ES2395496B1 - METHOD FOR HANDLING MICROSCOPIC SAMPLES AND MEASURING THE FORCES THAT ACT ON THEM - Google Patents

Abstract

Method for manipulating microscopic samples and measuring the forces acting on them. # It comprises polarizing a laser beam in a direction that is substantially different from the direction of the extraordinary refractive index of a diffractive light spatial modulator that modulates the polarized laser beam to generate a pattern of holographic optical traps that allows manipulating a plurality of microscopic samples; create an optical trap, different from the holographic trap pattern, for a different microscopic sample using the polarized beam component that is orthogonal to the direction of the extraordinary refractive index; polarize the light scattered by the pattern of holographic traps and the different optical trap; capture the image of the light scattered by the different optical trap with a detector; and measure the optical forces acting on the different microscopic sample from the signals provided by the detector.

Description

Method to manipulate microscopic samples and measure the forces that

act on them

The invention is related to optical tweezers, and more specifically with

5

a method to manipulate microscopic samples and also to measure the

forces that are exerted on them, which includes the stages of generating a

laser beam and modulate the laser beam using a diffractive space modulator,

in order to generate a pattern of holographic traps to manipulate a

plurality of microscopic samples

10

STATE OF THE TECHNIQUE

Optical tweezers use light to manipulate microscopic samples using

the creation of an optical trap for small particles. Optical trap

fifteen

It is created at a point where a laser beam is highly focused. The

radiation pressure is able to trap dielectric particles because the

transverse beam profile grows towards its center (the light is brighter in the

center than at the edges of the beam), so there is a strong gradient of the

electric field that attracts dielectric particles to the center of the beam and

2nd

can compensate for the force acting on the particles in the direction of

light propagation (the so-called scattering force, which can act in

opposite directions, depending on whether the light is reflected or refracted by the

particle). The dispersion force and the gradient force are proportional to the

light intensity but, as mentioned, the gradient force

2 5

acts in the direction of the intensity gradient while the force of

dispersion acts in the direction of the incident light.

The basic principle of optical tweezers is moment transfer

associated with the light that changes direction. The light takes a moment that is

3rd

proportional to its energy and points in the direction of propagation. Any

change in the direction of light, by reflection or refraction, results in a

Change of the moment of light. If an object deflects the light, changing its

moment, the conservation of the moment requires that the object must suffer a

same and opposite moment change. This results in a force acting

3 5

about the object

With the optical tweezers, small ones can be detected and measured

displacements of a particle, which allows to measure the force exerted

on the particle. The standard methods for measuring trap forces

Unique conventional optics are based on the linear relationship between force

exerted (F) and the position of the sample in the trap (x), F = -kx. In this

5

harmonic approach, which is valid for small displacements

with respect to the center, the trap acts like a spring, with a constant

elastic k, normally referred to as stiffness of the

trap. Among the different methods of precise position detection, the

focal plane interferometry ("BFP, back focal plane")

1st

achieved great acceptance because it provides both a width of

Large time band as a high spatial resolution, and it is easy to

implement. The stiffness is determined by analyzing the power spectrum of the

Brownian movement of a trapped microsphere. A position sensor

reads the intensity of interference between the laser and the light scattered by the

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trapped sample, which is related to the displacement x when the

sensor is placed in a conjugate plane of the focal plane of the condenser or of

the lens that collects the light from the laser and the sample.

Optical tweezers have been widely used as a powerful tool

2nd

to grab and exert precise forces on microscopic samples, and have

found application in numerous fields of science, from the physics of

colides up to cellular and molecular biology. In a conventional system of

optical tweezers, a highly focused laser beam is a trap

single optics In the last ten years, the use of holograms represented

2 5

in a spatial light modulator before targeting has allowed the

30 programmable trap pattern generation. These optical tweezers

holographic have greatly increased the handling capacity of

The technique.

3rd

US patent 6055106 describes the arrangement of an optical element

diffractive ("OOE, diffractive optical element" in English) on a conjugate plane

of the entrance pupil of the objective lens used to focus the laser beam.

The input beam incident on the OOE is divided into an output beam

structured characteristic of the nature of the OOE. The OOE may include

3 5

Computer generated holograms that divide the input beam into a

   pattern 30, to form a three-dimensional arrangement of optical traps. He

OOE can be a phase liquid crystal matrix that undergoes changes

Engravings with computer generated holographic patterns.

However, the applications of holographic optical tweezers are

restricted due to the inability to accurately measure the force exerted

5

when more than one trap is used because, when multiple are generated

traps with holographic optical tweezers, the signal received by the

photodetector contains mixed information of the light scattered by more than

a trapped sample, and from it the positions cannot be extracted

individual.

10

There are techniques to calibrate more than one trap that consist of capturing

images of the entire sample with a camera and follow the movement of each

object individually. However, conventional cameras have a

limited temporal resolution to be used in spectrum analysis of

fifteen

power, which is therefore replaced by other methods of measurement less

accurate. Recently high speed cameras with width have been used

of band comparable to that of photodetectors, although they have less

resolution than these and they are very expensive.

2nd

EXPLANATION OF THE INVENTION

It is the object of the present invention to provide a method for manipulating

a plurality of microscopic samples that allows both a measurement

Precise and economical of the force exerted by the optical tweezers. The method

2 5

compatibilizes the manipulation of microscopic samples with optical tweezers

Holographic with the precise measure of strength.

The invention includes the generation of holographic optical traps with

orthogonal polarizations, as detailed below. In the system of

3rd

position detection for force measurement, a curler selects the

light of a single trapped particle, while the light of all other particles

Trapped samples are voided. This allows multiple manipulation while

one of the traps acts as a force sensor, using a method

Precise as BFP interferometry and power spectrum analysis.

35

Optical traps are generated by a spatial light modulator

   diffractive, through which light propagates with different index of

refraction (ordinary or extraordinary) depending on the polarization of the light

incident. The inventors have pointed out that, while the component

propagated in the direction of the extraordinary refractive index is modulated

by the diffractive light spatial modulator, the orthogonal component in

5

Ordinary refractive index direction remains named after

of going through the device.

According to one aspect of the invention, the method further comprises

the following stages:

1st

polarize the incident laser beam in a direction that is substantially

different from the address of the extraordinary refractive index of the modulator

spatial diffractive light (this step is done before beam modulation

To be);

create an optical trap, distinct from the pattern of holographic optical traps

fifteen

generated by said modulation, for a microscopic sample

distinct using the polarized beam component that is orthogonal to the

Direction of the extraordinary refractive index of the spatial light modulator

diffractive and, thus, not modulated (this is the trap that acts

as a force sensor);

2nd

separate the polarization of the light scattered by the optical trap pattern

holographic and by the different optical trap;

capture the image of the light scattered by the different optical trap with a

detector after being separated by polarization; Y

measure the optical forces acting on the microscopic sample other than

2 5

from the signals provided by the detector.

The resulting polarization in the distinct optical trap is orthogonal to the

polarization of the holographic optical trap pattern, allowing easy

separation of the light scattered by the different optical trap.

30

In one embodiment, the diffractive light spatial modulator is a device.

of parallel-aligned nematic liquid crystal, as a device for

liquid crystal on silicon ("LCoS-liquid crystal on silicon" in English).

35

In one embodiment, the incident laser beam is linearly polarized and its

   direction can be selected using a half-wave sheet located

before the space diffractive light modulator.

5

In one embodiment, the selectable polarization direction of the laser beam is inclined 30 ° -60 ° relative to the direction of the extraordinary refractive index. In one embodiment, the light scattered by the pattern of holographic optical traps and by the different optical trap is collected with a condensing lens.

1st

In one embodiment, the detector is a photodetector of positions.

In one embodiment, the position photodetector is a four quadrant detector or a side effect position sensor.

fifteen

In one embodiment, said detector is a camera.

In one embodiment, the movement of the different microscopic sample in the different optical trap is analyzed by focal plane interferometry.

2nd

In one embodiment, the polarization separation is performed by a linear polarizer.

25

In one embodiment, polarization separation and detection are integrated into a single device. The method may further comprise the step of correcting the aberrations of the different optical trap.

3rd

In one embodiment, the diffractive light spatial modulator is placed on a mobile platform that allows moving the different unmodulated trap in the sample plane.

35

The measurement of the optical forces can be done indirectly by measuring positions or directly from changes in the moment of light.

   Preferably, the size of the microscopic samples is located in the

range between O, 1 and 1 O microns in diameter.

In summary, the invention uses a spatial diffractive light modulator (DSLM)

of the type that modulates polarized light in an E direction, in which the light is

5

It propagates with the extraordinary refractive index, and does not modulate the light

polarized in the O direction, in which the light propagates with the index of

ordinary refraction, orthogonal to E. The incident light is polarized in a

address other than E. The orthogonal component to E, i.e. parallel to O, is

simply reflected without suffering any phase delay, while the phase of the

1st

component parallel to E is properly delayed to form traps

Holographic

The component of the orthogonal light to E forms, when reflected in the DSLM, a

fixed point of spurious light that can generate an uncontrollable optical trap

fifteen

different and unaffected by controllable holographic optical traps. The

polarization of this additional optical trap is also orthogonal to the

polarization of holographic optical traps, which allows their

discrimination in the detection stage by means of a suitable polarizer.

With the additional optical trap formed by spurious light, the force can be

2nd

precisely measured because the light scattered by the trapped sample

in the fixed trap it can be separated by polarization of the light scattered by

samples trapped in holographic traps.

Therefore, the invention allows multiple traps generated with

2 5

holographic optical tweezers while performing precise force measurements.

Therefore, it combines the advantages of holographic traps (stability,

flexibility, control 30 ...) with those of the force measurement methods of a

Unique optical trap (precision and simplicity). Or alternatively extend

precise force measurement methods developed for a single

3rd

trap to manipulate multiple traps.

Throughout the description and the claims the word "comprises" and

its variants are not intended to exclude other technical characteristics, additives,

components or steps. For experts in the field, other objects, advantages

3 5

and features of the invention will be derived in part from the description and

   in part of the practice of the invention. The following examples and drawings are

provided by way of illustration, and are not intended to be limiting of

The present invention. The present invention covers all possible

combinations of particular and preferred embodiments indicated herein. He

subject matter expert will be able to introduce modifications and replace

any technical element thereof for others that are equivalent

5

technically, depending on the specific requirements in each case, without

out of the scope of protection defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

1st

FIG. 1 is a diagram of a diffractive optical tweezers system

known.

FIG. 2A is a scheme of a liquid crystal cell.

FIG. 2B is another scheme of a liquid crystal cell.

FIG. 2C shows two orthogonal components of an electric field.

fifteen

FIG. 3 is a scheme of a plurality of optical traps.

FIG. 4 shows some calibration graphs.

FIG. 5 is another calibration chart.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

twenty

For a better understanding of the invention, FIG. 1 shows a montage

simplified known diffractive optical tweezers, comprising a

DOE1 02 diffractive optical element, a telescope 104 with L 1 and L2 lenses, and a

objective 1 05. An incident beam 1 01 is converted by DOE 1 02 in the

2 5

diffracted beams 1 03; beams 103 pass through telescope 104 and are

focal hoisted by objective 105 in different optical traps 106. The beam of

entry 101 is shown more intensively in the central region and less

Intense at the edges.

3rd

In holographic optical tweezers, the DOE is a spatial light modulator

("Spatiallight modulator-SLM 'in English) that spatially modifies the

incident wavefront to generate the desired 3D trap pattern in the

Sample plane. Normally, LCoS devices are used. In these,

a parallel-aligned nematic liquid crystal cell behaves

35

like a uniaxial crystal whose optical axis rotates with the electric field applied.

   Light propagates through the cell with the ordinary refractive index

(no) or extraordinary (nE) depending on the direction of field vibration

electrical (i.e., the polarization of the incident light), as represented

in FIG. 2A, which schematically shows an incident light beam 1 O and

an LCoS cell that includes liquid crystal molecules 15. These tend to

orient with the applied voltage V and accordingly rotate an angle e

5

(from e = 0 ° without voltage to e ::::: 90 ° for high voltages). While it is not

independent of the angle of rotation e, nE changes with the orientation of the

molecules and, therefore, with the gray level of the corresponding pixel of the

hologram A linearly polarized incident beam of light in the direction of the

extraordinary refractive index leaves the cell with the same polarization

1st

(vertical in the figure) and with a lag or (e) = 2n nE (e) d 1A., where d is the

cell thickness and A. is the wavelength of light. This corresponds to the

proper configuration of the device for phase-only modulation, which

used in conventional holographic optical tweezers. It is possible to generate

multiple traps with a phase ramp for positioning

fifteen

two-dimensional and a quadratic phase term for axial positioning

of each point of light.

FIG. 28 illustrates the reflective nature of an LCoS device showing

schematically the path of a ray of light 1 O, first through a

2nd

polarizer input 11 and cell LCoS 12, and then at the output of

said cell and through an output polarizer 13. Cell 12 consists

basically three layers: a mirror substrate 12a, the liquid crystal layer

12b and a glass cover 12c.

2 5

As shown in FIG. 2C, linearly polarized light has in the case

general two components of the electric field, one spreading with the

ordinary refractive index no and the other propagating with the index of

extraordinary refraction nE. The two components have polarizations

orthogonal Therefore, if the incident light has a different polarization than

3rd

the address of nE, one of the components of the electric field will propagate

with no and it will not be affected by changes in the gray level. This one

corresponds to unmodulated light that will focus on a small point in the

center of the sample plane. The polarization of this unmodulated trap and

fixed (FIG. 3) is perpendicular to the polarization of the modulated traps or

3 5

holographic 22, which can be positioned three-dimensionally for the

   Dynamic sample manipulation.

In one embodiment, the method of the present invention comprises the step

to modify the polarization of the incident light of a clamp assembly

conventional holographic optics rotating the polarization of the laser beam with

A half wave sheet. This results in unmodulated light and light

5

modulated, with perpendicular polarizations. The fixed trap is used for

measure of force while controllable traps are used for

manipulation and can be modified depending on the hologram

represented on the LCoS device. In FIG. 3, a unique trap, no

modulated and uncontrollable 21 is used for force measurement, and has a

1st

horizontal polarization 24. A set of holographic traps 22, which

they can position dynamically and independently at 30, they have the same

polarization 25, which is orthogonal to polarization 24 of the measuring trap

21. All of these optical traps are used to grab and manipulate the sample 20

through the handling of samples 23.

fifteen

When analyzing the movement of the trapped samples for the measurement of

force, a linear polarizer located before the detection system of

positions select only the fixed trap light. Scattered light

by the samples trapped in other points it is canceled. Therefore, the

2nd

standard methods of precise measurement of force developed for a

single optical trap can be used even in the presence of multiple

traps

Several experiments have been performed to demonstrate the invention. First

25

it was found that (for small displacements) all traps

they have a harmonic potential, that is, there is a linear relationship between the

strength and position of the sample in the trap, F = -kx. This is a requirement.

to apply standard measures of forces. The flatness of the devices

LCoS introduces optical aberrations at the incident wavefront that

3rd

deteriorates the quality of the focal light points raised. For the tweezers

holographic, these distortions can be corrected, in principle, with one's own

SLM adding appropriate phase values to the hologram. However, the

Fixed trap is not modulated and no compensation is possible.

35

FIG. 4 shows the calibration results of both a trap not

   modulated as a modulated trap. The analysis of the movement of a

Sample in one of these traps has been performed with BFP interferometry. The

first row shows the power spectra, which should be a function

Lorentziana, corresponding to a Gaussian distribution of positions

(second row) or, equivalently, to a harmonic potential (third row). The

graphs on the left correspond to the unmodulated trap, and those of the

5

right to one of the modulated or holographic traps. Both types of

trap present a harmonic behavior, as required in the

precise standard force measurements: the power spectrum is adjusted

either to a Lorentzian function, the histogram of positions of the sample to

a Gaussian distribution and the potential to a parabola of the form U =

1st

1 / 2kx2

It has also been proven that, when performing force measurements, the signal from the

light scattered by the fixed trap does not contain information of the light coming

of holographic traps, which would otherwise distort the results.

fifteen

In other words, the linear polarizer should effectively isolate the

light used to measure the strength of the rest, which has a polarization

orthogonal. To show this, a trap pattern consisting of

a modulated (holographic) trap and by the unmodulated trap. He

polarizer located before the photodetector in the measurement assembly was oriented

2nd

to select the light of the unmodulated trap. A microsphere was fixed to

a coverslip that was moved by a piezoelectric platform.

When the microsphere passed through the unmodulated beam, the photons were

dispersed throughout the sample and a change in the signal of the

photodetector (wavy line in FIG. 5). The detector hardly registered any

25

signal for the modulated trap (almost horizontal line in FIG. 5), since the

Light was canceled by the polarizer. The box in the graph shows a zone

expanded of the unwanted signal, which is only about 1-2% of the

significant signal

3rd

Therefore, the present invention makes multiple manipulation compatible

samples with holographic optical tweezers with the precise measurement of force. He

Assembly is simple and easily adaptable to a commercial microscope.

It is also compatible with direct force measurement methods, which

directly measure the force exerted by the optical tweezers, in contrast to

35

indirect methods, which are based on a model for the trap

   (harmonic potential), as well as for the sample (spherical) and the medium

surrounding (fluid with homogeneous refractive index and viscosity and with

low Reynolds number). Indirect methods also require

a previous calibration of the stiffness k, which depends on the conditions

Experimental The present invention allows extending the advantages of

Direct force measurement methods to multiple holographic traps.

5

Although only particular embodiments of

the invention in the present specification, the person skilled in the art will be able

of introducing modifications and replacing any technical element of the

same for others that are technically equivalent, depending on the

1st

specific requirements in each case, without going beyond the scope of protection

   defined in the appended claims.

Claims (6)

1. Method to manipulate microscopic samples and measure the forces that

they act on them, which comprises the steps of generating a laser beam and

5

modulate the laser beam using a space diffractive light modulator to

generate a pattern of holographic optical traps to manipulate a

plurality of microscopic samples; characterized because in addition

It comprises the steps of: polarizing the incident laser beam in one direction

which is substantially different from the direction of the refractive index

1st

extraordinary space diffractive light modulator, before the stage of

modulation; create an optical trap, other than the optical trap pattern

holographic, for a different microscopic sample using the component

of the polarized beam that is orthogonal to the direction of the index of refraction

extraordinary space diffractive light modulator and that, in that way, not

fifteen

be modulated; polarize the light scattered by the pattern of

holographic optical traps and by the different optical trap; capture the image

of the light scattered by the distinct optical trap with a detector after

be separated by polarization; and measure the optical forces acting on the

different microscopic sample from the signals provided by the

2nd

detector.

2. Method according to claim 1, wherein the spatial light modulator

diffractive is a parallel-aligned nematic liquid crystal device,

as a liquid crystal device on silicon, LCoS.

25

3. Method according to any of claims 1 or 2, wherein the laser beam

incident is linearly polarized and its direction can be selected

by means of a half wave sheet located before the spatial modulator of

diffractive light

30

4. Method according to claim 3, wherein the polarization direction

Selectable laser beam is tilted 30 ° -60 ° with respect to the direction of the

extraordinary refractive index.

35

5. Method according to any of the preceding claims, wherein the light

dispersed by the pattern of holographic optical traps and the optical trap

Different is collected with a condensing lens.

6. Method according to any of the preceding claims, wherein the detector is a photodetector of positions.

5

7. Method according to claim 6, wherein the photodetector of positions is a four quadrant detector or a side effect position sensor.

10

8. Method according to any of claims 1 to 5, wherein the detector is a camera. 9. Method according to any of the preceding claims, wherein the movement of the different microscopic sample in the different optical trap is analyzed by focal plane interferometry.

fifteen

1O. Method according to any of the preceding claims, wherein the polarization separation is performed by a linear polarizer.

2nd

11. Method according to any of claims 1 or 1 O, wherein the polarization separation and detection are integrated into a single device

12. Method according to any of the preceding claims, further comprising the step of correcting the aberrations of the different optical trap.

2 5

13. A method according to any of the preceding claims, wherein the diffractive light spatial modulator is placed on a mobile platform that allows the different unmodulated trap to be moved in the sample plane.

3rd

14. Method according to any of the preceding claims, wherein the measurement of the optical forces is made directly from changes in the moment of light.

35

15. Method according to any of claims 1 to 13, wherein the measurement of the optical forces is made indirectly by the measurement of positions.