JPH0989774A - Fine-substance microscopic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fine-substance microscopic apparatus which makes use of a magnetic force and by which a fine substance such as a magnetic sample particle or the like can be fixed surely. SOLUTION: A permanent magnet 21 is installed at the tip part of an objective lens 13. The permanent magnet 21 is inserted into a through hole 101a in a table 101 on which a dish 18 used to house fluorescent magnetic beads 20 is placed, and it is arranged so as to be sufficiently close to the outside bottom face of the dish 18.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscopic substance microscopic apparatus for fixing a microscopic substance having magnetism by using magnetic force and performing a speculum.

[0002]

2. Description of the Related Art Recently, researches on DNA and proteins have been actively conducted, and in these researches, fluorescence observation or the like for observing fluorescence reaction of DNA or protein is used. FIG. 7 shows an example of a microscopic substance microscopic apparatus using a microscope for performing such fluorescence observation, in which a liquid in which sample particles such as DNA and protein are suspended is stored in a table 1 of a microscope main body (not shown). The dish 2 was placed. The objective lens 3 is arranged below the table 1 on which such a dish 2 is mounted, and a magnet 5 is provided around the objective lens 3 by a fixed frame 4.

Then, the sample particles floating in the liquid of the dish 2 are bound to the magnetic beads 6, and in this state, a magnetic field is formed in the magnet 5 arranged around the objective lens 3 to place the magnetic beads 6 on the bottom surface of the dish 2. Observation by a microscope through the objective lens 3 is performed from the state of being attracted and two-dimensionally distributed.

In this case, fluorescence observation is employed for the observation with a microscope. In addition to magnetic beads, fluorescent beads are bound or the magnetic beads are made to have fluorescence, and evanescent illumination is limited to the vicinity of the bottom surface of the dish 2. Based on the above, the state of fluorescence generated from the magnetic beads having fluorescence is observed.

[0005]

The magnetic beads 6 used for such fluorescence observation have a diameter of about 1 μm. However, when the size of the sample particles is reduced, for example, when the particle size is about several tens nm, the size of the sample is significantly different from the size of the magnetic beads 6, and a plurality of sample particles are bound to one magnetic bead 6. In some cases, an accurate fluorescent reaction may not be observed. In addition, in the magnetic beads 6 having a diameter of 1 μm, the dish 2
It is also difficult to determine the location of the sample particles in order to locally disperse them on the bottom surface and observe the fluorescence.

Therefore, it is conceivable that the diameter of the magnetic beads 6 is made approximately the same as that of the sample molecules and one sample molecule is bound to one magnetic bead 6, but such a magnetic bead having a small diameter is used. Then, even if the magnetic field of the magnet 5 causes polarization of S and N in the magnetic beads 6 themselves, the attraction force due to the gradient of the magnetic flux density is small because the distance between these poles is extremely small. Since it is provided around the objective lens 3 and is far away from the bottom surface of the dish 2, a large attractive force cannot be expected, and it is difficult to apply an effective attractive force to the magnetic beads 6.
In some cases, the magnetic beads 6 could not be constrained by drawing them to the bottom of the dish 2. Further, in the case of particles having a small diameter, the particles tend to float in the liquid due to Brownian motion, which makes it more difficult to attract the magnetic beads to which the sample particles are bound.

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a microscopic substance microscopic apparatus capable of reliably immobilizing a microscopic substance such as sample particles having magnetism using magnetic force. To aim.

[0008]

According to the first aspect of the present invention,
In a microscopic substance microscopic device for fixing a microscopic substance having magnetism by using magnetic force and performing a microscopic examination through an objective lens, a magnet is provided at a tip portion of the objective lens located on the microscopic substance side. There is.

According to a second aspect of the present invention, in the first aspect, the magnet is a permanent magnet. Claim 3
In the first aspect of the present invention, the magnet is an electromagnet.

As a result, according to the present invention, since the magnet can be arranged close to the magnetic micro substance, a sufficiently large attracting force can be expected even for an extremely micro substance corresponding to sample particles such as DNA and protein. As a result, an effective suction force can be applied, and Brownian motion of these minute substances can be limited and restrained by a strong force.

[0011]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows a schematic structure of an inverted microscope applied to a microscopic substance microscopic apparatus of the present invention.
In the figure, 10 is a microscope main body, and this microscope main body 10
Includes a transillumination light source 11, an excitation light source 12, an objective lens 13, a condenser lens 14, a lens barrel 15, an eyepiece lens 16, and the like.

In this case, the excitation light from the excitation light source 12 is passed through an optical system in the microscope body 11, the objective lens 13 and a prism (not shown) for evanescent illumination to accommodate the magnetic beads having fluorescence on the table 101. Irradiation is performed from the outer bottom surface of a dish (not shown), an evanescent field is formed near the inner bottom surface of the dish by evanescent illumination, and the evanescent field is used to generate fluorescence in the magnetic beads. Then, this fluorescent state is sent to the eyepiece lens 16 through the optical system in the microscope body 11 and the lens barrel 15 as an observation image by the objective lens 13 by the transmitted illumination light given from the transmitted illumination light source 11 through the condenser lens 14. It is possible to observe the fluorescence reaction of.

In this case, the objective lens 13 is the revolver 1
7 is provided with a plurality of various magnifications, and a desired magnification can be selected. FIG. 2 shows a schematic configuration of a main part of the present invention, and the same parts as those in FIG. 1 are denoted by the same reference numerals.

In this case, the table 10 of the microscope body 10
1 has a through hole 101a, and this through hole 101a
The dish 18 is placed on the top. This dish 18
As described above, the magnetic beads 20 having fluorescence are housed together with the liquid 19 in which the sample particles such as DNA and protein are suspended, and the magnetic beads 20 are bound to the sample particles suspended in the liquid 19. . Further, the magnetic beads 20 here have a diameter of 3 nm if the sample particle diameter is several tens nm.
The diameter is about 0 nm.

Below the table 101 of the microscope body 10,
The objective lens 13 is arranged. This objective lens 13
Is integrally provided with a cylindrical permanent magnet 21 around the optical lens 131 located at the tip. This permanent magnet 21
Is made of a magnetic material such as ferrite, and has a polarity at both ends of the cylindrical body, that is, both ends in the vertical direction in the drawing. The permanent magnet 21 can be made detachable by, for example, a screwed structure.

The objective lens 13 as described above is driven in the vertical direction to move the focal point and focus on the outer bottom surface of the dish 18. In this case, by using an objective lens 13 having a high magnification and a short working distance (WD), the permanent magnet 21 portion at the tip of the objective lens 13 is inserted into the through hole 101a of the table 101 and the dish 18 is inserted. It is close enough to the outer bottom surface.

Further, an evanescent illumination prism 181 is arranged between the dish 18 and the objective lens 13. This evanescent illumination prism 181 is
A matching oil is detachably fixed to the outer bottom surface of the dish 18 on the objective lens 13 side. As a result, the excitation light from the excitation light source 12 that has passed through the objective lens 13 is
The light enters the prism 18 for evanescent illumination and is totally reflected, and an evanescent field is formed near the inner bottom surface of the dish 18.

The magnetic beads 20 having a fluorescence property of 30 nm diameter used in the present invention are specifically manufactured by the following preparation method. (1) First, polyacrylamide beads are prepared by the Water in Oil microemulsion method.

(2) Treatment with ethylenediamine to introduce amino groups into the beads. (3) Fluorescent dyes are prepared by reacting the amino group-reactive fluorescent dye with the beads. Next, (4) 100 μl of 1.4M FeCl ₃ and 600 μl of polyethylene glycol (MW = 200) are mixed.

(5) 100 μl of ammonia water is added to the solution of (4) with vigorous stirring to prepare magnetite. (6) Add 1 ml of water to the magnetite of (5) and add 400
Collect the magnetite by applying a centrifugal force at 0 rpm for 2 minutes.

(7) Further, a few ml of water is added to the precipitate, centrifugal force is applied in the same manner, and this operation is repeated a plurality of times for washing. (8) The precipitate obtained in (7) was added with 1 ml of 0.2M nitric acid.
Suspended for 30 minutes and left at 15,000 rpm for 30 minutes.
Centrifuge for minutes and collect the supernatant.

(9) Absorbance of supernatant (at 310 nm)
To measure. The absorbance at this time is 40. (10) Fluorescent beads prepared by (3) (about 1 m
(Adjusted to g / ml) 0.3 ml of polyethylene glycol (MW = 200) and 15 μl of magnetite (ODat310 = 40) dissolved in 0.2 M nitric acid are mixed.

(11) 1.5 Tris-HCl buffer (pH
8.8) Add 6 μl to (10). With this neutralization of pH, magnetite is introduced into the beads. (13) Centrifugal force is further applied at 4000 rpm for 2 minutes,
Collect the magnetic beads.

(14) The magnetic beads are suspended in 0.3 ml of water and dialyzed against water. By such preparation, the magnetic beads with a diameter of 30 nm having final fluorescence in (14) can be obtained. These beads retain their strong magnetism for a few days, but due to the gradual oxidation, magnetite becomes maghemite and the magnetism becomes weak. Even magnets with weak magnetism can be sufficiently captured by a magnet provided on the microscope side. In order to prevent oxidation, oxygen may be replaced with an enzyme for storage. In addition, since the magnetite entered the beads, the protein
The beads are more likely to be absorbed, but to avoid this, a small amount of medieval activator may be added.

Thus, according to this, the permanent magnet 21 provided at the tip of the objective lens 13 is inserted into the through hole 101a of the table 101 on which the dish 18 containing the magnetic beads 20 having fluorescence is mounted. Dish 1
8 is close enough to the outer bottom surface of magnetic beads 2
It is possible to expect a sufficiently large attracting force to the magnetic beads 20 from the permanent magnet 21 even if a sample having a diameter of 30 nm which is almost the same as the sample particle size of 0 to 30 is used. These magnetic beads 20 are caused to act on each other, and these magnetic beads 20 are attracted to the bottom surface of the dish 18 and can be restrained with a strong force. Further, the restraint of the strong force at this time can also limit the Brownian motion of the magnetic beads 20 and the sample particles. As a result, the sample particles can be reliably fixed, and highly accurate fluorescence observation can be realized.

When the objective lens 13 having the permanent magnet 21 at the position facing the dish 18 is temporarily removed by operating the revolver 17, the magnetic beads 20 in the dish 18 are released from the magnetic field of the permanent magnet 21. Since it is possible to remove the objective lens 13, the magnetic beads 20
Are freely scattered in the liquid, and then the objective lens 13 is returned to the position opposite to the dish 18 so that the magnetic beads 20 restrain the movement of the magnetic beads 20 by the magnetic field of the permanent magnet 21. It becomes possible to bind to the bottom surface, and by repeating such an operation, it becomes possible to bind only the target sample particles, and it is possible to observe the fluorescence reaction of the sample particles after a lapse of time. (Second Embodiment) FIG. 3 shows a schematic configuration of the second embodiment. The same parts as those in FIG. 2 are designated by the same reference numerals. It should be noted that although the illustration of the evanescent illumination prism 181 between the dish 18 and the objective lens 13 described in FIG. 2 is omitted in FIG.
8 is detachably fixed to the outer bottom surface on the objective lens 13 side with matching oil.

By the way, in fluorescence observation, even if the sample size is small, a high-magnification objective lens is not always necessary.
Rather, a low-magnification (for example, about 40 times) bright objective lens may be used.

In such a case, the working distance (WD) of the objective lens becomes long, but as a countermeasure against this, as shown in the drawing, the cylindrical connecting cylinder 2 is provided at the tip of the objective lens 13.
2 is provided, and a cylindrical permanent magnet 21 is integrally provided at the tip end portion of the connecting tubular body 22, and the permanent magnet 21 portion is inserted into the through hole 101a of the table 101 so that the outer bottom surface of the dish 18 can be sufficiently covered. Configure to be in close proximity. The permanent magnet 21 here can also be made detachable from the connecting cylinder body 22 by, for example, a screw-in structure.

Even in this case, the same effect as that of the first embodiment can be expected. (Third Embodiment) FIG. 4 shows a schematic configuration of the third embodiment. The same parts as those in FIG. 2 are designated by the same reference numerals. It should be noted that although the illustration of the evanescent illumination prism 181 between the dish 18 and the objective lens 13 described in FIG. 2 is omitted in FIG.
8 is detachably fixed to the outer bottom surface on the objective lens 13 side with matching oil.

In this case, the permanent magnet 21 provided at the tip of the objective lens 13 is replaced with an electromagnet 23 having a coil, and a drive current is supplied from the drive circuit 24 to the electromagnet 23 to generate a predetermined attractive force. ing. In this case, assuming that the number of turns of the coil of the electromagnet 23 is n and the current flowing through the coil is I, the strength H of the magnetic field is H = n × I 2.

Even in this case, however, the same effect as that of the first embodiment can be expected, and the current I flowing in the coil can be made variable while the number of coil turns n is constant, so that the drive circuit can be made variable. By changing the current I flowing through the coil by means of 24, the strength of the attraction force of the electromagnet 23 can be changed according to the size of the magnetic beads 20, or the electromagnet 23
It is also possible to adjust the strength of the suction force of the above to adjust the concentration of particles on the bottom surface of the dish 18. In addition, the drive circuit 2
By turning on / off the bias of the electromagnet 23 by No. 4, it is possible to obtain the same effect as temporarily removing the objective lens 13 by operating the revolver 17 described in the first embodiment.

Although the electromagnet 23 is provided at the tip of the objective lens 13 in the above description, part or all of the objective lens 13 is made of a magnetic material, and a coil is wound around the magnetic material to form an electromagnet. You may do it. (Fourth Embodiment) FIG. 5 shows a schematic configuration of the fourth embodiment, and the same parts as those in FIG. 3 are designated by the same reference numerals. In FIG. 5, the illustration of the evanescent illumination prism 181 between the dish 18 and the objective lens 13 described in FIG. 2 is omitted.
8 is detachably fixed to the outer bottom surface on the objective lens 13 side with matching oil.

In this case, in addition to the electromagnet 23 provided at the tip of the objective lens 13, an electromagnet 25 is arranged above the bottom surface of the dish 18 so that the attraction force of the magnetic beads 20 toward the bottom surface of the dish 18 is further increased. I have to.

The electromagnet 25 here is one in which a hollow cone (conical shape or the like) -shaped iron core (a magnetic metal such as Ni is also possible) 252 is provided inside a ring-shaped coil portion 251. The tip is brought close to the magnetic beads 20 at the bottom of the dish 18, and the magnetic flux is concentrated near the magnetic beads 20 to strengthen the magnetic field.

The iron core 252 of the electromagnet 25 as described above is used.
May be immersed in the liquid of dish 18, so
The surface of the iron core is rustproofed and eluted. Further, the electromagnet 25 is attached to the support base 26,
The support 28 is provided so as to be vertically movable via a vertical drive mechanism 27. This is dish 18 on table 101
This is because it is easy to move in and out, and in detail is to adjust the vertical position of the distribution boundary surface P of the lines of magnetic force in relation to the electromagnet 23 on the side of the objective lens 13 described later.

A drive current is supplied from the drive circuit 24 together with the electromagnet 23 to the electromagnet 25. It is to be noted that the transmitted illumination light 30 applied to the bottom surface of the dish 18 through the condenser lens 29 passes through the central portion of the iron core 252.

Therefore, in such a structure, the magnetic poles of the electromagnets 23 and 25 are reversed from the drive circuit 24, for example, as shown in FIG. If currents are made to flow through the coils of the electromagnets 23 and 25 so as to face each other, a distribution boundary surface P formed by the magnetic force lines of these electromagnets 23 and 25 is formed. It is possible to create a space d with a large density gradient. The space d is located on the dish 1 where the magnetic beads 20 are located.
If the height of the electromagnet 25 is adjusted so as to coincide with the inner bottom surface of 8, the magnetic beads 20 can be restrained with a strong force, and further, the restraint at this time also limits the Brownian motion of the magnetic beads 20 and the sample particles. Therefore, it is possible to realize more accurate fluorescence observation.

Further, the drive circuit 24 causes the electromagnets 23, 2
If the magnetic field directions of 5 are the same or the supplied current is small, the attracting force to the magnetic beads 20 can be weakened. Therefore, once the attracting force to the magnetic beads 20 is weakened, the magnetic beads 20 are freely scattered in the liquid, afterwards,
If the magnetic beads 20 are constrained by strengthening the attracting force to the magnetic beads 20, new sample particles can be constrained to the bottom surface of the dish 18. By repeating such an operation, the target sample particles can be restrained. It becomes possible to restrain only those, and it is also possible to observe the fluorescence reaction of the sample particles after a lapse of time.

If the magnetic fields of the electromagnets 23 and 25 maintain the current in the same direction, for example, in the case of rod-shaped samples, these samples can be oriented in the direction of magnetic force lines. Although the embodiments have been described above, the present invention also includes the following inventions.

(1) In the first aspect, a part or all of the objective lens is made of a magnetic material, and a coil is wound around the magnetic material to form an electromagnet. By doing so, the structure of the electromagnet can be simplified.

(2) In the first aspect, another magnet is further provided with a minute substance sandwiched between the magnets on the objective lens side, and a magnetic field in the opposite direction is generated from these magnets. By doing so, it is possible to create a space having a large density gradient of magnetic force lines on the distribution boundary surface of the magnetic force lines of these magnets.

(3) In (2), these magnets are composed of electromagnets, and it is possible to generate magnetic fields in the same direction or opposite directions from these electromagnets. This makes it possible to increase or decrease the attracting force to the minute substance, and by repeating this operation, it is possible to restrain only the target minute substance and observe the reaction of the minute substance over time. .

(4) The electromagnet has a hollow cone-shaped iron core inside the ring-shaped coil portion. By doing so, the magnetic flux can be concentrated near the minute substance and a strong magnetic field can be given.

In the above-described embodiment, the case where the sample particles are bound to the bottom surface of the dish 18 and the fluorescence reaction of the sample particles is observed has been described. However, the sample particles were bound to the bottom surface of the dish in the scanning probe microscope. In the state, it can be applied to attaching a reaction substance to specific particles, and observing shape change and electrical change of sample particles.

[0045]

As described above, according to the present invention, since the magnet can be arranged in the vicinity of the minute substance having magnetism, a sufficiently large attraction force can be expected even for an extremely minute substance,
An effective suction force can be applied, and Brownian motion of these minute substances can be restricted and constrained by a strong force, which enables secure fixation of sample particles and realizes highly accurate fluorescence observation. it can.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of an inverted microscope applied to a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of a main part of the first embodiment.

FIG. 3 is a diagram showing a schematic configuration of a second embodiment of the present invention.

FIG. 4 is a diagram showing a schematic configuration of a third embodiment of the present invention.

FIG. 5 is a diagram showing a schematic configuration of a fourth embodiment of the present invention.

FIG. 6 is a diagram for explaining a fourth embodiment.

FIG. 7 is a diagram showing a schematic configuration of an example of a conventional microscopic substance microscope apparatus.

[Explanation of symbols]

 10 ... Microscope main body, 101 ... Table, 101a ... Through hole, 11 ... Transmitted illumination light source, 12 ... Excitation light source, 13 ... Objective lens, 131 ... Optical lens, 14 ... Condenser lens, 15 ... Lens barrel, 16 ... Eyepiece lens , 17 ... Revolver, 18 ... Dish, 19 ... Liquid, 20 ... Magnetic beads, 21 ... Permanent magnet, 22 ... Connection cylinder, 23 ... Electromagnet, 24 ... Drive circuit, 25 ... Electromagnet, 251 ... Coil part, 252 ... Iron core , 26 ... Supporting base, 27 ... Vertical drive mechanism, 28 ... Posts, 29 ... Condenser lens, 30 ... Transmitted illumination light.

Claims (3)

[Claims] 1. A micro-substance microscope apparatus for fixing a micro-substance having magnetism by using magnetic force and performing microscopic examination through an objective lens, wherein a magnet is provided at a tip portion of the objective lens located on the micro-substance side. A microscopic substance microscopic device characterized by being provided with. 2. The microscopic substance inspection apparatus according to claim 1, wherein the magnet is a permanent magnet. 3. The microscopic substance inspection apparatus according to claim 1, wherein the magnet is an electromagnet.