JPH052136A - Particulate observing method and microscope used for the same - Google Patents

Abstract

PURPOSE:To provide a microscope which is easily focused even on a moving particulate. CONSTITUTION:This microscope is provided with an ocular 15, an objective lens 7, a stage 9, a condenser lens 10, a mirror 11, an optical element combination 12, and a visible light source 13, which are an optical system and an illuminating system for observation. Furthermore, it is provided with a laser light source 1, a field lens 5, and a half mirror 6 to seize the particulate. The field lens 5, the half mirror 6 and the objective lens 7 are the optical system forming a trap. Thus, a laser beam is condensed to form the trap, the microscope is focused on the trap, and the moving 'particulate to be observed' is seized by the trap and observed with the microscope.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an observation method for collecting and observing non-resting particles, organisms, or cells by converging and irradiating light such as laser light, and a microscope used therefor.

[0002]

2. Description of the Related Art When observing moving microorganisms such as placton moving under a microscope, or when observing particles floating in a solution and flowing, the object to be observed is highly magnified in the center of the observation field of view. In order to observe, the method is to first find the target at low magnification, move the stage on which the target is placed, bring it to the center of the field of view, and then increase the magnification.

[0003]

The above-mentioned conventional method has the following problems.

(1) Since the observation object is moving, it is difficult to accurately bring it into the center of the visual field even at low magnification. Therefore, at a high magnification, the observation target does not enter the field of view, making observation impossible. Further, even at a low magnification, it is difficult to focus the observation target in the visual field.

(2) Even when the observation object is in the center of the field of view at low magnification and the focus is achieved, the object of observation is kept in the field of view unless the operation of converting the objective lens and observing at high magnification is performed very quickly. It is difficult to put in. Further, even when the image is in the visual field, it immediately goes out of the visual field, and it is difficult to perform sufficient observation.

(3) When a lens is exchanged from a low magnification to a high magnification, the focus which is in focus at the low magnification may be deviated. The observation target goes out of the visual field while adjusting this. Further, even if the object is within the field of view, if the object to be observed moves in the optical axis direction, the focus will shift and the observation will be difficult. Furthermore, it is almost impossible to focus on an arbitrary surface of the observation target.

[0007] In order to solve the above problems, the viscosity of the surrounding solution is increased to reduce the moving speed of the observation target. However, this method has the following problems.

(A) Since it must be carried out within a range that does not affect living organisms, the viscosity is considerably limited and the effect is small. In addition, it is necessary to set the solute and concentration to match the observation target, which makes the work complicated and expensive.

(B) By increasing the viscosity of the solution, the refractive index is increased, that is, the reflectance is decreased, and the reflected light entering the eyes of the observer is decreased. In addition, the irregular reflection that occurs due to the irregular shape is reduced,
Observation becomes difficult. Further, even when the method of observing by the phase difference caused by refraction is adopted, the difference in the refractive index with the observation target becomes small, and the observation becomes difficult.

In view of the above problems, it is an object of the present invention to provide a microscope with which the moving fine particles can be easily focused.

[0011]

In order to solve the above problems, the present invention enables microscopic observation of non-stationary organisms or particles by collecting and irradiating a light beam to capture an observation target. I chose

Therefore, a microscope for observing fine particles has a light source and an optical system for condensing a light beam from the light source to form a trap.

[0013]

The optical system collects the luminous flux and forms a trap. Then, by focusing the microscope on the trap, the moving "fine particles to be observed" are captured by the trap and observed.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is an explanatory view of a microscope with a fine particle capturing function by laser light according to the present invention.

This microscope includes an eyepiece lens 15, an objective lens 7, a stage 9, a condenser lens 10, a mirror 11, and an optical element combination 12, which are an optical system and an illumination system for observation. , Visible light source 13. Further, a laser light source 1, an aperture stop 3, a field stop 4, a field lens 5, and a half mirror for capturing fine particles.
6 and. Aperture stop 3, field stop 4, field lens 5, half mirror or dichroic mirror 6
The objective lens 7 is an optical system that forms a trap.

The object to be observed is placed in the cell 8 placed on the stage 9.

The laser light source is installed either in the reflection light source section of the reflection / transmission illumination type microscope or in the excitation light source section in the case where an epi-illumination device is combined with the transmission microscope. In this embodiment, it is installed in the reflection light source section of the microscope for both reflection and transmission illumination.

The laser light from the laser light source 1 enters the collector lens 2, passes through the field lens 5, is reflected by the half mirror 6 and is condensed by the objective lens 7, and captures the observation object at the position of 14. .

On the other hand, the illumination for observation passes from the visible light source 13 through the optical element combination 12, is reflected by the mirror 11, is condensed by the condenser lens 10, and is irradiated to the observation object at the position 14. This visible light is observed through the objective lens 7 and the eyepiece lens 15. The observation may be performed directly with eyes or on a monitor through an imaging system.
The object to be observed is captured in the cell 8 at the position 14 and is suspended in a solution such as water. The cell 8 is, for example, a transparent container such as glass. In the present invention, moving means moving in a medium or fluid, or moving with a fluid.

As the laser light source 1, an argon ion laser and a semiconductor laser are suitable. Laser light source 1
Alternatively, the positions of the collector lens 2 and the field lens 5 are variable. Therefore, the capture position can be shifted from 14 on the optical axis. Examples of the observation target include various organisms and particles such as cells such as lymphocytes suspended in an aqueous solution, chromosomes, plankton, and the like.

As an actual observation method, a cell filled with a solution containing organisms and particles is set in a microscope, and the stage 9 is moved to bring an observation object roughly to the center of the visual field and irradiate and capture a laser. The observation is easy.

Capture by laser beam focused irradiation will be described.

FIG. 4 shows a state in which the particle X is irradiated with the laser light focused at the point Q by the objective lens 7. The incident laser light a is refracted on the surface of particles having different refractive indices, and the vector of the momentum of light (the vector having a magnitude of 2π / λ in the direction of the pointing vector) changes.
The amount of change in the momentum of light is equal to the amount of change in the momentum of the particle, and the force corresponding to the amount of change in the momentum acts on the particle.
This is designated as Fta1. Similarly, when the laser light is emitted from the particle, Fa2 force acts. The total force of Fta1 and Fta2 is F
At ta, the force Ftb1 exerted by the laser light b incident at the same time
And the total force of Ftb2 is Ftb. As a result of the combination of the forces Fta and Ftb, an upward force Ft with respect to the optical axis acts on the particle. A part of the laser light a is reflected on the surface of the particle to generate Fra1, Fra2, Frb1 and Frb2 shown in FIG. The resultant force is Fr. The particles X are captured at a position where Ft, Fr, gravity, buoyancy in the solution, and molecular force due to heat balance.

FIG. 2 shows a state in which the laser light is focused on the lower part inside the particle. In this case, the force F acting on the particle due to refraction
Becomes downwards. Based on these principles, microscopic observation can be performed with stationary organisms and particles stationary.

Further, as shown in FIG. 3, a laser beam focusing position A
Is shifted with respect to the focal point B of the objective lens, a part of the particles (often the upper surface) is focused at the same time as the capturing.

Further, the present microscope can shift the condensing position of the laser light on the optical axis by changing the position of the light source or moving the condensing lens, and can observe any surface of the particles. .

By the laser light used in the present invention, flowing or moving particles or organisms and cells are trapped and become stationary. If the laser light is focused coaxially with the observation optical system, it can be sufficiently observed without being out of the visual field.

Further, if the laser beam and the observation optical system are focused on a predetermined position, the observation system is focused at the same time as capturing, and it is not necessary to move the stage up and down.

Using this microscope, lymphocytes taken out from human peripheral blood were observed in a system displaced from the focus position and the focus of the objective lens by about 5 μm, and they were in focus immediately when laser light was applied.

When the collector lens was moved on the optical axis, the focus height of lymphocytes changed, and multifaceted observation was confirmed.

Similar results were obtained for the chromosomes extracted from lymphocyte cells.

The peripheral blood lymphocytes and chromosomes used here were obtained in accordance with a conventional method after collecting blood, adding a cell division inducer and culturing for 3 days.

By the method of the present invention described above, cells and chromosomes that are floating in a solution and flowing, and even plankton that are swimming are trapped and allowed to stand still, without leaving the visual field.
It can be easily observed.

When the stage is moved to the left and right, and the laser is irradiated when the observation target is brought to the approximate center of the visual field, even if the stage is not exactly at the capture position, it will be carried to the capture position if it is within the irradiation optical path. At this time, by focusing the laser light and the focus of the observation optical system on a predetermined position,
The observation target can be automatically focused without moving the stage up and down.

This is not only easy to observe, but also has the advantage of being compatible with automatic inspection.

If the position of the collector lens or the field lens is changed without moving the stage, the capture position can be raised or lowered. This means that the observation system can be focused on an arbitrary position in the optical axis direction of the observation target, whereby the contour of each vertical surface of the observation target can be observed.
You can also obtain dimensional information.

Further, in this method, it is not necessary to increase the viscosity of the solution, the sample preparation is easy, and the cost can be suppressed. Further, since it is possible to prevent a decrease in the difference in refractive index due to an increase in viscosity, there is an effect that observation becomes very easy.

[0038]

As described above, according to the present invention, it is possible to provide a microscope in which the microscope can be easily focused on moving fine particles.

[Brief description of drawings]

FIG. 1 is a block diagram of a microscope with a function of capturing fine particles by laser light according to the present invention.

FIG. 2 is an explanatory diagram of a state in which laser light is focused on the lower part of a particle according to the present invention.

FIG. 3 is an explanatory diagram of a state in which a laser beam focusing position A according to the present invention is shifted with respect to a focal point B of an objective lens.

FIG. 4 is an explanatory diagram showing a state in which a laser beam focused on a point Q by an objective lens is applied to a particle X according to the present invention.

[Explanation of symbols]

1 ... Laser light source, 2 ... Collector lens, 3 ... Aperture stop, 4 ... Field stop, 5 ... Field lens, 6 ... Half mirror, 7 ... Objective lens, 8 ... Cell, 9 ... Stage, 10 ...
Condenser lens, 11 ... Mirror, 12 ... Optical element combination, 13 ... Visible light source, 14 ... Capture position, 15 ... Eyepiece.

Claims (6)

[Claims] 1. A light beam is condensed in a movable space of "fine particles to be observed" to form a trap, and moving "fine particles to be observed" is trapped in the trap, a microscope. A method of observing fine particles, which comprises observing with a microscope, focusing on the trap. 2. The method for observing fine particles according to claim 1, wherein the focus position of the light beam is set to a position different from the focus position of the microscope, and the part other than the focus position of the light beam is observed. Method. 3. The fine particle observing method according to claim 1, wherein the fine particle to be observed is captured at an arbitrary position, with the light collecting position of the light flux being an arbitrary position. A method for observing fine particles, which comprises observing an arbitrary surface of. 4. In a microscope for observing fine particles, a light source and a light beam from the light source are "fine particles to be observed".
Has an optical system that forms a trap by condensing it in a movable space, and captures moving "fine particles to be observed" in the trap, and observes the microscope focus in accordance with the trap. A microscope characterized by that. 5. The microscope according to claim 4, wherein the light beam is focused on a position different from the focus position of the microscope, and the position other than the light beam focusing position is observed. 6. A microscope for observing fine particles, comprising a light source and an optical system capable of condensing a light flux from the light source to form a trap at an arbitrary position, and moving "fine particles to be observed" The trap can be captured in the trap to observe any surface of the "fine particles to be observed".