DETAILED DESCRIPTION
10 [0018] Conventionally various techniques have been developed for
analyzing properties and behaviors of trapped particles suspended in a
medium. For example, different types of microfluidic devices have been
developed to study physical and chemical properties of particles suspended
in a fluid. In a conventional device, microfluidic device includes microfluidic
15 chips having one or more microchannels. Using dripping methods, a
solution comprising microscopic particles, such as cells, may be added drip
by drip to the microchannels. The cells may then be manipulated or
arranged in required manner and displayed or printed in different
arrangements for analysis of the cells. However, such devices are quite
20 complex, time consuming, and cumbersome to operate.
[0019] Another technique for particle analysis involves using an
optical tweezer. Since the first use for optical trapping of micron-sized latex
spheres suspended in water, the use of optical tweezers has grown
exponentially. The functioning of optical tweezers generally revolves around
25 the principle that light carries linear and angular momentum which can be
harvested to manipulate microscopic particles. Optical tweezers have been
used for understanding molecular forces (or torques) and mechanical
properties of proteins (DNA and RNA). In addition, Optical tweezers have
applications in sorting cells, sorting colloidal spheres, and understanding
30 single-molecule dynamics. In recent years, many variants of optical tweezers have been developed. For example, optical traps have been
realized by a variety of optical means such as, scanning point-like traps
where microscopic particles are trapped, or manipulated one by one, in a
single point-like trap. However, there are various limitations and
5 complexities associated with single point-like traps. For example, such
techniques are inherently slow as the microscopic particles are analyzed
one by one. Further, the techniques need precise alignment of the
microscopic particles, and are complex in nature. Also, since a single
microscopic particle is trapped in a point-like trap, multiple microscopic
10 particles cannot be arranged in a desired manner for analysis, for example,
to understand growth of cells and communication between multiple cells.
Therefore, the conventional optical tweezers are not efficient and effective
to perform the analysis.
[0020] The present subject matter relates to an optical tweezer
15 system. In particular, the present subject matter uses light-sheet for trapping
of the particles and therefore, are alternatively referred to as light-sheet
optical tweezer system. With the present subject matter, for example,
particles, such as dielectric particles, silica beads, live cells, live HeLa cells,
model organisms, such as Caenorhabditis elegans (C. elegans), and the
20 like, can be optically trapped and analyzed to study different properties
thereof.
[0021] In an example, an optical tweezer system may include a first
light source to emit a first light beam. The first light beam may be a laser
light beam. The optical tweezer system may include a beam expansion
25 element, a cylindrical lens, an objective lens, and a coverslip.
[0022] The beam expansion element may include at least one beam
expansion lens. The at least one beam expansion lens may include a
concave lens, a convex lens, a convex-concave lens, a concave-convex
lens, or a combination thereof. The beam expansion element may expand
30 the first light beam received from the first light source to form an expanded light beam. The expanded light beam may be formed on a back aperture of
the cylindrical lens.
[0023] The cylindrical lens may focus the expanded light beam on a
back aperture of objective lens, thereby forming a light-sheet at a working
5 distance of the objective lens. As is understood, the light-sheet may be a
sheet or line of light formed along one of a lateral axis.
[0024] The objective lens may transform the light-sheet received from
the cylindrical lens to generate a tightly-focused diffraction-limited lightsheet on the cover slip. As will be understood, the diffraction-limited light10 sheet may be the light-sheet formed with a resolution that is limited by
numerical aperture of the objective lens. In an example, the objective lens
may perform two-dimensional Fourier transform of the light-sheet received
from the cylindrical lens to generate the tightly-focused diffraction-limited
light-sheet. To enable the generation of the tightly-focused diffraction15 limited light-sheet on the cover slip, the cover slip may be disposed at a
focus of the objective lens. In an example, the tightly-focused diffractionlimited light-sheet may be generated perpendicular to a direction of
propagation of the first light beam. The tightly-focused diffraction-limited
light-sheet will be referred to hereinafter as the diffraction-limited light-sheet.
20 [0025] The coverslip may include a sample. The sample includes a
medium, such as a fluid medium, that has a plurality of microscopic
particles. The microscopic particles may be, for example, dielectric particles,
silica beads, live cells, live HeLa cells, model organisms, such as
Caenorhabditis elegans (C. elegans), and the like. The medium may be, for
25 example, deionized water, cell medium, buffer medium, such as phosphatebuffered Saline (PBS), and the like. The plurality of microscopic particles is
to be trapped in a predetermined pattern. During the operation, the
generation of the diffraction-limited light-sheet may cause the trapping of
the plurality of microscopic particles of the sample in the predetermined
30 pattern in the diffraction-limited light-sheet.
[0026] In an example, the generation of diffraction-limited light-sheet
may cause one or more forces acting on the plurality of microscopic particles
of the sample to enable the trapping of the plurality of microscopic particles
of the sample in the diffraction-limited light-sheet in the predetermined
5 pattern. The one or more forces may include a scattering force and a
gradient force. The scattering force may be caused due to reflection of the
diffraction-limited light-sheet. The scattering force may push the plurality of
microscopic particles away from a centre of the diffraction-limited lightsheet. The gradient force may be caused due to refraction of the diffraction10 limited light-sheet. The gradient force pulls the plurality of microscopic
particles of the sample towards the diffraction-limited light-sheet. In
particular, the gradient force may pull the plurality of microscopic particles
towards a centre of the diffraction-limited light-sheet. In an example,
magnitude of the gradient force may have to be greater than magnitude of
15 the scattering force to enable trapping of the plurality of microscopic
particles of the sample in the diffraction-limited light-sheet in the
predetermined pattern.
[0027] In an example, the rotation of the cylindrical lens about an
optical axis may cause rotation of the diffraction-limited light-sheet. For
20 instance, rotation of the cylindrical lens may cause corresponding rotation
of the light-sheet formed on the objective lens. The rotation of the light-sheet
formed on the objective lens may cause the rotation of the diffraction-limited
light-sheet. The rotation of the diffraction-limited light-sheet may enable
obtaining different patterns of trapping of the plurality of microscopic
25 particles. In addition, in some scenarios, the diffraction-limited light-sheet
may be translated. The rotation of the diffraction-limited light-sheet and the
translation of the diffraction-limited light-sheet may enable obtaining
different patterns of the trapping of the plurality of microscopic particles.
[0028] In an implementation, the optical tweezer system enables
30 analysis of the plurality of microscopic particles using a camera. In thisregard, the optical tweezer system includes an illumination unit and a
camera. The illumination unit to illuminate the coverslip to enhance the
visibility of the plurality of microscopic particles of the sample. The camera
may capture images and/or videos corresponding to the plurality of
5 microscopic particles of the sample. The capturing of the images and/or
videos may enable analysis of the plurality of microscopic particles.
Particularly, the images and/or videos may enable analysis of movements
and arrangement of the plurality of microscopic particles in the medium. The
camera may be, for example, a still camera, a video camera, a
10 Complementary Metal Oxide Semiconductor (CMOS) camera, a scientific
Complementary Metal Oxide Semiconductor (CMOS) camera, an array
detector, a charge-coupled Device (CCD) camera, an Electron Multiplying
Charge-Coupled Device (EMCCD) camera, or a combination thereof.
[0029] In an example, the illumination unit may include a second light
15 source, a condenser lens, an illuminator objective lens, a first mirror, a
dichroic mirror, and a tube lens. The second light source may generate a
second light beam. In an example, the second light source may be a white
light source and the second light beam may be a white light beam. The use
of the second light beam may enhance visibility of the coverslip with a large
20 field-of-view (FOV). The condenser lens may guide the second light beam
received from the second light source towards the illuminator objective lens.
The illuminator objective lens may direct the second light beam on the
sample. The first mirror may obtain a magnified reflection of the sample.
The dichroic mirror may allow only visible light from the magnified reflection
25 of the sample received from the a tube lens. The tube lens may direct the filtered light towards the camera.
[0030] In an example, the dichroic mirror and the first mirror may also
be used to form the light-sheet on the objective lens. In this regard, the
optical tweezer system may include a second mirror that may receive the
30 expanded light beam reflected from the beam expansion element. The
8
second mirror may reflect the expanded light beam towards the cylindrical
lens. The cylindrical lens may guide the expanded light beam towards the
dichroic mirror. The dichroic mirror may allow the expanded light beam to
pass through thereof and to direct the expanded light beam towards a first
5 mirror. The first mirror may reflect the expanded laser beam to focus on the
objective lens.
[0031] Since the present subject matter uses the light-sheet for
trapping of the plurality of microscopic particles, the optical tweezer system
may be referred to as the light-sheet optical tweezer system. The present
10 subject matter thus provides tunable and easy to align technique for
simultaneously trapping multiple particles by forming the diffraction-limited
light-sheet. Further, the light-sheet optical tweezer system may provide
capability to manipulate the particles via translation and rotation in the
transverse plane. For example, the cylindrical lens may be rotated along an
15 optical axis thereof to rotate the light-sheet in order to trap or arrange the
microscopic particles in a desired manner. By rotating the cylindrical lens,
the diffraction-limited light-sheet, formed by the objective lens, may also get
rotated. As a result, the one or more microscopic particles may be arranged,
or trapped, in a desired manner or at a desired angle.
20 [0032] Therefore, the present subject matter provides techniques for
trapping particles in a desired pattern. For example, one or more particles
may be arranged in a line by generating the diffraction-limited light-sheet on
the coverslip (therefore, on the sample). In traditional point-based traps,
light is focused by spherical optics (such as high numerical aperture (NA)
25 objectives) that produce point focus with maximum intensity at the center
giving rise to a well-defined point trap. Therefore, a single particle is trapped
in the point-like trap and multiple particles cannot be arranged in a desired
manner for any analysis. However, the present subject matter discloses
forming light along a line (i.e., forming light-sheet) rather than a point.
30 Therefore, in the present subject matter, a stable diffraction-limited light9
sheet is realized, that may be used to manipulate, pattern, and culture the
microscopic particles.
[0033] In the present subject matter, a camera is used for capturing
images and/or videos of the sample having the plurality of microscopic
5 particles that are arranged, or trapped, in the diffraction-limited light-sheet.
Therefore, from the captured images and/or videos, movement and
arrangement of the one or more microscopic particles can be analyzed. For
example, the particles may be arranged in a specific pattern to analyze
mechanical properties, molecular forces, and intercellular communication.
10 The particles, such as cells, can also be arranged in a specific pattern to
analyze growth of the cells. For instance, in cancer, the irregular or first mirror and may
reflect the light towards unbounded growth of cells, may be observed in any direction. However,
healthy cells may grow in a preferential direction. Therefore, by analyzing
growth of cells using the present subject matter, it may become possible to
15 distinguish between cancer cells and healthy cells. As a result, the present
subject matter may be used in medicine and healthcare industry for
analyzing growth of microscopic particles, such as cells or Live HeLa cells,
for identification of any abnormality, for example, cancer. The particles, such
as dielectric particles, can also be arranged in a specific pattern to analyze
20 particle to particle communication, intermolecular forces, and the like.
[0034] The present subject matter is further described with reference
to Figs. 1 to 6. It should be noted that the description and figures merely
illustrate principles of the present subject matter. Various arrangements
may be devised that, although not explicitly described or shown herein,
25 encompass the principles of the present subject matter. Moreover, all
statements herein reciting principles, aspects, and examples of the present
subject matter, as well as specific examples thereof, are intended to
encompass equivalents thereof.
[0035] Fig. 1 illustrates a block diagram of an optical tweezer system
30 100, according to an example implementation of the present subject matter.
10
In one example, the optical tweezer system 100 may be used for aligning a
plurality of microscopic particles in a desired manner. The microscopic
particles may be, for example, dielectric particles, silica beads, live cells,
live HeLa cells, model organisms, such as Caenorhabditis elegans (C.
5 elegans), and the like. The optical tweezer system 100 may generate lightsheet to optically trap the plurality of microscopic particles. Accordingly, the
optical tweezer system 100 is also referred to as light-sheet optical tweezer
system 100. Hereinafter, the optical tweezer system 100 will be referred to
as the light-sheet optical tweezer system 100. In an example
10 implementation, the light-sheet optical tweezer system 100 may include a
first light source 102 to emit a first light beam. The first light source 102 may
be, for example, a trap laser (e.g., a trap Infra-Red (IR) laser). The first light
beam may be, for example, the laser beam. Hereinafter, the first light source
102 will be explained with reference to the trap laser and the first light beam
15 will be explained with reference to the laser beam. The light-sheet optical
tweezer system may include a beam expansion element 104, a cylindrical
lens 106, an objective lens, and a coverslip 110.
[0036] In operation, the trap IR laser 102 may emit a laser beam and
transmit the beam towards the beam expansion element 104. The beam
20 expansion element 104 may expand the laser beam received from the trap
laser to form an expanded laser beam. The beam expansion element 104
may further guide the expanded laser towards the cylindrical lens 106. A
back aperture of the cylindrical lens 106 may receive the expanded laser
beam from the beam expansion element 104. The cylindrical lens 106 may
25 then form a light-sheet on a back aperture of an objective lens 108. The
light-sheet may be formed at a working distance of the objective lens 108.
As will be understood, the light-sheet may be a sheet or line of light formed
along one of a lateral axis. In an example, the cylindrical lens 106 may form
the light-sheet, by focusing the expanded laser beam on the back aperture
30 of the objective lens 108. The objective lens 108 may further transform the
light-sheet into a tightly-focused diffraction-limited light-sheet. The tightly11
focused diffraction-limited light-sheet will be referred to hereinafter as the
diffraction-limited light-sheet.
[0037] The diffraction-limited light-sheet may be the light-sheet
formed with a resolution that is limited by the numerical aperture of the
5 objective lens 108. In one example, the diffraction-limited light-sheet may
be formed at a focus of the objective lens 108. The objective lens 108 may
have a high Numerical Aperture (NA). Further, in an example, the coverslip
110 may be placed at the focus of the objective lens 108. The diffractionlimited light-sheet may thus be formed on the coverslip 110.
10 [0038] In an example, the coverslip 110 may include a sample. The
sample may have a medium, such as a fluid medium, having the plurality of
microscopic particles. The medium may be, for example, deionized water,
cell medium, buffer medium, such as phosphate-buffered Saline (PBS), and
the like. The plurality of microscopic particles may experience one or more
15 forces due to formation of the diffraction-limited light-sheet. In an example,
the plurality of microscopic particles may experience a gradient force and a
scattering force.
[0039] The scattering force may be due to reflection and may push
the plurality of microscopic particles away from the diffraction-limited light20 sheet. On the other hand, the gradient force may be due to refraction and
may exert a restoring force on the plurality of microscopic particles. Thus,
the gradient force pushes the plurality of microscopic particles towards high
intensity, which is a center of the diffraction-limited light-sheet. In general,
gradient force may have to be stronger than scattering force for a stable
25 trapping of the microscopic particles. Further, the gradient force increases
towards the center of the diffraction-limited light-sheet due to large intensity
at the center of the diffraction-limited light-sheet as compared to a periphery
of the diffraction-limited light-sheet. Practically, large gradient force is
ensured by the high NA objective lens 108. Therefore, when the gradient
30 force becomes more than the scattering force, the plurality of microscopic
12
particles may be attracted towards the diffraction-limited light-sheet. The
plurality of microscopic particles may thus be trapped, or arranged, in the
light-sheet.
[0040] The coverslip 110 may be observed, for example, using a
5 camera. In particular, the coverslip 110 may be observed under a
microscope equipped with a Complementary Metal Oxide Semiconductor
(CMOS) camera, to analyze patterns or arrangements of the plurality of
microscopic particles trapped in the diffraction-limited light-sheet, as will be
discussed with reference to Fig 2 and Fig. 3A.
10 [0041] Fig. 2 illustrates a block diagram of the light-sheet optical
tweezer system 100, according to another example implementation. As
mentioned earlier, the light-sheet optical tweezer system 100 may be used
for generating a diffraction-limited light-sheet for trapping the plurality of
microscopic particles, to form various patterns and shapes for analyzing
15 different properties of the plurality of microscopic particles. The light-sheet
optical tweezer system 100 may be used in different industries for analysis
of the plurality of microscopic particles. For example, the light-sheet optical
tweezer system 100 may be used in medicine and healthcare industry to
study cellular growth, cellular forces, cellular bonds, and intercellular
20 communication of live HeLa cells. The cells may be patterned in various
geometrical shapes and successfully cultured for a prolonged period of time.
In another example, the light-sheet optical tweezer system 100 may be used
for counting, arranging, and sorting of microscopic particles, for example,
dielectric silica beads and cells.
25 [0042] As mentioned earlier, the trap laser 102 may emit a laser
beam. In one example, a trap laser of wavelength 1064 nm may be used to
trap the microscopic particles. In another example, a trap laser of a
wavelength ranging from 705 nm to 2000 nm may also be used.
[0043] In an example, the beam expansion element 104 may include
30 at least one beam expansion lens 202. The at least one beam expansion
13
lens 202 may be, for example a concave lens, a convex lens, a convexconcave lens, a concave-convex lens, or a combination thereof for
expanding the laser beam. In an example, the beam expansion element
may include only a single beam expansion lens 202. In another example,
5 the beam expansion element 104 may include a plurality of beam expansion
lenses 202 arranged proximate to each other to expand the laser beam. For
instance, the beam expansion element 104 may include two beam
expansion lenses 202 arranged proximate to each other to expand the laser
beam, as illustrated in Fig. 3A. The laser beam may be expanded to spread
10 on a back aperture of the cylindrical lens 106, as illustrated in Fig. 3A.
[0044] In an example, the back aperture of the cylindrical lens 106
may receive the expanded laser beam and focus the expanded laser beam
on a back aperture of the objective lens 108. The objective lens 108 may be
placed at a focus of the cylindrical lens 106. The focal length of the
15 cylindrical lens may be, for example, 150 mm. The cylindrical lens 106 may
focus the expanded laser on the back aperture of the objective lens 108.
The light-sheet extending along an axis may thus be formed on the back
aperture of the objective lens 108. For example, the light-sheet may be
formed along y-axis, as illustrated in Fig. 3B.
20 [0045] The objective lens 108 may then transform the light-sheet into
a diffraction-limited light-sheet. In one example, the objective lens 108 may
perform Fourier transform of the light-sheet to form the diffraction-limited
light-sheet at focus of the objective lens 108. In one example, the objective
lens 108 may be a high numerical aperture (NA) objective lens with high
25 magnification capability. For example, the objective lens 108 have a
magnification factor of 100x and numerical aperture of 1.25. The rotation of
the light-sheet may cause rotation of the diffraction-limited light-sheet.
[0046] In an example, to obtain various patterns of trapping of the
plurality of microparticles, the cylindrical lens 106 may be rotated along an
30 optical axis thereof. The rotation of the cylindrical lens 106 may cause
14
rotation of the light-sheet 310 formed on the back aperture of the objective
lens 108. For example, when the cylindrical lens is rotated 15 degrees,
along the optical axis, in a clockwise direction, the light-sheet may
accordingly rotate. Therefore, by rotating the cylindrical lens 106 in a
5 desired manner, light-sheets oriented in different directions may be obtained
that may enable trapping of microscopic particles in a desired manner. [0047] Further, as previously described, the coverslip 110 may be
placed at the focus of the objective lens 108. The diffraction-limited lightsheet may thus be formed on the coverslip 110. The coverslip 110 may be
10 a transparent microscope coverslip. On formation of diffraction-limited lightsheet, the microscopic particles may experience one or more forces. For
example, the microscopic particles may experience a gradient force and a
scattering force.
[0048] In general, on formation of the diffraction-limited light-sheet,
15 two cases may arise. In a first case, the plurality of microscopic particles
may be much smaller than the wavelength of light (Rayleigh regime). In a
second case the plurality of microscopic particles may be larger than the
wavelength of light (Geometric regime). In the Rayleigh regime,
corresponding force along y-axis, 𝐹̅
𝑦
𝑔 =
𝜕𝑈
𝜕𝑦 = −𝛼
𝜕
𝜕𝑦 𝐼(𝑥, 𝑦,𝑡) =
−𝛼
𝜕
𝜕𝑦
〈𝐸⃗ (𝑦,𝑡)
2
〉 = −
𝛼
2
𝜕
𝜕𝑦
| 𝐸0
(𝑦,𝑡)|
2 = 𝛽
𝜕
𝜕𝑦 20 𝐼0(𝑦,𝑡) , where, 〈… 〉 is the time
average, and 〈𝐸⃗ (𝑥, 𝑦,𝑡)〉 = |𝐸0|/2. Variation of intensity along x-axis may
be negligible and does not change appreciably, except at far ends. Hence,
𝐹
𝑥 =
𝜕𝑈
𝜕𝑥 = 𝛽
𝜕
𝜕𝑥 𝐼0
(𝑥,𝑡) = 0. In the above expression, the refractive index
and permittivity has been absorbed in a single parameter, 𝛽 = 𝛼⁄2𝑐𝑛𝜖0,
25 where 𝑛 is the refractive index of the particle and 𝜖0 is the permittivity of
vacuum. Accordingly, the plurality of microscopic particles may be trapped
when the polarizability of the particle is greater than the surrounding media,
i.e., the medium of solution placed on the coverslip 110.
15
[0049] In the geometric regime, where the plurality of microscopic
particles may be larger in size than the wavelength of light, such as dielectric
beads, ray-optics may be employed to understand forces acting on the
plurality of microscopic particles. Classically, force on the particle can be
defined as the rate of change of momentum, 𝐹 =
𝜕𝑝
𝜕𝑡 5 , where 𝑝 is the
momentum of a microscopic particle. The conservation of momentum
necessitates exchange of momentum between light and the microscopic
particle. However, off-focal dielectric beads, i.e., dielectric beads that may
not be near the diffraction-limited light-sheet formed at focus of the objective
10 lens 108, may experience a net force towards a trap-center (high-intensity
region) of the diffraction-limited light-sheet due to gradient force, as
explained by the force diagram in Fig. 3D. A similar force but in the opposite
direction appears when the particle is on the other side of the diffractionlimited light-sheet.
15 [0050] Further, the scattering force 𝐹𝑆 may be primarily due to
reflection of light, and the scattering force in Rayleigh regime may be
expressed as, 𝐹
𝑆 =
𝑛𝑚 𝜎𝑆
𝑐
, where, 𝜎𝑆
is the cross-section of the microscopic
particle. The scattering forces are directly proportional to the cross-section
of microscopic particle. So, bigger microscopic particles experience a
20 greater scattering force. Fig. 3C shows elemental forces and the resultant
scattering force (𝐹
𝑧
𝑠
) due to the reflection of light at a surface of the
microscopic particle, such as a surface of a dielectric bead. Accordingly,
scattering (due to reflected light at the bead surface) results in momentum
transfer between light and the microscopic particles that tend to push the
particle out of focus of the objective lens 108 with force, 𝐹
𝑧
𝑠 25 (as illustrated in
Fig. 3C). The gradient force with its maximum at the trap-center leads to a
stable trap. Thus, a condition for a stable trap along the focus of the
objective lens 108 may be realized when the gradient force overcomes the
scattering forces and other forces, for example, buoyant force and forces
30 due to Brownian motion and gravity.
16
[0051] Therefore, the scattering force may be primarily due to
reflection and may push the plurality of microscopic particles away from the
diffraction-limited light-sheet. On the other hand, the gradient force may be
primarily due to refraction and may exert a restoring force on the plurality of
5 microscopic particles. So, the gradient force pulls the plurality of
microscopic particles towards high intensity, which is center of the
diffraction-limited light-sheet. Thus, when the gradient force becomes more
than the scattering force, the plurality of microscopic particles may be pulled
towards the diffraction-limited light-sheet. As a result, the plurality of
10 microscopic particles may be trapped in the diffraction-limited light-sheet.
[0052] Further, the coverslip 110 may be observed to analyze the
plurality of microscopic particles trapped in the diffraction-limited light-sheet.
In one example implementation, the light-sheet optical tweezer system 100
may include an illumination unit 204 to illuminate the coverslip 110, thereby
15 enhancing visibility of the plurality of microscopic particles. The light-sheet
optical tweezer system 100 may also include a camera 206 to capture
images of the coverslip 110. The camera 206 may be, for example, a still
camera, a video camera, a Complementary Metal Oxide Semiconductor
(CMOS) camera, a scientific Complementary Metal Oxide Semiconductor
20 (CMOS) camera, an array detector, a charge-coupled Device (CCD)
camera, an Electron Multiplying Charge-Coupled Device (EMCCD) camera,
or a combination thereof.
[0053] In one example implementation, the illumination unit 204 may
include a light source 208, a condenser lens 210, an illuminator objective
25 lens 212, a first mirror 214, a dichroic mirror 216, and a tube lens 218. The
light source may generate a light towards the condenser lens 210. In one
example, the light source 208 may be a Light Emitting Diode (LED) that may
emit white light. The condenser lens 210 may receive and guide the light
towards the illuminator objective lens 212. In one example, the condenser
17
lens 210 may be a convex lens that may guide light on the illuminator
objective lens 212.
[0054] The illuminator objective lens 212 may illuminate a large fieldof-view of the coverslip by allowing the light to pass through it and fall on
5 the coverslip 110. The coverslip 110 may thus be illuminated for better
viewing of the plurality of microscopic particles. In one example, the
illuminator objective lens 212 may be a low numerical aperture objective
lens and may have magnification capability. For example, the illuminator
objective lens 212 may have a numerical aperture of 0.25 and a
10 magnification factor of 10x to illuminate large field-of-view of the coverslip
110.
[0055] As the coverslip 110 is illuminated, a magnified reflection of
the coverslip 110 may be obtained on the first mirror 214 through the
objective lens 108. As discussed above, the objective lens 108 may be of a
15 magnification factor 100x. Therefore, the objective lens 108 may provide the
magnified reflection. The first mirror 214 may further reflect the magnified
reflection towards the dichroic mirror 216. In an example, the first mirror 214
may be placed at an angle to reflect the magnified reflection towards the
dichroic mirror 216.
20 [0056] As would be understood by a person skilled in the art, the
dichroic mirror 216 allows light of a certain wavelength to pass through,
while light of other wavelengths is reflected. The dichroic mirror 216, also
known as dichroic reflector, are commonly used behind a light source to
reflect visible light forward while allowing the invisible infrared light to pass
25 out from its rear. In an example, the dichroic mirror 216 may be a high-pass
dichroic mirror with a cut-off at 805 nm. The dichroic mirror 216 may filter
light and allow only visible light from the magnified reflection of the sample
and direct it towards the tube lens 218. In one example, the dichroic mirror
216 may be placed at an angle to reflect the saturated light towards the tube
30 lens 218.
18
[0057] The tube lens 218 may focus the saturated light at a focus
distance thereof. In one example, the tube lens 218 may be a convex lens
that may focus the saturated light at a focus thereof. Further, the camera
206 may be placed at the focus of the tube lens 218 to receive the white
5 light. The camera 206 may thus capture images and/or videos of the
plurality of microscopic particles trapped in the diffraction-limited light-sheet
formed on the coverslip 110. In one example, the camera 206 may capture
the images at predefined regular intervals of time. In another example, the
camera 206 may capture a video indicating movement of the microscopic
10 particles for being trapped into the diffraction-limited light-sheet. In one
example, the camera 206 may be a high-speed CMOS camera that may
capture 230 frames/second. Subsequently, the travel time (t) can be
calculated from the number of frames (of the video) between initial position
(microscopic particles undergoing free Brownian motion) to a final position
15 (trapped in the diffraction-limited light-sheet). The images or the videos may
then be analyzed, for example by a user, to characterize the system 100
and analyze different properties of the microscopic particles.
[0058] Fig. 3A illustrates a schematic diagram of the light-sheet
optical tweezer system 100, according to an example implementation.
20 During operation, the trap laser 102 may emit a laser beam towards the
beam expansion element 104, placed adjacent to the trap laser 102. The
beam expansion element 104 may include the beam expansion lenses 202-
1 and 202-2, collectively referred as the beam expansion lenses 202, to
expand the laser beam to form the expanded laser beam. For illustration
25 purposes, only two beam expansion lenses have been illustrated. However,
there may be less or more number of the beam expansion lens(es) 202 that
may be used for expanding the laser beam.
[0059] The expanded laser beam may be directed towards the
cylindrical lens 106 placed proximate to the beam expansion element 104.
30 The expanded laser beam may be guided towards a second mirror 302 that
19
may reflect the expanded laser beam towards the cylindrical lens 106. The
cylindrical lens 106 may receive and focus the expanded laser beam on the
back aperture of the objective lens 108. The cylindrical lens 106 may focus
the expanded laser beam on the back aperture of the objective lens 108
5 through the dichroic mirror 216. The cylindrical lens 106 may guide the
expanded laser beam towards the dichroic mirror 216. The dichroic mirror
216 may allow the expanded laser beam to pass through its rear side and
be directed towards the first mirror 214. The first mirror 214 may reflect the
expanded laser beam to focus on the back aperture of the objective lens
10 108. Thus, the cylindrical lens 106 may focus the light-sheet on the back
aperture of the objective lens 108, as illustrated in Fig. 3B.
[0060] The objective lens 108 may then transform the light-sheet into
the diffraction-limited light-sheet that may be focused on the coverslip 110.
In one example, the coverslip 110 may include a solution having one or
15 more microscopic particles 304. On formation of diffraction-limited lightsheet, the microscopic particles 304 may experience the gradient force and
the scattering force that may trap the microscopic particles 304 into the
diffraction-limited light-sheet. The scattering force may be primarily
experienced due to reflection and may push the microscopic particles 304
20 away from the diffraction-limited light-sheet, as illustrated in Fig. 3C. On the
other hand, the gradient force may be primarily due to refraction and may
exert a restoring force on the microscopic particles 304. So, the gradient
force pushes the microscopic particles towards high intensity, which is
center of the diffraction-limited light-sheet, as illustrated in Fig. 3D. Thus,
25 when the gradient force becomes more than the scattering force, the
microscopic particles 304 may be pulled towards the diffraction-limited lightsheet and trapped in the diffraction-limited light-sheet, as illustrated in Fig.
4.
[0061] Further, the coverslip 110 may be observed to analyze the
30 trapped microscopic particles 304. The illumination unit 204 may illuminate the coverslip 110 to provide a clear view of the plurality of microscopic
particles 304 present at the coverslip 110. The illumination unit 204 may
include the light source 208 to generate a light towards the condenser lens
210 placed adjacent to the light source 208. The condenser lens 210 may
5 receive and guide the light towards the illuminator objective lens 212. The
illuminator objective lens 212 may illuminate a large field-of-view of the
coverslip 110. The coverslip 110 may thus be illuminated for better viewing
of the microscopic particles 304.
[0062] Further, a magnified reflection of the coverslip 110 may be
10 obtained on the first mirror 214 through the objective lens 108. The first
mirror 214 may be placed at a desired angle to reflect the magnified
reflection towards the dichroic mirror 216. The dichroic mirror 216 may filter
light from the magnified reflection to produce saturated light that may be
highly saturated in color. In one example, the dichroic mirror 216 may be
15 placed at an angle to reflect the saturated light towards the tube lens 218.
[0063] The tube lens 218 may focus the saturated light towards the
camera 206 placed at the focal distance of the tube lens 218. The camera
206 may receive the saturated light and may thus capture one or more
images of the microscopic particles 304 trapped in the diffraction-limited
20 light-sheet formed on the coverslip 110. The camera 206 may capture the
one or more images at a predefined regular interval of time. The camera
206 may capture a video indicating movement of the microscopic particles
for being trapped into the diffraction-limited light-sheet. The images and the
videos captured by the camera 206 may be viewed by the user to analyze
25 properties related to the microscopic particles 304.
[0064] Fig. 3B illustrates a schematic view of the diffraction-limited
light-sheet, according to an example implementation. In one example, a
back aperture 306 of the cylindrical lens 106 may receive an expanded laser
beam 308, such as the expanded laser beam discussed above, from the
30 beam expansion element 104. In one example, the expanded laser beam
21
308 may propagate along an axis, such as z-axis, perpendicular to the plane
of the cylindrical lens 106. The cylindrical lens 106 may focus the expanded
laser beam 308 at the focus to form a light-sheet 310, such as the lightsheet discussed above. The light-sheet 310 may be formed on a back
5 aperture 312 of the objective lens 108 placed at the focus of the cylindrical
lens 106. In one example, the light-sheet 310 may be formed in a plane,
such as along y-axis, that may be perpendicular to direction of propagation
of the expanded laser beam 308.
[0065] The objective lens 108 may transform the light-sheet into a
10 diffraction-limited light-sheet 314, such as the diffraction-limited light-sheet
discussed above, and may be formed at a focus of the objective lens 108.
The coverslip 110 may be placed at the focus of the objective lens 108 to
form the diffraction-limited light-sheet 314 on the coverslip 110 to trap the
microscopic particles 304. Further, the cylindrical lens 106 may be rotated
15 along its axis to rotate the light-sheet 310. As a result, the diffraction-limited
light-sheet 314 may also be rotated and the microscopic particles 304 may
be trapped, arranged, or printed in a desired arrangement for analysis of
different properties.
[0066] Fig. 3C illustrates a schematic view of scattering force
20 experienced by the microscopic particles 304, according to an example
implementation. Fig. 3D illustrates a schematic view of gradient force
experienced by the microscopic particles 304, according to an example
implementation. As discussed above, the microscopic particles 304 may
experience a gradient force and a scattering force. The microscopic
25 particles 304 may be trapped when the polarizability of the microscopic
particles 304 is greater than the surrounding media, i.e., the medium of
solution placed inside the coverslip 110. In the geometric regime, where the
microscopic particles 304 may be larger in size than the wavelength of light,
such as dielectric beads, ray-optics may be employed to understand forces
30 acting on the microscopic particles 304. Classically, force on the
22
microscopic particles 304 can be defined as rate of change of momentum,
𝐹 =
𝜕𝑝
𝜕𝑡 , where 𝑝 is momentum of a microscopic particle. The conservation
of momentum necessitates exchange of momentum between light and the
microscopic particle. However, off-focal microscopic particles 304, such as
5 dielectric beads that may not be near the diffraction-limited light-sheet 314
formed at focus of the objective lens 108, may experience a net force (𝐹
𝑧
𝑔
)
towards a trap-center 316 (high-intensity region) of the diffraction-limited
light-sheet 314 due to gradient force, as explained by the force diagram in
Fig. 3D.
10 [0067] A similar force but in the opposite direction appears when the
particle is on the other side of the diffraction-limited light-sheet 314. The
scattering force 𝐹𝑆 may be primarily due to reflection of light, and the
scattering force in Rayleigh regime may be expressed as, 𝐹
𝑆 =
𝑛𝑚 𝜎𝑆
𝑐
,
where, 𝜎𝑆
is the cross-section of the microscopic particles 304. The
15 scattering forces are directly proportional to the cross-section of microscopic
particle. So, bigger microscopic particles experience a greater scattering
force. Fig. 3C shows elemental forces and the resultant scattering force (𝐹
𝑧
𝑠
)
due to reflection of light (illustrated by arrows 318 and 320) at a surface of
the microscopic particles 304, such as a surface of a dielectric bead.
20 Accordingly, scattering (due to reflected light at the bead surface) results in
momentum transfer between light and the microscopic particles 304 that
tends to push the particle out of focus with force, 𝐹
𝑧
𝑠
(as illustrated in Fig.
3C). The gradient force with its maximum at the trap-center 316 leads to a
stable trap. Thus, a condition for a stable trap along the focus of the
25 objective lens 108 may be realized when the gradient force overcomes the
scattering force.
[0068] Therefore, the scattering force may be primarily due to
reflection and may push the microscopic particles 304 away from the
diffraction-limited light-sheet 314. On the other hand, the gradient force may
23
be primarily due to refraction and may exert a restoring force on the
microscopic particles 304. The gradient force may push the microscopic
particles 304 towards high intensity, which is center of the diffraction-limited
light-sheet 314. When the gradient force becomes more than the scattering
5 force, the microscopic particles 304 may be pulled towards the diffractionlimited light-sheet 314. As a result, the microscopic particles 304 may be
trapped in the diffraction-limited light-sheet 314.
[0069] Fig. 4 illustrates a method 400 for trapping microscopic
particles using an optical tweezer system, according to an example
10 implementation. The order in which the method 400 is described is not
intended to be construed as a limitation, and any number of the described
method blocks may be combined in any order to implement the method 400,
or an alternative method. The optical tweezer system may correspond to
the optical tweezer system 100.
15 [0070] At step 402, a first light beam may be emitted from a first light
source of the optical tweezer system. The first light source may correspond
to the first light source 102. The first light source may be, for example, a trap
laser and the first light beam may be, for example, a laser beam.
[0071] At step 404, a light-sheet is formed by a cylindrical lens of the
20 optical tweezer system on an objective lens of the optical tweezer system
based on the first light beam. The cylindrical lens may correspond to the
cylindrical lens 106 and the objective lens may correspond to the objective
lens 108.
[0072] At step 406, the light-sheet received from the cylindrical lens
25 may be transformed by the objective lens to generate the tightly-focused
diffraction-limited light-sheet on the coverslip. The coverslip may include a
sample, and the sample may include a medium having a plurality of
microscopic particles that are to be trapped in a predetermined pattern. The
coverslip may correspond to the coverslip 110. In an example, the method
30 includes performing two-dimensional Fourier transform on the light-sheet
24
received from the cylindrical lens to generate the tightly-focused diffractionlimited light-sheet on the cover slip. In an example, the tightly-focused
diffraction-limited light-sheet may be generated perpendicular to a direction
of propagation of the first light beam.
5 [0073] At step 408, the plurality of microscopic particles of the sample
may be trapped in the predetermined pattern in the tightly-focused
diffraction-limited light-sheet. At step 410, the coverslip may be illuminated
by an illumination unit of the optical tweezer system to enhance the visibility
of the plurality of microscopic particles of the sample. The illumination unit
10 may correspond to the illumination unit 204.
[0074] At step 412, at least one of: images and videos corresponding
to the plurality of microscopic particles of the sample may be captured by a
camera of the optical tweezer system. The camera may correspond to the
camera 206.
15 [0075] At step 414, movements and arrangement of the plurality of
microscopic particles in the medium may be analyzed based on the
capturing of the at least one of: images and videos.
[0076] In an example, prior to the forming of the light-sheet by
cylindrical lens, the method includes receiving, by a beam expansion
20 element of the optical tweezer system, the first light beam from the first light
source. The first light beam may be expanded by beam expansion element
to form an expanded light beam. The expanded light beam received froM the beam expansion element may be focused by the cylindrical lens on the
objective lens. The beam expansion element may correspond to the beam
25 expansion element 104.
[0077] In an example, to enable the trapping of the plurality of
microscopic particles of the sample, the method includes causing one or
more forces to act on the plurality of microscopic particles of the sample due
to generation of the tightly-focused diffraction-limited light-sheet on the
25
coverslip. The one or more forces may include a scattering force caused
due to reflection of the tightly-focused diffraction-limited light-sheet. The
scattering force may push the plurality of microscopic particles away from
the tightly-focused diffraction-limited light-sheet. The gradient force may be
5 caused due to refraction of the tightly-focused diffraction-limited light-sheet.
The gradient force may push the plurality of microscopic particles of the
sample towards the tightly-focused diffraction-limited light-sheet. The
magnitude of the gradient force may have to be greater than magnitude of
the scattering force to enable trapping of the plurality of microscopic
10 particles of the sample in the tightly-focused diffraction-limited light-sheet in
the predetermined pattern.
[0078] In an example, to form of the light-sheet on the cylindrical lens,
the method includes disposing the objective lens at a focus of the cylindrical
lens. Further, to form the tightly-focused diffraction-limited light-sheet on the
15 coverslip, the method includes disposing the coverslip at a focus of the
objective lens.
[0079] The method includes rotating the cylindrical lens about an
optical axis thereof. The generated tightly-focused diffraction-limited lightsheet may be rotated based on the rotation of the cylindrical lens.
20 [0080] In an example, the method includes generating a second light
beam by a second light source of the illumination unit. The second light
source may be, for example, a white light source. The second light beam
may be, for example, white light beam. The second light source may
correspond to the second light source 208. The second light beam received
25 from the second light source may be guided by a condenser lens of the
illumination unit towards an illuminator objective lens of the illumination unit.
The condenser lens may correspond to the condenser lens 210 and the
illuminator objective lens may correspond to the illuminator objective lens
212. The second light beam may be directed on the sample by the
30 illuminator objective lens. A magnified reflection of the sample may be
26
obtained by a first mirror of the illumination unit. The first mirror may
correspond to the first mirror 214. Light from the magnified reflection of the
sample received from the first mirror may be filtered by a dichroic mirror of
the illumination unit. In an example, the filtering may include allowing only
5 visible light to pass through from the magnified reflection of the sample. The
dichroic mirror may correspond to the dichroic mirror 216. The filtered light
may be reflected towards a tube lens of the illumination unit by the dichroic
mirror. The tube lens may correspond to the tube lens 218. The filtered light
(i.e., the filtered visible light) may be directed towards the camera by the
10 tube lens.
[0081] In an example, the method includes receiving, by the beam
expansion element, the first light beam from the first light source. The first
light beam may be expanded by the beam expansion element to form an
expanded light beam. The expanded light beam reflected from the beam
15 expansion element may be received by a second mirror of the optical
tweezer system. The expanded light beam may be reflected towards the
cylindrical lens by the second mirror. The second mirror may correspond to
the second mirror 302. The expanded light beam may be guided towards
the dichroic mirror by the cylindrical lens. The expanded light beam may be
20 allowed by the dichroic mirror to pass through thereof. The expanded light
beam may be directed towards the first mirror by the dichroic mirror. The
expanded laser beam may be reflected by the first mirror to focus on the
objective lens and to form the light-sheet.
[0082] Since the present subject matter uses the light-sheet for
25 trapping of the plurality of microscopic particles, the optical tweezer system
may be referred to as the light-sheet optical tweezer system. The present
subject matter thus provides tunable and easy to align technique for
simultaneously trapping multiple particles by forming the diffraction-limited
light-sheet. Further, the light-sheet optical tweezer system may provide
30 capability to manipulate the particles via translation and rotation in the
27
transverse plane. For example, the cylindrical lens may be rotated along an
optical axis thereof to rotate the light-sheet in order to trap or arrange the
microscopic particles in a desired manner. By rotating the cylindrical lens,
the diffraction-limited light-sheet, formed by the objective lens, may also get
5 rotated. As a result, the one or more microscopic particles may be arranged,
or trapped, in a desired manner or at a desired angle.
[0083] Therefore, the present subject matter provides techniques for
trapping particles in a desired pattern. For example, one or more particles
may be arranged in a line by generating the diffraction-limited light-sheet on
10 the coverslip (therefore, on the sample). In traditional point-based traps,
light is focused by spherical optics (such as high numerical aperture (NA)
objectives) that produce point focus with maximum intensity at the center
giving rise to a well-defined point trap. Therefore, a single particle is trapped
in the point-like trap and multiple particles cannot be arranged in a desired
15 manner for any analysis. However, the present subject matter discloses
forming light along a line (i.e., forming light-sheet) rather than a point.
Therefore, in the present subject matter, a stable diffraction-limited lightsheet is realized, that may be used to manipulate, pattern, and culture the
microscopic entities, such as cells.
20 [0084] In the present subject matter, a camera is used for capturing
images and/or videos of the sample having the plurality of microscopic
particles that are arranged, or trapped, in the diffraction-limited light-sheet.
Therefore, from the captured images and/or videos, movement and
arrangement of the one or more microscopic particles can be analyzed. For
25 example, the particles may be arranged in a specific pattern to analyze
mechanical properties, molecular forces, and intercellular communication.
The particles, such as cells, can also be arranged in a specific pattern to
analyze growth of the cells. For instance, in cancer, the irregular or
unbounded growth of cells, may be observed in any direction. However,
30 healthy cells may grow in a preferential direction. Therefore, by analyzing
28
growth of cells using the present subject matter, it may become possible to
distinguish between cancer cells and healthy cells. As a result, the present
subject matter may be used in medicine and healthcare industry for
analyzing growth of microscopic particles, such as cells or Live HeLa cells,
5 for identification of any abnormality, for example, cancer. The particles, such
as dielectric particles, can also be arranged in a specific pattern to analyze
cell to cell communication, intermolecular forces, and the like.
VALIDATION AND RESULTS
[0085] Fig. 5A illustrates trapping of dielectric beads 502 in the
10 diffraction-limited light-sheet 314 over a period of time, according to an
example implementation. For the purpose of validation, a coverslip, such as
the coverslip 110, having a solution comprising the dielectric beads 502 was
used. The diffraction-limited light-sheet 314 was formed on the coverslip
110.
15 [0086] Experimental determination of trap-stiffness may begin with
the initial condition that the dielectric beads 502 are free in the solution and
exhibit Brownian motion. To visualize the functioning of light-sheet optical
tweezer system 100, dielectric beads 502 were suspended in deionized
water as a sample. A drop of the solution was dropped on the coverslip 110.
20 The coverslip 110 was then placed on an oil-dipped 100X (magnification
factor) objective lens, such as the objective lens 108. Subsequently, the
diffraction-limited light-sheet 314 was generated over the coverslip 110
comprising the solution having the dielectric beads 502. The dielectric
beads 502 may initially be randomly distributed. In the presence of
25 diffraction-limited light-sheet 314 (at travel time, t=0 seconds), some of the
randomly moving dielectric beads 502 feel the gradient force and are
directed towards the trap-center 316. The entire journey of the dielectric
beads 502 from time t=0 seconds (exhibiting Browning motion) to the trapcenter 316 was recorded by the camera 206. In one example, the camera
30 206 may be a high-speed CMOS camera (Gazelle, Pointgray, United States
29
of America (USA)). Subsequently, the travel time (t) can be calculated from
the number of frames (of the recorded video) between the initial position of
the dielectric beads 502 to the final position (the trap-center 316). From the
video, several free dielectric beads 502 are tracked on their way to the trap5 center 316 to calculate the time.
[0087] As illustrated in Fig. 5A, a plurality of frames, including a first
frame 504-1, a second frame 504-2, a third frame 504-3, a fourth frame 504-
4, a fifth frame 504-5, and a sixth frame 504-6 have been captured or
recorded at different times after initiation (t=0 seconds). The plurality of
10 frames 504-1, 504-2, 504-3, 504-4, 504-5, and 504-6 may be collectively
referred to as frames 504 or frame 504. The first frame 504-1 captured or
recorded at 16.04 seconds illustrates two dielectric beads 502 trapped in
the trap-center 316 of the diffraction-limited light-sheet 314. Also, a dielectric
bead 502-1 can been seen being pulled towards the trap-center 316 due to
15 the gradient force. In the second frame 504-2 (recorded at 16.30 seconds),
the dielectric beads 502-1 can be seen trapped by the trap-center 316. The
third frame 504-3 (taken at t=20.90 seconds) shows an approaching free
dielectric bead 502-2 which is eventually attracted by the gradient force and
the dielectric bead 502-2 can been seen trapped in the trap-center 316 in
20 the fourth frame 504-4 (taken at 21.62 seconds). Subsequently, the
dielectric beads 502 slide down to the other end of the diffraction-limited
light-sheet 314 (as can been seen in fifth frame 504-5 captured at 22.02
seconds) due to slight tilt in a sample holder. Over time a, number of
dielectric beads 502 are arranged in a line (the diffraction-limited light-sheet
25 314), as seen from the image in the sixth frame 504-6 recorded at 37.60
seconds. Therefore, the microscopic particles 304 may be arranged in a
line. Also, an average time taken by the microscopic particles 304 to reach
the trap-center 316 can be calculated. In this case, the dielectric beads 502
took, on an average, 0.70 seconds to reach the trap-center 316. This may
30 enable calculation of trap-stiffness.
30
[0088] Fig. 5B illustrates trapping of live HeLa cells 506 in the
diffraction-limited light-sheet 314 over a period of time, according to an
example implementation. Similar to dielectric beads 502, the live HeLa cells
506 were trapped. The live HeLa cells 506 were suspended in a cell medium
5 and thoroughly pipetted. A small amount (about 10 lit) was dropped on the
coverslip 110, which was attached to a 3-axis nanopositioning stage (for
example, MAX3SLH, Thorlabs, USA). Subsequently, the diffraction-limited
light-sheet 314 was generated over the coverslip 110 for trapping,
patterning, and culturing the live HeLa cells 506. The live HeLa cells 506 in
10 proximity of the trap-center 316 were immediately attracted by the gradient
force of the diffraction-limited light-sheet 314. Generally, the live HeLa cells
506 are heavier than the dielectric beads 502. Therefore, the live HeLa cells
506 are relatively slow to move and take more time to reach the trap-center
316. Experiments show that the following relation holds good: m / t= po,
15 where m is the mass of the microscopic particle 304 (live HeLa cells 506 in
this case), t is the time taken by the microscopic particle 304 to reach the
trap-center 316, and po is a constant related to the trap-stiffness. It may thus
be concluded that a large mass takes more time to reach the trap-center
316 and vice-versa.
20 [0089] As can be seen in Fig. 5B, at the time t=0, the live HeLa cells
506 are freely moving in the medium. However, the live HeLa cells 506 start
moving towards the trap-center 316 of the diffraction-limited light-sheet 314
as the trap laser 102 was turned on. Using the camera 206, it can be
observed that the live HeLa cells 506 took an average of 8.57 seconds to
25 reach the trap-center 316 which is approximately 12.25 times slower than
dielectric beads 502. This may be due to relative large weight of HeLa cells
as compared to dielectric beads.
[0090] Fig. 5C illustrates patterning of the live HeLa cells 506 at
varying angles, according to an example embodiment. As discussed above,
30 the cylindrical lens 106 may form the light-sheet 310 on back aperture of the
31
objective lens 108. The objective lens 108 may transform the light-sheet 310
into the diffraction-limited light-sheet 314 that may be focused on the
coverslip 110.
[0091] In one example implementation, the cylindrical lens 106 may
5 be rotated along its optical axis to rotate the light-sheet 310, and thereby
rotating the diffraction-limited light-sheet 314. Rotating the cylindrical lens
106 may provide an advantage. For example, the cylindrical lens 106 may
be rotated to a desired angle along its axis and, as a result, the diffractionlimited light-sheet 314 may accordingly rotate. Therefore, the microscopic
10 particles 304 may be aligned, or trapped, in the diffraction-limited light-sheet
314 formed at a desired angle.
[0092] In Fig. 5C, a cartesian plane 508 has been illustrated for
visualization of different angles at which the diffraction-limited light-sheet
314 may be formed on the coverslip 110. The angles indicated in the
15 cartesian plane 508 are only for illustration purposes and the diffractionlimited light-sheet 314 may be rotated at other angles also. A plurality of
frames 510 were captured using the camera 206. The frames 510 indicate
patterning of microscopic particles 304, such as the live HeLa cells 506,
using the diffraction-limited light-sheet 314 formed at different angles on the
20 coverslip 110. The plurality of frames 510 may include a first frame 510-1,
a second frame 510-2, a third frame 510-3, and a fourth frame 510-4.
[0093] The first frame 510-1 illustrates formation of the diffractionlimited light-sheet 314 at 15° angle. Therefore, the live HeLa cells 506 get
trapped in the diffraction-limited light-sheet 314 and are patterned at 15°
25 angle. Similarly, the second frame 510-2 illustrates formation of the
diffraction-limited light-sheet 314 at 90° angle. Therefore, the live HeLa cells
506 get trapped in the diffraction-limited light-sheet 314 and are patterned
at 90°.
[0094] In the third frame 510-3, the diffraction-limited light-sheet 314
30 is formed at 0° angle. Therefore, the live HeLa cells 506 get trapped in the
32
diffraction-limited light-sheet 314 and are patterned at 0° angle. Similarly, inthe fourth frame 510-4, the live HeLa cells 506 may be patterned at 150°.
[0095] Patterned cell growth plays a critical role during the early
development of multicellular organisms and may be essential for cells, such
5 as the live HeLa cells 506, to communicate with each other that control its
growth at a healthy rate. Uncontrolled growth is known to occur in cancer.
In the present study, live HeLa cancer cells have been considered. The live
HeLa cancer cells were thawn and grown in a 35 mm disc supplemented
with cell medium (DMEM+FBS). To detach them from the surface, the live
10 HeLa cancer cells were tripsinated, followed by centrifugation and
resuspension according to standard sample preparation protocols.
Subsequently, the floating live cells (spherical shape) were subjected to the
diffraction-limited light-sheet 314. One-by-one the live HeLa cancer cells
were trapped by the diffraction-limited light-sheet 314 and aligned in a line
15 as displayed in Fig. 5B. The corresponding timeline is also indicated. The
results show that the technique can pattern cells in a preferential direction
(such as, along a line). In addition, the diffraction-limited light-sheet 314 can
be rotated in the transverse plane, facilitating patterning at any desired
angle, as illustrated in Fig. 5C. Furthermore, the technique may allow
20 patterned growth of cells in specific shapes and enables sustained culturing
for long hours (for example, up to 18 hours). The experimental results, as
discussed above, show that the light-sheet optical tweezer system 100 may
be a promising technique for cell trapping, patterning, and culturing, all on a
single platform.
25 [0096] Fig. 5D illustrates patterning of the live HeLa cells 506 for
writing “IISC”, according to an example embodiment. As discussed above,
by using the diffraction-limited light-sheet 314, the microscopic particles 304
may be arranged or patterned in any desired orientation. For illustration
purposes, Fig. 5D illustrates patterning of the live HeLa cells 506 to print
30 “IISC”, as illustrated in frames 512-1, 512-2, 512-3, and 512-4 in Fig. 5D.
33
The microscopic particles 304 may be arranged in any specific manner by
changing orientation of the diffraction-limited light-sheet 314, as discussed
above.
[0097] Fig. 5E illustrates both patterning (as, C and T) and culturing
5 of the live HeLa cells 506, according to an example embodiment. In one
example, the live HeLa cells 506 were patterned in L shape, as illustrated
in frame 513-1. Subsequently, the patterned live HeLa cells 406 were
cultured for a longer duration, for example for 8 hours and 18 hours, as
illustrated in frames 513-2 and 513-3, in a standard cell culture incubator
maintained at a temperature of 37 10 o C and supplemented with 5% carbon
dioxide (CO2).
[0098] Similarly, the live HeLa cells 506 were patterned in T shape,
as illustrated in frame 514-1. Subsequently, the patterned live HeLa cells
506 were cultured for a duration of 8 hours and 18 hours, as illustrated in
15 frames 514-2 and 514-3, in a standard cell culture incubator maintained at
a temperature of 37o C and supplemented with 5% carbon dioxide (CO2).
[0099] In one example, by patterning and culturing the live HeLa cells
506 for a prolonged period of time, different properties related to cells may
be analyzed. For example, normal physiology and biochemistry of the live
20 HeLa cells 506 may be analyzed. In another example, by patterning and
culturing the live HeLa cells 506 for a prolonged period of time, effects of
drugs and toxic compounds on the live HeLa cells 506 may be analyzed.
Moreover, the technique may enable artificial tissue engineering and
engineered human tissues using light-sheet optical tweezer system 100.
25 The technique may also be used in the pre-clinical screening of new drugs.
[00100] Fig. 6 illustrates a graph indicating trap-stiffness at various
light intensities, according to an example embodiment. Spherical silica
beads (size ~ 2 m) suspended in deionized water were used as a sample
to estimate trap-stiffness. In general, suspended microscopic particles, or
30 beads, undergo random Brownian motion but follow a directed motion
34
(towards the trap-center) under the influence of gradient force. To a good
approximation, an optical trap behaves like a harmonic potential, and it can
exert a restoring force. Specifically, near the trap-center, the force can be
approximately modeled by Hooke's law, and the restoring/gradient force is
5 given by F(x) = -kx, where k is the trap-stiffness (N/m) and x is the
displacement from trap-center. The second force acting on the particle is
viscous drag force. Assuming spherical beads, the particles moving through
the medium, such as a solution or a fluid, experiences a viscous force of,
𝐹𝑣𝑖𝑠 = −6𝜋𝜂𝑟𝑏𝑣. For simplicity and calculating approximate trap-stiffness,
10 effect of gravity on the silica bead may be neglected. As a result, forces due
to weight and buoyancy may also be ignored. Thus, motion of the silica
beads may be governed by the forces, for example, gradient and
viscous/drag force, which are opposite. Balancing optical (gradient) forces
with drag force produces, −𝑘𝑥 = −6𝜋𝜂𝑟𝑏𝑣 ⇒ 𝑘 = 6𝜋𝜂𝑟𝑏 (
𝑣
𝑥
) ⇒ 𝑘 =
15 6𝜋𝜂𝑟𝑏/𝑡 , where 𝑥 = 𝑣𝑡 . Here, 𝑣 is the dragging velocity, 𝑥 is the
displacement, 𝑡 is the time, 𝜂 is the medium viscosity, and 𝑟𝑏 = 𝑑/2 is the
bead radius. Given the dynamic condition (internal flow and cell dynamics)
of the study, the proposed technique has given a good estimate. Here, this
relation may be used to determine the trap-stiffness. Further, it may be
20 noted that the dielectric beads 502 take a large time or equivalently more
number of frames to reach the trap-center 316.
[00101] In the graph illustrated in Fig. 6, number of black dots indicate
a number of frames taken during the trapping process and an insets 602-1,
602-2, 602-3, 602-4, and 602-5 indicate time taken by a single microscopic
25 particle 304 to reach the trap-center 316 at varying intensities. From the
graph, it may be evident that on increase in power (at which the laser beam
is emitted by the trap laser 102), reduction in the number of black dots is
observed. Therefore, with increase in power, the intensity at the trap-center
316 increases, resulting in large trap-stiffness. Hence, the microscopic particles 304 may take less time to reach the trap-center 316. As a result,
35
less number of frames may be captured as the trapping process may
complete in less time due to strong trap, necessitating quick trapping and
alignment of the microscopic particles 304. The same can also be observed
from the insets 602-1, 602-2, 602-3, 602-4, and 602-5.
5 [00102] Although examples for the present subject matter have been
described in language specific to structural features and/or methods, it
should be understood that the present subject matter is not limited to the
specific features or methods described. Rather, the specific features and
methods are disclosed and explained as examples of the present subject
10 matter.
36
I/We Claim:
1. An optical tweezer system comprising:
a coverslip comprising a sample, the sample comprising a
medium having a plurality of microscopic particles that are to be
5 trapped in a predetermined pattern,
a first light source to emit a first light beam;
a beam expansion element to expand the first light beam
received from the first light source to form an expanded light beam;
a cylindrical lens to focus the expanded light beam received from
10 the beam expansion element on an objective lens and to form a lightsheet on the objective lens; and
the objective lens to transform the light-sheet received from the
cylindrical lens to generate a tightly-focused diffraction-limited lightsheet on the coverslip to trap the plurality of microscopic particles of
15 the sample in the predetermined pattern in the tightly-focused
diffraction-limited light-sheet.
2. The optical tweezer system as claimed in claim 1, wherein the
generation of tightly-focused diffraction-limited light-sheet is to cause one or
more forces acting on the plurality of microscopic particles of the sample to
20 enable the trapping of the plurality of microscopic particles of the sample in
the tightly-focused diffraction-limited light-sheet in the predetermined
pattern.
3. The optical tweezer system as claimed in claim 2, wherein the one
or more forces comprises:
25 a scattering force caused due to reflection of the first light beam,
wherein the scattering force pulls the plurality of microscopic particles
away from the tightly-focused diffraction-limited light-sheet; and
37
a gradient force caused due to refraction of the first light beam,
wherein the gradient force pulls the plurality of microscopic particles of
the sample towards the tightly-focused diffraction-limited light-sheet.
4. The optical tweezer system as claimed in claim 3, wherein magnitude
5 of the gradient force is to be greater than magnitude of the scattering force
to enable trapping of the plurality of microscopic particles of the sample in
the tightly-focused diffraction-limited light-sheet in the predetermined
pattern.
5. The optical tweezer system as claimed in claim 1, wherein the tightly10 focused diffraction-limited light-sheet is generated perpendicular to a
direction of propagation of the first light beam.
6. The optical tweezer system as claimed in claim 1, wherein the
objective lens is disposed at a focus of the cylindrical lens.
7. The optical tweezer system as claimed in claim 1, wherein the
15 coverslip is disposed at a focus of the objective lens.
8. The optical tweezer system as claimed in claim 1, wherein the
objective lens is to perform Fourier transform of the light-sheet received
from the cylindrical lens to generate the tightly-focused diffraction-limited
light-sheet.
20 9. The optical tweezer system as claimed in claim 1, wherein rotation of
the cylindrical lens about an optical axis of the cylindrical lens is to cause
rotation of the tightly-focused diffraction-limited light-sheet.
10. The optical tweezer system as claimed in claim 1, wherein the beam
expansion element comprises at least one beam expansion lens, wherein
25 the at least one beam expansion lens comprises a concave lens, a convex
lens, a convex-concave lens, a concave-convex lens, or a combination
thereof.
38
11. The optical tweezer system as claimed in claim 1, wherein the first
light source is a trap laser and the light emitted by the first light source is a
laser beam.
12. The optical tweezer system as claimed in claim 1, comprising:
5 an illumination unit to illuminate the coverslip to enhance visibility
of the plurality of microscopic particles of the sample; and
a camera to capture at least one of: images and videos
corresponding to the plurality of microscopic particles of the sample.
13. The optical tweezer system as claimed in claim 12, wherein the
10 illumination unit comprises:
a second light source to generate a second light beam;
a condenser lens to guide the second light beam received from
the second light source towards an illuminator objective lens;
the illuminator objective lens to direct the second light beam on
15 the sample;
a first mirror to obtain a magnified reflection of the sample;
a dichroic mirror to filter light from the magnified reflection of the
sample received from the first mirror to allow only visible light and to
reflect the filtered visible light towards a tube lens; and
20 the tube lens to direct the filtered visible light towards the camera.
14. The optical tweezer system as claimed in claim 13, wherein the
second light beam is a white light beam.
15. The optical tweezer system as claimed in claim 1, comprising:
a second mirror to receive the expanded light beam reflected
25 from the beam expansion element and to reflect the expanded light
beam towards the cylindrical lens;
a dichroic mirror, wherein the cylindrical lens is to guide the
expanded light beam towards the dichroic mirror, wherein the dichroic
39
mirror is to allow the expanded light beam to pass through thereof and
to direct the expanded light beam towards a first mirror; and
the first mirror, wherein the first mirror is to reflect the expanded
light beam to focus on the objective lens.
5 16. The optical tweezer system as claimed in claim 1, wherein the
plurality of microscopic particles comprises dielectric particles, silica beads,
live cells, live HeLa cells, and model organisms.
17. A method for trapping microscopic particles using an optical tweezer
system, the method comprising:
10 emitting, from a first light source of the optical tweezer system, a
first light beam;
forming, by a cylindrical lens of the optical tweezer system, a
light-sheet on an objective lens of the optical tweezer system based
on the first light beam;
15 transforming, by the objective lens, the light-sheet received from
the cylindrical lens to generate tightly-focused diffraction-limited lightsheet on a coverslip of the optical tweezer system, wherein the
coverslip comprises a sample, and wherein the sample comprises a
medium having a plurality of microscopic particles that are to be
20 trapped in a predetermined pattern;
trapping the plurality of microscopic particles of the sample in
the predetermined pattern in the tightly-focused diffraction-limited
light-sheet;
illuminating, by an illumination unit of the optical tweezer system,
25 the coverslip to enhance visibility of the plurality of microscopic
particles of the sample;
capturing, by a camera of the optical tweezer system, at least one
of: images and videos corresponding to the plurality of microscopic
particles of the sample; and
40
analysing, based on the capturing of the at least one of: images
and videos, movements and arrangement of the plurality of
microscopic particles in the medium.
18. The method as claimed in claim 17, wherein prior to the forming of
5 the light-sheet by the cylindrical lens, the method comprises:
receiving, by a beam expansion element of the optical tweezer
system, the first light beam from the first light source;
expanding, by the beam expansion element, the first light beam
to form an expanded light beam; and
10 focusing, by the cylindrical lens, the expanded light beam
received from the beam expansion element on the objective lens.
19. The method as claimed in claim 17, wherein to enable the trapping
of the plurality of microscopic particles of the sample, the method
comprises:
15 causing one or more forces to act on the plurality of microscopic
particles of the sample due to generation of the tightly-focused
diffraction-limited light-sheet on the coverslip, wherein the one or more
forces comprises:
a scattering force caused due to reflection of the first light
20 beam, wherein the scattering force pushes the plurality of
microscopic particles away from the tightly-focused diffractionlimited light-sheet; and
a gradient force caused due to refraction of the first light
beam, wherein the gradient force pushes the plurality of
25 microscopic particles of the sample towards the tightly-focused
diffraction-limited light-sheet.
20. The method as claimed in claim 19, wherein magnitude of the
gradient force is to be greater than magnitude of the scattering force to
41
enable trapping of the plurality of microscopic particles of the sample in the
tightly-focused diffraction-limited light-sheet in the predetermined pattern.
21. The method as claimed in claim 17, wherein the tightly-focused
diffraction-limited light-sheet is generated perpendicular to a direction of
5 propagation of the first light beam.
22. The method as claimed in claim 17, wherein to form of the light-sheet
on the cylindrical lens, the method comprises:
disposing the objective lens at a focus of the cylindrical lens, and
wherein to form the tightly-focused diffraction-limited light-sheet on the
10 coverslip, the method comprises:
disposing the coverslip at a focus of the objective lens.
23. The method as claimed in claim 17, wherein to generate the tightlyfocused diffraction-limited light-sheet on the coverslip, the method
comprises:
15 performing, by the objective lens, fourier transform of the lightsheet received from the cylindrical lens.
24. The method as claimed in claim 17, comprising:
rotating the cylindrical lens about an optical axis thereof to rotate
the generated tightly-focused diffraction-limited light-sheet.
20 25. The method as claimed in claim 24, comprising:
generating, by a second light source of the illumination unit, a
second light beam;
guiding, by a condenser lens of the illumination unit, the second
light beam received from the second light source towards an
25 illuminator objective lens of the illumination unit;
directing, by the illuminator objective lens, the second light beam
on the sample;
42
obtaining, by a first mirror of the illumination unit, a magnified
reflection of the sample;
filtering, by a dichroic mirror of the illumination unit, light from the
magnified reflection of the sample received from the first mirror to allow
5 only visible light;
reflecting, by the dichroic mirror, the filtered visible light towards
a tube lens of the illumination unit; and
directing, by the tube lens, the filtered visible light towards the
camera.
10 26. The method as claimed in claim 17, comprising:
receiving, by a beam expansion element of the optical tweezer
system, the first light beam from the first light source;
expanding, by the beam expansion element, the first light beam
to form an expanded light beam;
15 receiving, by a second mirror of the optical tweezer system, the
expanded light beam reflected from the beam expansion element;
reflecting, by the second mirror, the expanded light beam
towards the cylindrical lens;
guiding, by the cylindrical lens, the expanded light beam towards
20 a dichroic mirror of the optical tweezer system;
allowing, by the dichroic mirror, the expanded light beam to pass
through thereof;
directing, by the dichroic mirror, the expanded light beam towards
a first mirror of the optical tweezer system; and
25 reflecting, by the first mirror, the expanded light beam to focus on
the objective lens and to form the light-sheet.