Laser trapping apparatus

A laser trapping apparatus for optically trapping an optional micro-particle from a group of micro-particles such as microorganisms suspended in a medium by a laser beam focused at a focal point of an optical converging system at the focal point, the apparatus comprising: a parallel beam output device for outputting a plurality of laser beams around an optical axis in parallel with said optical axis, and an optical converging system having an objective lens for focusing the plurality of laser beams irradiated from the parallel beam output device to the focal point.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention concerns a laser trapping apparatus that optically, 
traps a certain particle from a group of particles, such as microorganisms 
dispersed and suspended in a medium by a laser beam focused at a focal 
point of an optical converging system. 
2. Description of the Prior Art 
The optical particle trapping technique has been developed by A. Ashkin, et 
al (Physical Review Letters, Vol. 54, 1985). 
As shown in FIG. 6, two objective lenses are arranged along an identical 
optical axis around a trapping position as a center, and two laser beams 
are irradiated along the identical optical axis from both opposing sides 
of the trapping position thereby trapping a particle 2 in a cell 3, which 
is called as "trapping by radiation pressure". 
Because the two objective lenses are opposed to each other in use, this 
system is problematic in that the optical axes of the lenses must be 
aligned accurately to each other at a 1 .mu.m order. 
Further, when a mechanical operation is conducted, such as, sucking a 
particle trapped between each of the objective lenses by a micropipette, 
the operation must be conducted by inserting the pipette in a narrow gap 
between each of the lenses. The top end however can not be moved freely, 
resulting in a problem of poor maneuverability. 
In order to overcome such a disadvantage, A. Ashkin et al have proposed a 
technique of trapping a particle by irradiating only one laser beam 
instead of irradiating two laser beams to the particle (Optics Letters 
Vol. 11 No. 5, 1986). 
In this technique, as shown in FIG. 7, a laser beam is converged by one 
objective lens thereby trapping a particle to a focal point, which is 
referred to as "single-beam gradient force optical trap". 
According to this method, since the particle can be trapped by irradiating 
light only from one direction, there is no requirement of aligning optical 
axes of two optical systems. Accordingly, if an optical system of an 
inverting microscope is utilized to invert the irradiation direction, and 
a laser beam is irradiated for trapping just beneath a cell 3 storing a 
liquid medium in which particles 2 are dispersed and suspended, a 
micropipette or the like can be operated freely since the upper surface of 
the cell is opened. Therefore, this method has attracted an attention as a 
technique capable of handling particles easily. 
This configuration of laser trapping, however, is problematic in that the 
optical power density at the focal point is extremely high (from 10.sup.6 
to 10.sup.7 times of the sunlight on the earth). Therefore the optical 
power may damage microorganisms when trapped as a particle (refer, for 
example, to Hong, Liang, et al. Biophysical Journal Vol. 70, 1996). 
An objective of laser-trapping a microorganism is to separate only one 
microorganism from a group of a number of microorganisms. 
For this purpose, it is necessary to move one microorganism in a trapped 
state to a position apart from other microorganisms of the group. 
Therefore a laser trapping apparatus must have a trapping force capable of 
enduring movement. 
Further, another objective of laser trapping is to irradiate a processing 
laser beam to a microorganism being trapped by a laser beam to apply 
processing such as cell fusion. A trapping force capable of enduring the 
radiation pressure of the processing laser beam is required. 
Therefore, it is important that the trapping force is great upon applying 
operation to the microorganism. 
Because the trapping force is in proportion with the optical power, if it 
is intended to obtain a large trapping force, the optical power must be 
increased by so much. The increase of the optical power, however, results 
in a problem of damaging the microorganism. 
In view of the above, it is a technical objective of the present invention 
to obtain a large trapping force without increasing the optical power. 
SUMMARY OF THE INVENTION 
The foregoing objective of the present invention can be attained by a laser 
trapping apparatus for optically trapping an optional micro-particle from 
a group of micro-particles such as microorganisms suspended in a medium by 
a laser beam focused at a focal point of an optical converging system at 
the focal point. The apparatus includes a parallel beam output device and 
an optical converging system. 
The parallel beam output device outputs a plurality of laser beams around 
an optical axis in parallel with the optical axis. The optical converging 
system has an objective lens for focusing the plurality of laser beams 
irradiated from the parallel beam output device to the focal points. 
According to the present invention, a plurality of parallel beams are 
incident to an optical converging system and focused to a focal point 
thereof. 
In this case, traces of optical rays focused to the focal point are 
substantially in symmetry with respect to a plane of symmetry. The plane 
is defined by an optical axis of the optical converging system and a line 
in perpendicular to the optical axis. The traces of optical rays are 
focused from the optical converging system to the focal point on both 
right and left sides of the plane of symmetry. 
Trapping forces of the laser beams are as follows. 
First, when single laser beam is irradiated to an optical converging system 
and focused at a focal point, a spherical particle positioned at the focal 
point undergoes a force F.sub.1 due to the laser beam in the direction of 
the optical axis and a force F.sub.2 due to the laser beam in the 
direction perpendicular to the optical axis, as shown below. 
F.sub.1 =Q.sub.1 nP/c 
F.sub.2 =Q.sub.2 nP/c 
n: refractive index of medium 
P: optical power 
c: velocity of light 
Q.sub.1, Q.sub.2 : coefficient 
In a case of irradiating two parallel laser beams L.sub.1, L.sub.2 to the 
optical converging system and focusing to the focal point, the trapping 
forces relative to a spherical particle placed on the focal point is 
determined, assuming each of the optical power as P/2 and the angle 
between each of the two laser beams L.sub.1, L.sub.2 and the optical axis 
as .phi.. 
Trapping force F.sub.z when an external force is exerted in the direction 
of the optical axis Z relative to the particle, trapping force F.sub.-z 
when an external force is exerted in the direction of the counter-optical 
axis -Z, trapping force F.sub.x when an external force is exerted in the 
direction of the axis X in perpendicular to the optical axis Z within a 
plane in which the traces of optical rays are present, and trapping force 
F.sub.y when an external force is exerted in the direction of an axis Y in 
perpendicular to the axis X and the axis Z are represented, respectively, 
by the following equations. 
F.sub.z =(Q.sub.1 cos .phi.+Q.sub.2 sin .phi.).sub.n P/c=Q.sub.z 'nP/c 
F.sub.-z =(-Q.sub.1 cos .phi.+Q.sub.2 sin .phi.).sub.n P/c=Q.sub.-z 'nP/c 
F.sub.x =(Q.sub.2 cos .phi.).sub.n P/C=Q.sub.x 'nP/c, and 
F.sub.y =(Q.sub.1.sup.2 +Q.sub.2.sup.2).sup.1/2 nP/c=Q.sub.y 'nP/c 
When the trapping forces are calculated by using the equations above and 
compared with those of the single-beam gradient force optical trap using 
the laser beam, they are shown in Table 1. 
TABLE 1 
______________________________________ 
Direc- Single beam gradient 
tion 2-beam trap force optical trap 
Ratio 
______________________________________ 
Z 0.63 0.49 1.28 
-Z 0.3 0.26 1.15 
X 0.25 0.31 0.81 
Y 0.6 0.31 1.94 
______________________________________ 
Trapping forces are calculated assuming such that .phi.=60.degree. in a 
case of the 2-beam trapping, and the NA of the objective lens as 1.25 
(=refractive index: 1.33.times.sin 70.degree. ) and the intensity 
distribution of the incident light is uniform in a case of the single-beam 
gradient force optical trap. Further, the specific refractive index of the 
particles is defined as 1.2 for each case. 
As apparent from the result, large trapping force can be obtained in the 
2-beam trapping in each direction except for the direction X. 
Particularly, when one particle, among a group of particles, is moved in a 
trapped state it should be taken into a consideration how the particle is 
transported at a high speed and it is important that the trapping force in 
the moving direction be large. 
In the case of the 2-beam trapping, if the particle is moved in the 
direction Y, the trapping force is about twice of the single beam gradient 
force optical trap and the particle can be moved at a twice speed. On the 
other hand, if it is moved at an identical speed, the power of the light 
for trapping the particle can be reduced to 1/2. 
Then, if two laser beams incident to the objective lens are rotated 
relative to the optical axis Z, the direction Y can be rotated around the 
axis Z. Accordingly, if the particle is moved in an optional direction, 
the direction Y of an intense trapping force can be aligned with the 
moving direction. 
Further, making the trapping force uniform in any of the directions on the 
plane X-Y can be attained by increasing the number of laser beams incident 
to the objective lens to three or more. In this case, the coefficient 
Q.sub.x, Q.sub.y of the trapping force approaches more to that of the 
single beam gradient force optical trap as the number of the beams is 
increased, and the coefficient Q.sub.z, Q.sub.-z, in the direction Z or 
direction -Z is greater by 15 to 30% compared with the single-beam 
gradient force. 
Since the trapping force is great in the direction of the optical axis Z as 
described above, it is extremely advantageous also in a case of 
irradiating a processing laser beam in the direction of the optical axis Z 
while trapping an microorganism to apply processing to the microorganism.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A laser trapping apparatus according to the present invention will be 
explained more concretely. 
A laser trapping apparatus 1 shown in FIG. 1 comprises a cell plate 3 for 
storing a liquid medium in which group of particles 2--such as Escherichia 
coli in the liquid medium, and a laser trapping optical system 5 for 
irradiating a laser beam to the cell plate 3 from below by utilizing an 
optical system of an inverting microscope 4, and moving a particle 2 a 
from a group of particles 2--distributed and suspended in the cell plate 3 
in a trapped state in an optional direction thereby separating the trapped 
particle from other particles 2--of the group. 
As shown in FIG. 2, the laser trapping optical system 5 comprises a 
parallel beam output device 6 for outputting two laser beams L.sub.1, 
L.sub.2 around an optical axis Z of the optical system 5 in parallel with 
the optical axis Z, a dichroic mirror DM for reflecting the two laser 
beams L.sub.1, L.sub.2 irradiated from the parallel beam output device 6 
and an objective lens (optical converging system)7 for focusing the laser 
beams L.sub.1, L.sub.2 onto a focal point. 
The parallel beam output device 6 comprises a laser light source 8 for 
outputting a single laser beam L.sub.0, and a prism 9 for splitting the 
laser beam L.sub.0 outputted from the laser light source 8 into a 
plurality of parallel laser beams L.sub.1, L.sub.2 - - - . 
As shown in FIG. 2, the prism 9 is configured such that each of an incident 
end 9.sub.in and an emitting end 9.sub.out is cut into a wedged shape 
(angled shape) to form two pairs of facets 10.sub.in and 10.sub.out. Each 
pair of facets has an apex at an intersection with the optical axis Z, and 
each of the facets 10.sub.in formed on the incident end 9.sub.in is made 
in parallel with each of the facets 10.sub.out formed on the emitting end 
9.sub.out. 
Further, in each of the facets 10.sub.in, 10.sub.out, when the laser beam 
L.sub.0 is irradiated from the laser beam source 8 is incident along the 
optical axis Z to the incident .sup.9 in, it is refracted at each facet 
10.sub.in, 10.sub.in formed at the incident end 9.sub.in into different 
directions and split into two optical beams L.sub.1, L.sub.2. Then, when 
they are emitted from each of the facets 10.sub.out, 10.sub.out of the 
emitting end 10.sub.out being paired with each of the facets 10.sub.in, 
10.sub.in on the incident end 9.sub.in, they are refracted again and 
emitted as two parallel beams around the optical axis Z as a center. 
Then, the prism 9 is disposed rotatably around the optical axis Z as the 
center to constitute a parallel beam rotating device 11. Therefore such 
that the incident position of each of the laser beams L.sub.1, L.sub.2 to 
the optical converging system 5 is around the optical axis Z as the center 
in accordance with the direction along which the one particle 2a is to be 
moved, by the rotation of the two laser beams L.sub.1, L.sub.2 around the 
optical axis Z. 
The cell plate 3 which contains the liquid medium comprises a plate main 
body 20 that serves as a cover glass for the inverting microscope 4, a 
first cell 21A for storing a liquid medium in which a number of 
micro-particles are dispersed and suspended and a second cell 21B for 
storing a liquid medium in which no micro-particles are suspended. Each of 
the cells is opened at the upper surface and formed by being spaced apart 
a predetermined distance from each other and in communication with each 
other by a narrow induction channel 22 for inhibiting free movement of 
micro-particles. 
Accordingly, the induction channel 22 has a bottom formed at a high 
accuracy as a cover glass for the inverting microscope 4 and has a buffer 
cell 21C formed with the upper surface being opened at the midway for 
storing a liquid medium in which no micro-particles are suspended. 
Thus, the induction channel 22 comprises a first induction channel 22a for 
communication between the first cell 21A and the buffer cell 21C and a 
second induction channel 22b for communication between the buffer cell 21C 
and the second cell 21B. 
Each of the cells 11A-11C is formed in each of recesses 23A-23C of a larger 
diameter as a liquid medium injection port formed on the surface of the 
plate main body 20. The upper opening for each of the cells 21A-21C is 
adapted to be opened/closed by each of covers 24A-24C which is moved 
slidably in the horizontal direction along the bottom of the recesses 
23A-23C. 
Faces of the recesses 23A-23C and the covers 14A-14C in contact with each 
other are polished at a high accuracy such that they are in sliding 
contact with a gap formed at an accuracy on the order of a wavelength of 
light. Then, each of the cells can be opened/closed without forming a 
stream in the induction channel 22 when each of the covers 24A-24C is 
caused to slide. 
In the cell plate 3 for separating Escherichia coli, each of the recesses 
23A-23C is about 9 mm diameter.times.2 mm depth and each of the cells 
21A-21C is about 2 mm diameter.times.1.8 mm depth. The length of the 
induction channel 22a and 22b for communication between the cells 21A and 
21C and between the cells 21C and 21B is about 9 mm. The cross section for 
each of the induction channels 22a, 22b is about 0.1 mm square. The 
thickness for the bottom of each of the cells 21A-21C and the induction 
channel 22 is about 0.17 mm. 
An objective lens 7 of the inverting microscope 4 is placed below a stage 
26 disposed moveably in the direction X-Y by a stage moving device 25. A 
CCD camera 27 for photographing the inside of the plate 3 is set on an 
optical axis of the microscope. The images taken up by the CCD camera 27 
are displayed on a display apparatus 28. 
A method of using the apparatus according to the present invention having 
the foregoing configuration will be explained with reference to an example 
of separating Escherichia coli as the particle 2. 
At first, as shown in FIG. 4(a), a clean liquid medium in which no 
Escherichia coli (micro-particles) are present is injected into the cells 
21B, 21C of the cell plate 3. When the covers 24B, 24C are caused to slide 
to close each of the cells 21B, 21C, the liquid medium is filled in the 
induction channels 22b, 22a by a capillary phenomenon. 
Then, as shown in FIG. 4(b), a liquid medium in which a great number of 
Escherichia coli are dispersed and suspended is injected into the cell 21A 
and the cell 21A is closed by sliding the cover 24A. Then, the stage 26 is 
moved while observing the inside of the first cell 21A by the display 
apparatus 28. When a laser beam is irradiated to one Escherichia coli 2a, 
as it is situated at a focal point, the one Escherichia coli 2a is 
trapped. 
Laser beams L.sub.1, L.sub.2 are emitted from the parallel beam emitting 
device 6 and focused at a focal point F. As shown in FIG. 2, the two laser 
beams L.sub.1, L.sub.2 are rotated by a predetermined angle around the 
optical axis Z by the rotation of the prism 9. The irradiation position of 
the two laser beams L.sub.1, L.sub.2 is determined such that the traces of 
optical rays of each of the laser beams L.sub.1, L.sub.2 focused by the 
objective lens 7 to the focal point are made substantially in symmetry 
with respect to a plane Z-Y. The Z-Y plane is defined with a line Y, 
(directional line) representing the direction of the induction channel 22 
along which Escherichia coli 2a which is moved from the focal point, and 
the optical axis Z such that the traces of the optical rays advance 
passing through the focal position F and along the plane Z-X in 
perpendicular to the directional line Y. 
That is, the prism 9 is rotated such that the directional line Y is in 
perpendicular to a plane on which the traces of the optical rays of the 
laser beams L.sub.1, L.sub.2 from the objective lens 7 to the focal point 
and the optical axis Z. 
Since this makes the trapping force greatest in the direction Y, that is, 
about twice of the single-beam gradient force optical trap, Escherichia 
coli 2a can be moved along the induction channel 22 at a twice speed for 
an identical optical power of the laser beam L.sub.0 irradiated from the 
laser beam source 8. In a case of moving at an identical speed, the 
optical power of the laser beam L.sub.0 irradiated from the laser beam 
source 8 can be reduced to one-half. 
Further, since the trapping force in the direction of the optical axis Z 
and in the direction -Z is larger than that in the single-beam force 
optical trap, if the optical power of the laser beam L.sub.0 is identical, 
Escherichia coli 2a can be moved faster along the direction of the optical 
axis Z or -Z. If it is moved at an identical speed, the optical power of 
the laser beam L.sub.0 can be reduced. 
Accordingly, Escherichia coli can be moved faster for a same level of 
allowable biological damages and biological damages can be reduced if 
moved about at the same speed. 
Then, as shown in FIG. 4(c), when the cover 24B is opened by sliding at the 
instance the Escherichia coli 2a is moved to the cell 21B, and the liquid 
medium in the cell 21B is sucked by a micropipette 30 or the like, since 
only one Escherichia coli moved by laser trapping is present in the cell 
21B, this one Escherichia coli 2a can be separated reliably from other 
Escherichia coli of the group 2 - - - . 
In the foregoing explanation, the parallel beam output device 6 has been 
explained to a case of using the prism 9 for splitting the laser abeam 
L.sub.0 irradiated from the laser beam source 8 into two laser beams 
L.sub.1, L.sub.2, but the present invention is not restricted only thereto 
but a plurality of laser beam sources may be used. 
The laser beam L.sub.0 is split by the prism 9 not only in to two laser 
beams L.sub.1, L.sub.2, but may be split into more than two laser beams. 
In this case, the prism 9 may be a normal tetragonal cylinder having an 
incident end 9.sub.in and an emitting end 9.sub.out on both ends in which 
normal tetragonal pyramidical slopes 10.sub.in, - - - , 10.sub.out - - - 
are formed to each of them as shown in FIG. 5(a). A normal hexagonal 
cylinder having an incident end 9.sub.in and an emitting end 9.sub.out on 
both ends in which normal hexagonal pyramidical slopes 10.sub.in, - - - , 
10.sub.out - - - are formed to each of them as shown in FIG. 5(b). A 
normal octagonal cylinder having an incident end 9.sub.in and an emitting 
end 9.sub.out on both ends in which normal octagonal pyramidical slopes 
10.sub.in, - - - , 10.sub.out - - - are formed to each of them as shown in 
FIG. 5(c). A circular cylinder having an incident end 9.sub.in and an 
emitting end 9.sub.out on both ends in which normal trigonal pyramidical 
slopes 10.sub.in, - - - , 10.sub.out - - - are formed to each of them as 
shown in FIG. 5(d). 
In a case of irradiating a laser beam for processing further to a trapped 
microorganism, a laser beam irradiated from a source of a processing laser 
beam (not illustrated) may also be introduced to the optical system of the 
inverting microscope 4, transmitted through the dichroic mirror D of the 
laser trapping optical system 5 and irradiated coaxially to the particle. 
As described above, according to the present invention in a case of 
trapping, for example, by two laser beams, the two laser beams L.sub.1, 
L.sub.2 are irradiated such that the traces of optical rays of each of the 
laser beams focused by the objective lens to the focal point are 
substantially in symmetry with respect to the plane Z-Y defined with the 
line Y in perpendicular to the optical axis Z, and the optical axis Z and 
such that the optical traces of the optical rays advance passing through 
the focal point and along the plane Z-X in perpendicular to the 
directional line Y. 
In this case, because the trapping force is greatest in the direction Y and 
it is about twice of the single-beam gradient force optical trap, the 
micro-particle can be moved in the direction Y at a twice speed for an 
identical optical power of the laser beam irradiated from the laser beam 
source. If the micro-particle is moved at an identical speed, the optical 
power of the laser beam irradiated from the laser beam source can be 
reduced to one-half. 
Further, if the optical power of the laser beam L.sub.0 irradiated from the 
laser beam source is identical, the micro-particle can be moved faster in 
the direction of the optical axis Z or -Z. On the other hand, if it is 
moved at the identical speed, the optical power of the laser beam L.sub.0 
can be reduced to suppress biological damages. 
Further, when the two laser beams incident to the objective lens are 
rotated relative to the optical axis Z, since the axis X rotates around 
the optical axis Z, the axis Y of intense trapping force rotates 
correspondingly around the optical axis Z and the direction Y can be 
aligned with a desired direction to move the micro-particle. 
Further, because the trapping force in the direction of the optical axis Z 
and -Z is also greater than that of the single-beam gradient force optical 
trap, it is extremely advantageous in a case of processing a microorganism 
by irradiating a laser beam for processing in the direction of the optical 
axis Z which trapping the microorganism.