Device for manipulation, exposure and observation of small particles, in particular biological particles

 

(57) Abstract:

The inventive first laser 4 emits a beam in the first wavelength range, using the first optical device 12, 13; 14, 15 focuses and forms an optical trap. Sample table 22 is used for placement of the respective particles. Next provided the source 17 lighting observations, which is used to observe and record the behavior of the particles. The second laser 3 emits a beam of a second wavelength range, which is focused in order to manipulate particles on the stage. Optical devices for each of the beams can be positioned and focused independently from each other, and to the beginning of the manipulation and exposure to rays, regardless of their wavelengths are focused on the same object plane of the object stage. 2 C. and 17 C.p. f-crystals, 1 Il.

The invention relates to a device for manipulation, exposure and observation of small particles, in particular biological particles, comprising at least a first laser generating radiation in the first wavelength range, then using the first optical device Fok the media object for receiving particles, the light source for observation device for observing and recording, allowing to observe particles in the media object and record their behavior.

The invention also concerns the method for manipulation, exposure and observation of small particles, in particular biological particles, in which objects are captured in the optical trap in the media object with at least the first laser radiating in the first wavelength range, and observed with a means of monitoring and recording and/or recorded the behavior of objects.

A device of this kind is known, for example, from U.S. patent US-PS 4 893 886, and working with the so-called optical trap, which uses a highly focused laser beam with an intensity profile that is close to the Gaussian distribution. In this optical trap are combined with each other components of the radiation pressure forces the scattering and gradient forces, in order to create a point of stable equilibrium, which is close to the focus of the laser beam. Power dissipation in this case is proportional to the optical intensity and acts in the direction of the incident laser light. The gradient force is proportional to the optical intensive pricheski traps and their physical basis is described, for example, in the publication "Optical trapping and manipulation of single living cells using infrared rays laser". auth. A. Ashkin and others in the publication: Reports Bunsen society of physical chemistry, March 1989, S. 254 - 260.

Using this kind of devices can capture, consolidate and manipulate small particles, in particular biological, which usually can move freely in the fluid media object. This raises the difficulty lies in the fact that in order to ensure accurate exposure, manipulation and observation must be done at the same time.

The present invention is a method and device, which is targeted and precise manipulation, exposure to and observation of small particles, in particular biological particles.

To solve this problem, the device of the above described type provided with at least one second laser, emitting a second wavelength range, the radiation which is focused by the second optical device with sufficient precision in order to influence inewsinn and for rays of light of the observations are executed separately with the possibility of positioning and focus independently of each other. The beams in the first wavelength, the rays in the field of the second wavelength and the rays of the light of the observations to the beginning of the manipulation and observation, regardless of their wavelengths are focused on the same object plane of the object. In addition, in the device according to the invention provides that the respective first laser has the ability to adjust the wavelength and performed, mainly, infrared (IR). The second laser is adjustable wavelength ultraviolet (UV) laser, mainly by a pulsed laser. This enables suitable for practical purposes fixation of the particles, during which own the UV laser, in this case, there is a danger that the energy input is too large, the particles will receive as a result of unwanted damage.

According to one special embodiment of the invention, the first laser is Ng-YAG laser, Nd-YLF laser or a titanium-sapphire laser and the second laser is a nitrogen laser, an infrared laser with a frequency multiplier or a dye laser pumped.

In addition, according to the invention the first and second lasers may be located n the zoom allows the location of the light sources in a smaller volume of space, in particular, when the circuit boards lasers are located one below the other. The components of the focusing and deflecting optics can be mounted for portability in one common circuit Board attached to the rack.

Light sources emitting in the respective wavelength ranges can be made in the form of individual lasers. In a preferred embodiment, the beam of the first laser is divided by a beam splitter, which forms at least first and second beams in the first wavelength range, and these beams are at least partially held separately and are then forwarded to the object in the media object. If necessary from the first laser beam with the same beam splitter can be separated and other rays, which are, at least in part, separately and are then forwarded to the object in the media object, if several such beams should be used as optical traps.

In one special embodiment of the invention provides that the beam splitter is made of polarization, which forms a first beam of S-polarized second beam is P-polarized and sets the phase position between the beams. While PR is.

In the device according to the invention is suitable to each beam from the first laser and the second laser passed through its own expanding optical system, each of which can be adjusted in three dimensions, particularly in three orthogonal axial directions.

It is also advisable to set on a course of rays of the first and second laser mirrors and splitters had the ability to rotate and accordingly the inclination regardless expanding optical systems. This provides the advantage of increasing the possibility of regulating rays in the X - y plane.

The following embodiment of the invention provides that the beam of light lighting the observations by adjusting the object and/or media object along the optical axis in the Z axis direction can be focused on the object in the media object, and that the point of observation for a beam of light lighting observations in the object plane can be adjusted by moving the media object within the object plane in the X - y plane.

Next is expedient that in the course of the rays from the first and second laser were installed svetoslavtsi through which rays in Auda on the object. This ensures that the purposeful regulation of the intensity of these rays, to avoid unwanted damage to the particles.

Particularly preferred is a variant of the invention in which the beams in the first wavelength range and the beams in the second wavelength range is directed to the object in the media object through a common mirror and a common lens. This makes it possible to achieve particularly compact installation. This simplifies the course of the rays and ensures more reliable operation of the device.

In one special embodiment of the invention provides that the rays from the radiation sources, converted, rejected and focused on the object, are all, in essence, in the same first plane, the media object is located in the second plane, perpendicular to the first plane, and that mirrors and splitters respectively for the variances of the separate beams equally are located in planes perpendicular to the first plane.

Thus, there is a particularly compact and easy-to-use device, which ensures reliable coordination rays.

The method according to the invention is characterized by the fact that IP is Ute with sufficient precision, to be exposed to particles present in the zone of the media object; that rays of light in the first wavelength range and the second wavelength range and the light rays of the light of the observations can be independently from each other, using separate optical devices, be adjusted in the object plane, the so - called X-Y plane, and the focus of this object in a perpendicular plane to the Z-direction; and that in the beginning all these rays, regardless of wavelength, focus in the same object plane of the object.

This way the user of the respective device has a stable initial position and may be oriented in any plane events occur and, accordingly, is it possible to influence them.

In the further development of the method according to the invention provides that a particle trapped in an optical trap of the laser can be moved (a) by adjusting at least one beam in the first wavelength range in the direction of the X - Y and/or (b) by adjusting the media object in the direction of X - Y in the object plane, and (a) in the trap move only one particle, and (b) in the trap move all particles is ovushka first laser, can be moved

a) by adjusting at least one beam in the first wavelength range in the Z axis direction and/or;

b) by adjusting the lens and/or media object in the Z axis direction, and in the case of (a) caught in the trap the particle comes out of the selected plane of observation and in the case of (b) remains in the plane of observation.

It is clear that it is possible not only moving particles. In the further development of the method, when using at least two separate beams in the first wavelength range, it is possible to make the rotation of the particle in the optical trap directly to the fact that (a) one beam will remain in its initial position, and the second beam should move in the direction of the X - Y or (b) the first beam will remain in its initial position, and the second beam moves in the Z-direction, or (c) at least two beams do the opposite motion and move in the Z-direction at different distance, or (d) make combinations of movements, illustrated above designations (a), (b) and (c).

In the further development of the method provides that the effect on particle beams in the second wavelength range to produce a randomly selected X - Y plane of nosetackle.

This kind of change in the plane of observation possible after busy starting position to the beginning of the process.

It is advisable to use for fixing the particles in an optical trap laser radiation of a visible or infrared spectrum, and for exposure to particles the use of ultraviolet laser, in particular a pulsed ultraviolet laser.

If in the method according to the invention all the rays are directed to the appropriate object in the media object at the same time through the same lens that allows particularly reliable regulation and control.

In conclusion, according to the invention all the rays are able to control the exposure and/or supervision independently from each other be adjusted in intensity and/or be switched on and off. This provides many opportunities for exposure, processing and monitoring of small particles.

Below is a detailed description of the invention with an indication of its features and benefits on the example of one of the embodiments with reference to the drawing attached to the description.

In the drawing is given a schematic representation of one in whom 2">

The device contains a laser rack 5, which are placed one above the other IR laser 4 as a first laser and a UV laser 5 as the second laser. In addition, the common circuit Board can be placed several tires designed lifting devices for the respective optical components to produce the alignment of these components in relation to lasers 3 and 4 so that the emitted laser beams in the respective wavelength ranges connected through additional optical device fell to the media object 22. While the beams of both lasers 3 and 4 are parallel. The optical components of the tire can be made modular.

In the depicted embodiment, the IR laser 4 emits a beam, which beam splitter 16 is divided into first and second beams. The first beam passes through an appropriate blend of 6 and extends optics 14, 15, and then declined reflecting the infrared rays by the mirror 9 and passes through a beam splitter, for example a prism or a semi-transparent mirror 20, and also through the following semi-transparent mirror 8 or the corresponding prism. Then this beam deflecting mirror 7 is supplied to the depicted schematically microscope 1 having a lens 21, the main part of the beam from the IR laser 4 passes through the beam splitter 16 to the mirror 19 and then passes through the appropriate blend and extend optics 12, 13. Then the second beam is deflected by the second beam splitter, for example a semitransparent mirror 20, and in the same way as the first beam from the infrared laser 4 is applied to the microscope 1.

To create a second beam in the first wavelength range may, of course, use a different IR laser, not shown in the drawing. Thus there are additional opportunities in terms of intensity, polarization, wavelength and controllability of such laser radiation, however, this also increases the cost of the equipment, so the decision should be made based on specific conditions.

In the present embodiment, savedelete 16 may be made in the form of a simple beam splitter so that a sufficient output power and intensity of radiation of the infrared laser part intensity was branched by the second beam. However, in the preferred embodiment as the beam splitter 16 is used polarized beam splitter, which forms a first beam of S-polarized light and the second beam of P-polarized light and sets the phase position between the two beams, and the percentage ratio between the intensity of the two beams in the first wavelength range of the first IR barking infrared light, to ensure accurate maintenance of the beam without unwanted loss. If necessary, during the first and/or second beam from the IR laser and can be installed svetislavian 25 in order to adjust the capacity of the used radiation. Alternative or additionally may be provided that the power of the infrared laser 4 is set independently within the specified limits.

Regardless of this as the first and second beams from the infrared laser 4 provides the means by which the respective beam can be interrupted. This can be used a blend of 6 overlapping or separate devices.

As the second laser has a UV laser 3, the beam of which is in the second range of wavelengths passes through svetislavian 18, hoods 6 and own expanding optics 10, 11, and then is deflected by the mirror 8 and the other mirror 7 is sent to the lens 21 and the subject table 22. UV laser 3 it is advisable to perform pulse to precisely regulate and control the applied energy ultraviolet light, in order to avoid damage to the object to which you are exposed.

In addition it allows the light of the fact or continuously. Svetislavian 18 may be in the form of an adjustable filter or beam splitter.

Of course, that the components of the rays of UV laser 3 are adapted to ultraviolet light, and the components 7, 8 and 21 suitable for infrared light and ultraviolet light and therefore enlightened.

The microscope 1 is equipped with 17 light observations, it is reasonable to visible light, as well as, on the one hand, the visual device 23 observations, it is advisable with an appropriate protective filter, and on the other hand the combined device 2 for monitoring and recording, which includes, for example, a camera, a monitor, and a video system. To perform protective functions between them has a variable filter 24.

In the device of this kind can be used in a variety of lasers that are associated with the microscope 1 via a compact optical system. As UV laser 3 (pulse), emitting, for example, near-ultraviolet zone, and IR laser 4 (continuously working), emitting, for example, near infrared zone, should be very well focused (diffraction-limited), and also to have a minimum beam divergence. Under these conditions may b the s for laboratory research.

For example, as UV laser 3 can be used a nitrogen laser or infrared laser with costochondritis, while as the infrared laser 4 using Nd-YAG laser, diode pumped or Nd-YLF laser, the capacity of which is respectively selected. It should be borne in mind that in the fluid moving particles do not have their own movement in the media object 22 can be detected already at low laser power, while particles with their own dynamics or particles in the high-viscosity solutions can be captured only when a high power laser. As already mentioned, for the introduction of two independently from each other moving the infrared rays can be equipped with two Nd-YAG laser, diode pumped, or one Nd-YAG-laser high power pumped by flash lamp, as is schematically shown on the drawing.

Important for the device according to the invention is that for each beam from the respective lasers 3 and 4 has its own expanding optics 10, 11; 12, 13; 14, 15, which can be adjusted in three dimensions, particularly in three mutually orthogonal directions, as schematically shown in the drawing. With this expanding optics bundles so expanded that section loaysa optics can be used two PLANO-convex lenses, or one convex and one-concave lens with an appropriate focal length.

Indicated in the drawing in connection with the expansion optics 10, 11; 12, 13; 14, 15 the coordinate directions are also referred to the situation in the media object 22, and the X - Y plane runs perpendicular to the plane of the drawing, and the direction Z perpendicular to it and lies in the plane of the drawing.

By changeover expanding optics 12, 13 or 14, 15 in the X - Y plane, you can change the point of fixation of the optical trap in the X - Y plane, while the movement in the Z axis direction means a change of focus perpendicular to the X - Y plane, so trapped in the optical trap, the particle emerges from the plane of observation (the original).

This also applies to the mode of action of expanding optics 10, 11 for UV laser 3. When changing expanding optics 10, 11 in the direction of the X - Y changes the point at which the influence on the particle in the object plane. Movement in the Z axis direction leads to defocusing with respect to the plane of observation in this direction. Thus, the processing can be carried out at the choice to either focal observation plane, or outside of it.

Additionally or alittle, to the respective beams to move in the X - y plane.

The media object 22 is accomplished in a known manner movable in the direction of the axes X, Y and Z, in order to make the necessary adjustments. The lens 21 is movable at least in the z direction.

In the present embodiment, a device monitoring and recording are so located that they can work in transmitted light. Of course, the arrangement may be such that the devices monitor and record 2 and 23 are installed on one side of the microscope 1 and the source 17 light surveillance installed in the beam direction behind the microscope 1 so that you can work in reflected light.

Using the above described device may be made of a large number of effects and manipulation of small particles that can be observed. Retention of particles at one or more points is turning on and off of both the infrared rays from the infrared laser 4, and the number of these rays can be increased by the fact that the device is similarly mistaken and, for example, a beam splitter 16 or 18 branches other rays, which with its own expanding optics are entered into the system.

After to wavelengths, focus to the beginning of the manipulations and observations on the same object plane, i.e. in particular the X - Y plane of the media object 22, the individual rays can be influenced independently of the other rays, in order to achieve the mobility of particles on the media object 22 and handle them purposefully selected location in three dimensions.

Movement in the X - Y plane can be accomplished by a movement of the media object 22 in the direction of X - Y, all the particles move in this plane at the same time. On the other hand, in the direction of X - Y can be arranged by the action of at least one extender optics 12, 13 or 14, 15, or tilting at least one of the mirror surfaces of the components 8, 9, 20, so that individual particles can be set in motion independently of each other.

Movement in the Z axis direction can be realized by various methods, for example by movement of the media object in the Z axis direction with respect to the lens 21, or the movement of the lens 21, or the movement of the lens 21 in the Z axis direction with respect to the media object. In both cases remains the focus of the visible light observation.

Regardless of this may be privada object on the media object 22 becomes possible. Thus, it is possible to monitor and processing a three-dimensional object in different sections.

So as an extender optics 10, 11 for UV laser 3 is movable independently of this, the processing may be performed on the selection or in the focal plane of observation, or outside of it.

In the above-described device using IR-laser radiation is possible positioning of the object in a different plane than the plane of the media object, without the need for rigid fastening of the object on the media object, because the recording is only the infrared beam from the infrared laser 4. It makes possible a simple and reliable (simultaneous) observation and processing of freely moving objects.

In the above described embodiment is the location at which all light rays emitted by the source in one plane are deflected and focused. Summarizing the different rays to the media object 22 through common deflecting mirror 7 and the lens 21.

Thus, it is possible a particularly compact arrangement, which also ensures reliable functioning of the device.

Of course, it is possible obhodimo to use spherical and parabolic mirrors, in order to be a desirable way to direct the rays to the media object 22. This option can be useful if the compactness of the installation is not the determining factor.

1. Device for manipulation, exposure and observation of small particles, in particular biological particles, comprising at least one first laser emitting beams in the first wavelength range, which with the help of the first optical device to focus with sufficient precision for education in a given field of optical traps, as well as the media object for placement of particles, the light source for illumination of the observation device for observing and recording the behavior of particles in the media object, and at least one second laser, emitting beams in the second wavelength range, with the help of the second optical devices focus with sufficient precision to affect particles in the media object, and for each of the beams of the first and second lasers it has its own expanding optical system which has a capability of adjustment in three dimensions, particularly in three orthogonal axial directions (X, Y, Z) such obrazets light rays of the light of the observations respectively positioned and focused separately and independently from each other, characterized in that each specified expanding optical system for the corresponding laser beam is provided with a separate pair of lenses, preferably two PLANO-convex lenses or one convex lens and one-concave with the respective focal distances, the position of which is adjustable independently from each other, and the laser beams in the first wavelength range, the laser beams in the second wavelength range and the light rays of the light of the observations to the beginning of the manipulation and observation focus on the same object plane (the X-Y plane) of the media object, regardless of the wavelength, during this period, some rays can be influenced independently of the other beams in order to ensure the movement of particles in the media object and process them purposefully in a specific location in three dimensions, while maintaining the ability to focus the visible light observation.

2. The device according to p. 1, wherein the first laser is made in the form of an adjustable in its wavelength range of the laser, mainly infrared laser and the second laser is made in the form of an adjustable in the range of wavelengths of ultraviolet laser, preeman Nd-YAG laser, or Nd-YLF laser or a titanium-sapphire laser and the second laser is made of nitrogen, or an infrared laser with costochondritis, or a dye laser pumped.

4. The device according to PP. 1 to 3, characterized in that the first and second lasers are hosted on the same rack as independent from each other, positioning and alignment.

5. The device according to PP. 1 to 4, characterized in that it is provided with a beam splitter, which divides the beam of the first laser and forms at least first and second beams in the first wavelength range, which at least partly conducted separately and sent to the object in the media object.

6. The device under item 5, characterized in that the beam splitter polarizing made, forming a first beam of S-polarized light and the second beam of P-polarized light, and regulating the phase position between the beams, while the percentage ratio between the intensity of the beams in the first wavelength range can be adjusted.

7. The device according to PP. 1 - 6, characterized in that the mirrors and splitters located along the light rays are made to rotate and tilt independently of the expansion of optical systems is the ability to focus the beam of light observations along the optical axis (Z-direction) on the object in objectville, when the media object is made with the possibility of changes in the object plane (the X-Y plane), to regulate the location in the plane of observation points for a beam of light observation.

9. The device according to PP. 1 to 8, characterized in that it is provided with svetoslavtsi established in the course of the rays of the first laser and the second laser, through which the beams in the respective wavelength ranges stepwise or gradually attenuated before they are sent to the object in the media object.

10. The device according to PP. 1 to 9, characterized in that the beams in the first and second wavelengths through a common mirrors are directed to the object in the media object through a common lens.

11. The device according to PP. 1 to 10, characterized in that all the rays emitted from lucabrasi device and then subjected to impacts, rejected and focused on the object, lie essentially in the same first plane, and the media object is in the second plane (the X-Y plane) perpendicular to the first plane, and mirrors and splitters to reject single-rays are also located in a plane essentially perpendicular to the first plane.

12. JV is AMI, in which objects in the media object is fixed in the optical trap by at least one of the first laser emitting in the range of the first wavelength, and see objects by means of observation and recording, and/or record the behavior of objects and which uses at least one second laser, emitting a second wavelength range, the radiation of which focus with sufficient precision to impact the particles in the zone of the media object, and for each beam of the first laser and the second laser use their own expanding optical system, made with the possibility of regulation in the direction of three orthogonal axes (X, Y, Z) so that the beams of both lasers and beams of light observation regulate independently from each other in the object plane and the focus in the axial direction, characterized in that each of the specified extender optical system uses a separate pair of lenses, mainly two PLANO-convex lens or a PLANO-convex lens and one-concave lens with the appropriate focal lengths, the position of which is adjustable independently from each other, and the laser beams in the first wavelength range, laser luckyyou on the same object plane (the X-Y plane) of the media object, regardless of the wavelength, the individual rays act independently of the other beams in order to ensure the movement of particles in the media object and process them purposefully in a specific location in three dimensions, while maintaining the ability to focus the visible light observation.

13. The method according to p. 12, characterized in that the particle in the optical trap of the first laser is moved by

(a) moving at least one beam of the first wavelength range in the direction of the X-Y and/or

b) by moving the media object in the direction of X-Y in the object plane,

moreover, in case (a) move only caught a particle, while in case b) move all particles, except for the trapped particles.

14. The method according to p. 12 or 13, characterized in that the particle in the optical trap of the first laser move

a) by changing the focus position of at least one beam of the first wavelength range in the Z axis direction and/or.

b) by moving the lens and/or media object in the Z axis direction,

moreover, in case (a) of the trapped particle moves out of the plane of observation, and sloucho when using at least two separated beams in the first wavelength range, carry out the rotation of the particle in the optical trap by one beam is left unchanged, and the second beam set into motion in the direction of X-Y, or the first beam is left unchanged, and the second beam set into motion in the Z-direction, or at least two beams are set in motion in opposite directions, or both of the beam result in movement in the Z axis direction, but at different distance, or perform a combination of these movements.

16. The method according to PP. 12 to 15, characterized in that exercise influence on the particle beam of the second wavelength range in a randomly selected X-Y plane of the media object and the plane of observation can be located in the same plane or in parallel planes.

17. The method according to PP. 12 to 16, characterized in that for fixing the particles in an optical trap using laser beams of the visible or infrared spectrum, and for exposure to particles using ultraviolet rays of the laser, in particular a pulsed ultraviolet laser.

18. The method according to PP. 12 to 17, characterized in that all the light rays directed to the appropriate object in the media object at the same time through a single lens.

19. Spot other, regulate the intensity, include and/or off.

 

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