JPH0641918B2 - Laser light irradiation method and apparatus for irradiating flowing chromosomes or cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method and apparatus for irradiating a slit-shaped laser beam perpendicular to the flowing axis of flowing chromosomes or cells.

[Prior Art] Conventionally, a device called a cell sorter has been used for cell chemistry,
Widely used in immunology, hematology, oncology, genetics and other fields.

A cell sorter is a device that irradiates cells or chromosomes that flow at high speed with light, detects optical or electrical signals from cells, and breaks those signals to distribute chromosomes or cells with defined characteristics. is there.

Various devices have been devised to date for cell sorters. In those devices, by irradiating cells or chromosomes with laser light, detecting the fluorescence intensity from the cells or chromosomes that have been fluorescently labeled in advance, and breaking the electric signal that is the detection result, Devices for selecting and collecting cell types or chromosomes of a given type are known.

The problem here is how to form the laser beam. The laser light of the interference fringe (flange pattern) is obtained by causing the two laser lights to interfere with each other by utilizing the coherence property. For example, the Young's interference method and the Mach-Zehnder's interference method can form fringe patterns with vertical stripes. Fabry Perot
In the -Perot interferometry, a ring-shaped fringe pattern can be formed.

It is known to irradiate flowing chromosomes with a fringe pattern formed by Mach-Zehnder interferometry (Richard
M. Norgren et al. “Method and Apparatus for Fringe-Scan
ning Chromosome Analysis ”, United States Patent No. 4,
596,036).

However, in the method of RM Nogren et al., The laser light having several fringe patterns with strong light intensity is applied to the chromosome, and thus the total fluorescence intensity of the light emitted from the several vertical stripe laser lights is measured. Therefore, which vertical stripe emits strong fluorescence is
It cannot be understood without analyzing the fluorescence intensity data. Therefore, it has been impossible to measure the fluorescent substance band pattern and length of the chromosome in real time.

[Problems to be Solved by the Invention] An object of the present invention is to solve the above-mentioned conventional problems and provide a laser light irradiation method capable of measuring the band pattern, length, etc. of a chromosome in real time. Especially.

[Means for Solving the Problems] In order to achieve such an object, in the method for irradiating a laser beam of the present invention, in the method for irradiating a flowing chromosome or a cell with a laser beam, at least one laser source emits light. The first half mirror splits the laser beam into a passing laser beam and a reflected laser beam, the reflected laser beam is re-reflected by at least one mirror, and the passing laser beam and the re-reflected laser beam are reflected by the second half mirror. To form a horizontal fringe interference laser light that is perpendicular to the flow axis of the chromosome or cell, narrows the horizontal fringe interference laser light in the flow axis direction with an optical lens, and the narrowed laser light is perpendicular to the flow axis. It is characterized in that it passes through a slit opened in a direction and irradiates the chromosome or cell with the passing laser light from a direction perpendicular to the flow axis.

The irradiation device of the laser light of the present invention, irradiation means for irradiating the flowing laser light from the laser source to the chromosomes or cells, fluorescence photometric optical means for measuring the fluorescence intensity from the chromosomes or cells, from the fluorescence photometric optical system In a laser light irradiation device having a selection and collection means for detecting a signal and selectively collecting chromosomes or cells, the irradiation means includes a first half mirror on which laser light emitted from a laser source is incident, and a first half mirror. Of at least one reflection mirror that re-reflects the laser light reflected by the half mirror, and the passing laser light and the re-reflection laser light that have passed through the first half mirror, and a flow axis through which the chromosome or cell flows. A second half mirror that forms vertical horizontal stripe interference laser light, an optical lens that narrows the horizontal stripe interference laser light to the flow axis, and an opening in the direction perpendicular to the flow axis. And, characterized by comprising a slit for passing the narrowed laser light.

[Operation] According to the present invention, a laser beam having several fringe patterns formed by the Mach-Zehnder interferometry is passed through a substantially linear slit, so that the light intensity is very strong and the thickness is thin. Only the slit-shaped laser light can be applied to the chromosome or cell flowing at high speed.

[Examples] Hereinafter, the present invention will be described in detail with reference to the drawings.

FIG. 1 is a configuration diagram of an optical system of an embodiment of the present invention that forms a slit-shaped laser beam.

In the figure, 101 is a laser oscillator that emits continuous light of visible light or ultraviolet light having a laser beam diameter of about 1.6 mm, 102 is a beam splitter (half mirror), 103 and 104 are mirrors respectively, and 105 is a beam splitter (half mirror). Mirror). 106 is a cylindrical lens, and 107 is a slit having a width of several hundreds of microns and a thickness of about 1 micron.

Reference numeral 108 denotes a flow chamber that forms an aqueous solution containing cells or chromosomes in a sheath fluid, and the chromosomes or cells pass through the chamber along the central axis of the chamber. The outer shape of the nozzle portion of the flow chamber 108 is a regular octagon.
The diameter of the pores opened in the nozzle through which cells or chromosomes pass one by one was set to about 50 to about 200 microns. Since the cells or chromosomes passing through the pores of the nozzle are irradiated with light, the material of the nozzle was selected such that synthetic laser such as quartz passes uniformly. Here, since oil for oil immersion or glycerin is applied between the outer wall of the regular octagonal nozzle portion and the slit 107, the outer wall of the nozzle portion and the slit are almost in contact with each other. Further, 109 is an objective lens, 110 is an interference filter such as a long pass filter, 111 is a convex lens, 112 is a pinhole, and 113 is a dichroic filter.

Next, the irradiation of laser light and the emission of fluorescence in the optical system shown in FIG. 1 will be described.

First, laser light 114 is emitted from the laser oscillator 101. Here, the traveling direction of the laser light 114 is taken as the x-axis. The direction of the central axis of the flow axis of cells or chromosomes flowing in the flow chamber 108 is taken as the y axis. The positive direction of the y-axis is opposite to the direction in which the chromosomes or cells flow. z-axis direction is x-axis, y-axis,
And the z-axis is oriented in the direction of the right-handed system. The laser beam 114 emitted in the positive direction of the x-axis is split by the beam splitter 102 into a passing laser beam 115 and a reflected laser beam 116. The reflected laser light 116 is reflected by the mirror 103 and the laser light 11
7 and is reflected by the mirror 104 to become laser light 118. The passing laser light 115 and the reflected laser light 117 are parallel to each other.

The passing laser light 115 passes through the neutral filter 125 provided between the beam splitters 102 and 105 and becomes the laser light 119. The neutral filter 125 is
The beam splitter 105 is for adjusting so that the light intensities of the two laser beams 118 and 119 are equal to each other. If the light intensities of the two laser lights are not equal to each other, the contrast between the light intensities of the peaks and the valleys of the fringe pattern described later becomes weak. Laser light 118 and 119 with beam splitter 105
Laser light with several fringe patterns that interferes with each other 12
It becomes 0.

Beam splitters 102 and 105 and mirrors 103 and 1
A method of forming an optical system with 04 and causing the two laser beams 118 and 119 to interfere with each other is called Mach-Zehnder interferometry. Beam splitters 102 and 105 optical axis and mirror 103
And 104 optical axes intersect to form a parallelogram.

The laser light 120 has its expansion in the y-axis direction reduced by the cylindrical lens 106, and becomes the laser light 121 having a strong light intensity distribution along the z-axis direction. The laser light 121 passes through the slit 10
After passing through 7, the laser light having several fringe patterns changes to the laser light having only one fringe pattern. The laser light having only one fringe pattern irradiates the chromosomes or cells flowing along the central axis of the flow chamber 108, and the emitted light including forward scattered light and fluorescence is emitted.
It becomes 2. The emitted light 122 is condensed by the objective lens 109.
The fluorescence passes through the interference filter 110, but the forward scattered light does not pass through the interference filter 110.

Note that the fluorescence appears on the longer wavelength side than the wavelength of the excitation light laser. When the fluorescent light is narrowed down by the convex lens 111 and passes through the pinhole 112, only the fluorescent light emitted from the chromosome or the cell becomes, and the diffused light and the escaped light are blocked by the pinhole 112. Further dichroic filter 113
Is used to disperse into fluorescence having a specific wavelength band. The dispersed fluorescence is detected by a photomultiplier tube (not shown), and the detection signal is analyzed using a high-speed processing device or the like to determine whether the chromosome (or cell) to be obtained or he is, If it is a chromosome (or cell) to be sought, a preparative signal is output.

FIG. 2 is a configuration diagram of the flow chamber and the fluorescence side light system. This figure is a view of the optical system of FIG. 1 as seen from below, that is, from the positive direction of the y-axis.

In the figure, the x-axis and the z-axis have the same meaning as described in the description of FIG. 206A and 206B are cylindrical lenses, and 207A and 207B are slits having openings. The size of the opening is the same as the size of the opening of the slit 107 shown in FIG. Reference numeral 226 is a pore through which a solution containing chromosomes or cells flows, and the diameter of the pore has already been described in the description of the nozzle section in FIG. Reference numeral 227 is a nozzle portion of the flow chamber, and the material and outer shape of the chamber are the same as those of the chamber 108 shown in FIG. 209A and 209B are objective lenses, and their focal points coincide with the central axes of the pores 226 in the chamber. 210A and 210B are interference filters that allow only fluorescence to pass through. 211A and 211B are convex lenses, and the focal points of these lenses coincide with the positions of the pinholes 212A and 212B, respectively. Pinholes 212A and 212B serve to separate only fluorescence from chromosomes or cells.

Next, the irradiation of laser light and the emission of fluorescence in the optical system shown in FIG. 2 will be described.

In this figure, two laser beams 220A and 220B having different wavelengths are applied to the chromosome or cell. The reason is that when fluorescently labeling a chromosome or a cell, two or more fluorescent probes are used. Now to simplify the explanation here,
It is assumed that 220A is laser light of ultraviolet light and 220B is laser light having a wavelength in the visible range. The laser light 220A is directed to the central axis of the pore 226 along the x-axis direction, and the laser light 220B is directed to the x-axis and the central axis of the pore 226.
It makes an angle of 5 degrees toward the central axis of the pore 226. Here, the laser light 220A and the laser light 220B are in the same plane. When the laser light 220A, 220B having several fringe patterns passes through the cylindrical lenses 206A, 206B, it has high light intensity and becomes thin laser light. Further slit 207A, 2
When passing through 07B, it becomes the laser light having one fringe pattern having the strongest light intensity and the thinnest among the laser light having several fringe patterns. When the two laser beams irradiate the chromosomes or cells flowing in the pores 226 formed along the central axis of the nozzle portion 227 of the flow chamber, they emit forward scattered light and fluorescence 218A, 218B.

Here, the light emitted by irradiating the cell or the chromosome with the laser light is observed in the direction perpendicular to the laser lights 220A and 220B. The reason is that the fluorescence to be measured appears in the wavelength measurement longer than the wavelength of the excitation light by the laser light irradiation, and the light intensity is weak. Light emitted from cells or chromosomes 218A and 218B becomes parallel light when passing through lenses 209A and 209B. Two lights are interference filters 210A, 210B
After passing through, only fluorescence will be emitted. Two fluorescence is a convex lens
After passing through 211A and 211B, they converge to form pinholes 212A and 212B.
Pass through. The following is separated by a dichroic filter (not shown), and the separated fluorescence is detected by a photomultiplier tube, and the detected signal is analyzed to separate the chromosome or cell.

FIG. 3 shows the fringe pattern of laser light and the intensity distribution of interference laser light.

FIG. 3 (A) is an enlarged view of a vertical stripe-shaped fringe pattern appearing in a circle having a diameter of about 1.3 mm, which indicates the outer edge of the spread of the laser light emitted from the laser source. The black part in the circle indicates the part where the light intensity is strong. Where the vertical stripe direction is the z-axis,
The vertical direction of the vertical stripes is the y-axis, that is, the flow axis direction in which the chromosome or cell flows.

FIG. 3 (B) shows the light intensity distribution in the y-axis direction of the laser light having the fringe pattern shown in FIG. 3 (A). From this figure, it can be seen that several band-shaped laser lights with high light intensity travel in parallel in the x-axis direction.

FIG. 4 is an explanatory diagram of a method of forming a thin band-shaped laser beam having a high light intensity from a laser beam having several fringe patterns.

In this method, laser light having several fringe patterns is passed through a slit having a substantially linear opening.

Here, the slit is composed of a slit substrate 429 made of synthetic quartz having a thickness of about 300 μm, and a chromium coating layer 428 having a thickness of about 0.1 μm coated on the slit substrate 429. The slit opening through which the laser light 421 passes has a thickness (thickness in the y-axis direction) of 1 μm by the photo-etching technology.
m, width (length in z-axis direction) about 100 microns. Here, the thickness of the opening of the slit is roughly determined by the full width at half maximum of the fringe pattern and the wavelength of the laser light.
It can be set within the range of 6 to 1.5 μm. Furthermore, the width of the slit should be wider than the diameter (50-200 μm) of the pore of the nozzle of the flow chamber so that the band-shaped laser beam can be easily irradiated to the flow chamber of the flow chamber where the chromosomes or cells flow. Usually, the range is 50 to 300 μm.

The distance between the slit and the inner wall of the nozzle portion of the flow chamber was set to 1 to 3 mm. Here, the laser beam 421 that has passed through a cylindrical lens (not shown) and has a fringe pattern of stripes having a strong light intensity in the lateral direction has a light intensity distribution indicated by a dotted curve 427. Therefore, the laser light 421 traveling in the x-axis direction has a thickness of 1 μm.
When passing through the slit of, only the laser beam having one fringe pattern having the highest light intensity is left out of the laser beams having several fringe patterns. The passing laser light irradiates the pores 426 of the nozzle portion of the flow chamber to the chromosomes or cells flowing at high speed along the flow axis 430 of the flow chamber.

As described above, one thin slit laser beam having a thickness of submicron is formed. Here, it is necessary to sufficiently adjust the accuracy of the cylindrical lens and the lateral width of the slit in order to form a laser beam having a high light intensity and a small thickness. According to this method, it is possible to reduce the thickness of the slit type laser light to about half the wavelength.

A problem with the method of this embodiment is that the laser light emitted from the linear slit is diffracted at the slit opening.

Keeping in mind that the opening of the slit is rectangular, the Frauhoffer diffraction (Frauhoffer Diffrac) occurs when the distance between the slit and the light source and the irradiation point of the stream axis is relatively long.
option) (eg Max Born & E
mil Wolf, “Principles of Optics”, Pergamon Pre
ss. 1975). In the case of Franhofer diffraction, the diffraction intensity I is generally Given in. Here, it depends on u ₁ and u ₂ , the thickness and width of the slit, the wavelength, the distance from the slit of the irradiation point to the light source and the stream axis, but mainly on the diffraction angle.

FIG. 5 shows the intensity distribution of the diffracted light by the linear slit. If the slit is linear, one of u _{1 and} u ₂ is 0. Therefore, the diffracted light intensity I at this time is Given in. u depends on the width and diffraction angle of the slit, the wavelength, the distance between the slit and the light source, and the irradiation point. The intensity of the diffracted light in the case of the slit having the rectangular opening has a distribution substantially proportional to the square of the curve 2 shown in FIG.

As shown in FIG. 5, the laser light is composed of a slit-shaped laser light having the highest light intensity in the lateral direction (z-axis direction) which is perpendicular to the traveling direction, and a laser light parallel to this laser light. I understand.

Therefore, the laser light traveling in the traveling direction of the laser light is such that only one laser light having a wide width and a small thickness excites the fluorescent substance having a band pattern in the chromosome or cell flowing at high speed along the flow axis of the flow chamber. become. Since the laser light parallel to the laser light having the highest light intensity has a weak light intensity, it does not contribute much to the excitation of the fluorescent substance. Even if there is fluorescence emitted by laser light irradiation, the fluorescence becomes background noise.

Fresnel diffraction occurs when the distance between the slit, the light source, and the irradiation point to the stream axis is relatively short. In this case, the light intensity at the irradiation point is similar to that shown in FIG. Therefore, a strong linear light intensity distribution with a thin width in the direction perpendicular to the flow axis can be obtained.

[Effects of the Invention] As described above, according to the present invention, a laser beam having several fringe patterns formed by the Mach-Zehnder interferometry is passed through almost linear slits, so that the light intensity is increased. Only a very strong and thin slit laser beam can be applied to the chromosome or cell flowing at high speed.

Therefore, it becomes possible to measure the time change of the fluorescence intensity from the chromosome or the cell at intervals of several tens of nanoseconds, and the band pattern, the length in the flow axis direction, the position of the constriction of the chromosome, and the like appearing with the fluorescent label can be measured. It can be measured in real time.

[Brief description of drawings]

FIG. 1 is a block diagram of an optical system for forming a slit-shaped laser beam according to an embodiment of the present invention, FIG. 2 is a block diagram of a flow chamber and a fluorescence photometric system, and FIG. 3 is a fringe pattern of a laser beam and an interference laser beam. FIG. 4 is an explanatory diagram of a method of forming a band-shaped laser beam, and FIG. 5 is a diagram showing an intensity distribution of diffracted light by a linear slit. 101 …… laser oscillator, 102 …… beam splitter, 103,104 …… mirror, 105 …… beam splitter, 106 …… cylindrical lens, 107 …… slit, 108 …… flow chamber, 109 …… objective lens, 110 …… interference Filter, 111 ... Convex lens, 112 ... Pinhole, 113 ... Dichroic filter, 114 ... Laser light, 115 ... Passed laser light, 116 ... Reflected laser light, 117,118,119,120,121 ... Laser light, 122 ... Fluorescence And scattered light, 123 …… fluorescence, 125 …… neutral filter, 206A, 206B …… cylindrical lens, 207A, 207B …… slit, 209A, 209B …… objective lens, 210A, 210B …… interference filter, 211A, 211B …… Convex lens, 212A, 212B …… Pinhole, 218A, 218B …… Outgoing light, 220A …… Ultraviolet laser light, 220B …… Laser light with wavelength in the visible range, 226 …… Pore, 227 …… flow Nozzle portion of Enba, 421 ...... laser light, 426 ...... pore, 427 ...... laser beam having a fringe pattern, 428 ...... chromium coating layer, 429 ...... slit substrate, the flow axis of 430 ...... flow chamber.

Claims (8)

[Claims] 1. A method of irradiating a flowing chromosome or cell with a laser beam, wherein the laser beam emitted from at least one laser source is branched into a passing laser beam and a reflected laser beam by a first half mirror, The reflected laser light is re-reflected by at least one mirror, and the passing laser light and the re-reflected laser light are interfered by the second half mirror to form an interference laser light having horizontal stripes perpendicular to the flow axis through which the chromosome or cell flows, The horizontal fringe interference laser light is narrowed down by the optical lens in the direction of the flow axis, the narrowed laser light is passed through the slit opened in the direction perpendicular to the flow axis, and the passing laser light is changed from the direction perpendicular to the flow axis. A method of irradiating a laser beam, which comprises irradiating a chromosome or a cell. 2. The laser beam irradiation method according to claim 1, wherein the thickness of the opening of the slit is 0.6 to 1.5 μm with respect to the flow axis direction. 3. The laser beam irradiation method according to claim 1, wherein the width of the opening of the slit perpendicular to the flow axis is 50 to 300 μm. 4. The two laser beams emitted from the two laser sources are radiated from a crossing angle of 45 degrees, forming a plane perpendicular to the flow axis of the chromosome or cell. The laser beam irradiation method according to any one of items 1 to 3. 5. The shape of the outer wall of the nozzle portion of the flux portion of the chromosome or cell irradiated with the narrowed laser light is regular 8.
The method for irradiating laser light according to any one of claims 1 to 4, wherein the method is a prism. 6. The method according to any one of claims 1 to 5, wherein the distance between the slit and the wall of the pore of the nozzle of the flow chamber through which the chromosomes or cells flow is 1 to 3 mm. The method of irradiating a laser beam according to the item. 7. The invention according to claim 1, wherein the slit and the outer wall of the nozzle portion of the flux portion are brought into close contact with each other.
Item 7. A laser beam irradiation method according to any one of items 6 to 6. 8. An irradiation means for irradiating a flowing chromosome or cell with laser light from a laser source, a fluorescence photometric optical means for measuring the fluorescence intensity from the chromosome or cell, and a signal from the fluorescence photometric optical system for detection. And a laser light irradiation device having a selection and collection means for selectively collecting chromosomes or cells, wherein the irradiation means includes a first half mirror on which laser light emitted from a laser source is incident, and the first half At least one reflection mirror that re-reflects the laser light reflected by the mirror, the passage laser light that has passed through the first half mirror and the re-reflection laser light are synthesized, and a flow axis through which a chromosome or a cell flows. A second half mirror that forms vertical horizontal stripe interference laser light, an optical lens that narrows down the horizontal stripe interference laser light with respect to the flow axis, and a perpendicular to the flow axis. It has an opening in direction, that equipped with a slit for passing the narrowed laser beam, the laser beam irradiation apparatus according to claim.