the buffer output fluid 240. Those objects that remain in the original input fluid, either because they are less diffusive or because they are deflected by the array of optical traps 230, may be collected separately in the output fluid 250. In this case, less diffusive or motile objects are deflected back into the mixed input fluid, while more mobile components will escape the trap and be collected across the dividing line. Also, objects that are less strongly affected by the light trap are more able to be collected across the boundary.

Although the optical fractionation method requires a sufficiently large number of optical traps to fill the entire mixed input fluid, the reverse process described above requires only enough optical traps to cover the area immediately surrounding the boundary between the fluids. Thus, the opposite optical fractionation method requires far fewer optical traps than conventional optical fractionation methods, and therefore more efficient use of the laser light required to create the optical traps.

In this respect, optical fractionation has proven well superior to other sorting techniques, and optical fractionation in contrast gives the same advantages. These advantages include: continuous operation rather than batch operation; continuously optimized by adjusting laser power, laser wavelength, optical tweezers geometry, driving force, and exponential sensitivity to size. In contrast, optical fractionation extends these advantages to systems where conventional optical fractionation is either not applicable or not practical. Since the opposite optical fractionation method can advantageously utilize the advantages of the polarization of light forming the wells or the advantages of the beam mode structure forming the wells by means of the conventional optical fractionation method, objects are classified according to their birefringence, optical rotation, elasticity, and properties such as size, light scattering cross section, light absorption rate, surface charge, and shape.

It is well known that objects are classified according to their diffusivity and that H-intersections of microfluidics are useful. Adding an array of optical tweezers to the opposite optical separation method greatly enhances the selectivity of the process and gives a large new physical basis for object classification.

In another aspect of the invention, a thermal ratchet is utilized. Fig. 6(a-D) illustrate the principle on which optical peristalsis operates and is used to explain the characteristics of the optical thermal ratchet. In FIG. 6A, a pattern of discrete optical traps is depicted to locate a single object. The pattern is schematically represented as two discrete potential energy wells, each having a width σ, separated by a distance L. In fact, a practical pattern may include many optical traps organized into conduits. Optical peristalsis and the optical thermal ratcheting method disclosed herein are directed to transferring objects from one optical trap conduit to another. The two methods differ in how they accomplish this.

In optical peristalsis, the initial pattern of traps is replaced by another in which the conduits are displaced by a distance comparable to σ (see fig. 6B). Because the new potential well overlaps the old, the particle is transferred deterministically to the nearest tunnel on the new pattern. Fig. 6C repeats the process with yet another shifted well pattern. One cycle of optical peristalsis is completed when the original pattern is projected (see fig. 6). The net effect of this cycle is to transport particles trapped in the traps from one trap's tunnel in the first pattern to the next tunnel also in the first pattern. In fact, there are many particles trapped in many optical traps; and in each cycle of optical peristalsis, all particles will be transported forward by a set of tubes. The direction of movement is unambiguously determined by the order of the sequence and can be reversed by reversing the order.

The difference between optical thermal ratchets and optical peristalsis is that the distance between the conduits in the direction of motion is substantially greater than the width of the individual wells. Therefore, when the second pattern is excited, the particles trapped in the first pattern can be freely diffused. Those particles that diffuse far enough to reach the nearest conduit in the second pattern are quickly localized. The located component is then again transported forward (also by diffusion) while projecting the third pattern, and again when cycling back to the first pattern. Unlike optical peristalsis, which is a deterministic migration in optical peristalsis, it is ensured that all objects trapped in the well move forward in each cycle, while the above-mentioned biased diffusion, only migrates a certain component of the sample forward.

However, the above described embodiments of the thermal ratchet lead to new opportunities. Particles that are too slow to catch up with the forward propagating wave may still be diffusing far enough to catch up with the trap retrograde to their starting point when illuminated by the third pattern of fig. 6C. These particles are transported backwards one third of the distance between the pipes in each cycle. Whether the population of the sequence of patterns forming the wells moves forward or backward is determined by the balance between the particle diffusion rate and the sequence circulation rate. Thus, changing the rate of circulation can change the direction of the average motion, a phenomenon also known as flow reversal.

Under the influence of the circulating optical tweezer pattern, the expected flux of particles can be calculated. At position x_jThe forceps can be simulated by Gauss potential well.

The potential well has a depth V_oAnd a width sigma. The potential well is apparently spatially symmetric. The pattern of wells establishes one of the three states of the cycle required for ratcheting. As an illustrative example, it is contemplated that the wells in the pattern are spaced apart by equal distances L, so the overall potential in state k is

Where k is 0, 1, or 2. Also, as shown in the illustrated example, the potential energy profile may be considered to be repeatedly cycled through the three states at equal time intervals T. This time can be related to the time of diffusion of the particles of diffusivity D through the system

And (4) the equivalent. The result of the equilibrium between T and τ determines the direction in which the sequence of potential energy states drives the particle through the system.

The probability p (x, t) dx to find a Brown particle in dx at time t, position x, under the combined influence of optical traps and random thermodynamic forces, is determined by the main equation:

p(y，t+τ)＝∫P(y，τ|x，0)p(x，t)dx (15)

the propagation function for each state k, here, is given by,

Pk(y，t|x，0)＝e^L (16)

for times τ < T, there are:

and here beta^-1Is the thermal energy ratio. The main equation for a complete tri-state cycle is,

p(y，t+3T)＝∫dy₃P₃(y₃，T\y₂，0)∫dy₂P₂(y₂，T\y₁，0)∫dy₁P₁(y₁，T\x，0)p(x，t) (18)

for symmetric optical tweezer potentials, our consideration is that the main equation has a steady state solution that results in:

p(x，t+3T)＝p(x，t) (19)

the average speed of this steady state is then given by:

FIG. 7 vs. beta V_oThe numerical solution of the system of equations is drawn as two representative values of 10 and σ/L. For very small values of cycle time T, the particles cannot follow the rapidly changing potential energy morphology, and thus diffuse randomly; the average speed is eventually equal to zero in this limit. If the wells in successive patterns overlap (shown as σ ═ 0.15L in fig. 7), the particles pass deterministically from well to well, producing a uniform positive drift velocity. This transfer reaches its maximum efficiency for moderate cycle times T, but no improvement for longer dwell times. As a result, the drift velocity decreases when 1/T is in the long time limit.

A wider split trap (shown as σ -0.10L in fig. 7) produces another operating condition. Here, for a sufficiently large value of T, the particle is able to keep up with the forward propagating wave. However, faster cycling results in flow reversal characterized by negative v values. This numerical result demonstrates the principle by which an array of optical tweezers can be used to implement a fully symmetric thermal ratchet with flux reversal.

As shown in FIG. 7, for beta V_oA 10 Gauss trap potential tri-state cycle, spanning from deterministic optical creep, with σ 0.15L, to flow reversed thermal ratcheting, with σ 0.10L.

Up to this point, the flow reversal due to the change in the cycle time T has been explained. The flux reversal effect can also be caused by different diffusion coefficients producing different values of τ for different populations in the non-uniform sample. As long as T is chosen to drive one population forward and the other population backward, the different populations may be caused to move in opposite directions simultaneously. In this manner, the optical thermal ratchet described is useful for separating and purifying small objects carried by a liquid.

A preferred optical approach to implementing a reversible thermal ratchet is advantageous over other ratchet-based separation schemes. For example, thermal ratchets based on arrays of interdigitated electrodes have been applied to the classification of DNA fragments. However, these thermal ratchets require complex microfabrication processes, while optical ratchets can be implemented inexpensively and can be readily assembled into microfluidic devices for lab-on-a-chip applications. It has previously been demonstrated that an optical ratchet based on a single time-shared scanning optical tweezer can cause flux reversal. Said approach relies on the creation of spatially asymmetric potential energy profiles in a time-averaged sense, and the system is therefore based on a different principle than the above-described process. In the preferred system described herein, each optical well in each pattern provides a spatially symmetric potential energy well; while the pattern itself is also spatially symmetric. Unidirectional migration is driven by breaking the spatiotemporal symmetry through a sequence of at least three patterns in each cycle.

One of the previously proposed examples of symmetric thermal ratchets also involves a tri-state sequence. However, this approach relies on particles that allow diffusion in only one state, and act as a deterministic ratchet for the other two states, biasing diffusion. The process described in this document, which involves diffusion and localization in all three states, therefore gives more selectivity and a more rapid classification of non-uniform samples.

While preferred embodiments have been shown and described, it should be noted that changes and modifications may be made by those skilled in the art without departing from the invention in its broader aspects. Various features of the invention are set forth in the following claims.

Claims (22)

1. An apparatus for sorting a population of small objects, comprising:

a first channel and a second channel;

a force source for driving a population of small objects through the passage;

a plurality of channels formed by laser beams at the convergence of the first and second channels to form a plurality of optical traps;

the light traps are organized in a plurality of patterns, wherein the patterns are arranged such that the conduits of one pattern are separated by conduits of the remaining patterns; and

the light traps are oriented at an angle with respect to the driving force, and each pattern of the light traps is arranged to be spaced apart from each other;

wherein the population of minute objects is classified into at least a desired component and an undesired component.

2. The apparatus of claim 1, wherein the population of small objects is dispersed in a liquid medium disposed in the first channel, and wherein a buffer is further disposed in the second channel.

3. The apparatus of claim 1, wherein the channel comprises an H-shaped cross.

4. The apparatus of claim 1 wherein the optical traps are created by holographic optical means.

5. The device of claim 1, wherein the undesired component is more diffusive or mobile than the desired component.

6. The device of claim 1, wherein the desired component is more diffusive or mobile than the undesired component.

7. Apparatus according to claim 1, wherein the distance between the conduits in the direction of the driving force is greater than the width of the wells.

8. The apparatus of claim 7, wherein the optical traps are adapted to move only a portion of the population of small objects forward when the patterns are activated in sequence.

9. The apparatus of claim 7, wherein the patterns are adapted to cause population spreading of small objects trapped in one pattern when the other pattern is activated.

10. A method of continuously separating a population of fine particles into at least a desired component and an undesired component, comprising the steps of:

providing an external force for driving the population of the fine particles;

providing a plurality of patterns each comprising at least one channel, each channel comprising at least one optical trap, and the distance between the channels in the direction of the driving force being greater than the width of the optical traps;

providing a laser beam to form a plurality of optical traps; and

the optical traps are organized so that unwanted components can leave the traps while the wanted components are retained.

11. The method of claim 10, further comprising dispersing the population of microparticles in the liquid fluid fed into the first channel, wherein a buffer is also fed into the second channel.

12. The method of claim 10, wherein the channel comprises an H-shaped cross.

13. The method of claim 10, further comprising creating the optical traps using holographic optical tweezers technology.

14. The method of claim 10, wherein only a certain component of the population of small particles moves forward through the optical trap when the pattern is sequentially activated.

15. The method of claim 10, wherein when one pattern is excited, the population of minute particles trapped in the other pattern is allowed to diffuse freely.

16. The method of claim 10, wherein some particles move forward through the array of optical traps and some particles move backward through the array of optical traps.

17. A method of continuously separating a population of small particles, comprising the steps of:

providing an external force for driving the population of the fine particles;

focusing the laser beam to form a plurality of optical traps; and

providing a plurality of patterns each comprising at least one conduit, each conduit comprising at least one optical trap; and

exciting each pattern at intervals, wherein when one pattern is excited, particles trapped in another previously excited pattern are allowed to diffuse freely;

wherein the population of minute particles is classified into at least a desired component and an undesired component.

18. The method of claim 17, further comprising creating the optical traps using holographic optical tweezers technology.

19. A method according to claim 17, wherein the distance between the conduits in the direction of the driving force is greater than the width of the wells.

20. The method of claim 17, wherein only a certain component of the population of small particles moves forward through the optical trap when the pattern is sequentially activated.

21. The method of claim 17, wherein when one pattern is excited, the population of microparticles trapped in the other pattern is free to diffuse.

22. The method of claim 17, wherein some particles move forward through the array of optical traps and some particles move backward through the array of optical traps.