JPS61165639A - Particle analyser - Google Patents

Abstract

PURPOSE:To enhance measuring accuracy, by detecting the focus matching state of a photometric objective lens from the image of a two-split element of the reflected image obtained by projecting photometric luminous flux to the boundary surface of the laser beam irradiation surface of a specimen particle flowing part and detected by a photoelectric detector. CONSTITUTION:The luminous flux passed through an opening 23 is projected to the front surface 2a and back surface 2b of a flow cell 1 by a light source 22 and two respective reflected opening images of the opening 23 are respectively formed onto a two-split element 26 through a convex lens 25 and, when the focus of an objective lens 8 is matched, two opening images of the opening 23 formed to the two-split surfaces of the element 26 in the same size. Because the lens 8 and a condensing lens 10 are combined so as to form parallel luminous flux between both lenses, an automatic focus unit 9 is moved even if the position of the center axis of the flow cell 1 slightly moves on an optical axis when the flow cell 1 is replaced and two outputs of the element 26 are only made equal to each other to enable the matching of a focus and measuring accuracy can be enhanced.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a particle analysis device that is capable of determining the in-focus state of a photometric objective lens in a flow cytometer or the like.

[Prior Art] In a conventional particle analysis device used in a flow cytometer or the like, for example, a particle size of 70 p-m x 2 in the center of a flow cell is
Irradiation light is irradiated onto a specimen such as blood cells wrapped in sheath liquid and passed through a flow section with a minute rectangular cross section of 0 lumen, and the resulting forward and side scattered light is used to determine the shape and size of the specimen. It is possible to obtain particle-like properties such as refractive index and refractive index. Further, for a specimen that can be stained with a fluorescent agent, important information for analyzing the specimen can be obtained by detecting the fluorescence of the specimen from side scattered light in a direction substantially perpendicular to the irradiation light.

In order to perform accurate measurements with a flow cytometer, etc., a photometric objective lens must be used to accurately focus only on the sample particles or their immediate vicinity to prevent contamination by contaminant signals from objects other than the sample particles. To do this, it is necessary to adjust the focus of the photometric objective lens, but with conventional equipment, the operator manually adjusts the focus by visual inspection while flowing a standard sample before measurement, making the operation much easier. In addition to being complicated, there are individual differences among operators, and it is currently difficult to perform sufficiently accurate focus adjustment.

Additionally, if the focal point moves during measurement, it is impossible to confirm this, so it is impossible to determine whether spurious signals have been mixed in during the measurement, and there are concerns about the reliability of the data. .

Furthermore, it requires focus adjustment every time the nozzle, flow cell, etc. are replaced, which has the disadvantage that measurement is time-consuming. In addition, when performing fluorescence measurements, it is necessary to strengthen weak fluorescence signals, and for this purpose it is necessary to use a photoelectric detector to detect fluorescence, improve the luminous efficiency of the fluorescent agent, and increase the power of the irradiation light source. It is being considered to increase the amount of light and to improve the light collection efficiency of the photometric objective lens. The luminous efficiency of fluorescent agents is currently being actively researched, and increasing the power of the irradiation light source can be considerably increased if manufacturing costs are ignored, but on the other hand, if the power is increased too much, the sample particles It is difficult to say that this is a good method as it can also cause damage to the person.

Improving the light collection efficiency of a photometric objective lens can be achieved by increasing the numerical aperture of the objective lens, but this comes with the opposite effect of decreasing the depth of focus. If the depth of focus becomes shallow, even if the distance between the flow section and the objective lens moves slightly, not only signals from the sample particles but also signals from other objects will be mixed in, making it difficult to make accurate measurements. As described above, the conventional apparatus has the disadvantage that focus adjustment is complicated, and sufficient fluorescence signal intensity cannot be obtained, so that analysis accuracy cannot be improved.

[Object of the Invention] An object of the present invention is to provide a focusing optical system for detecting the focusing state of a photometric objective lens, to easily and accurately perform focus adjustment, and to obtain sufficient fluorescence signal intensity. An object of the present invention is to provide a particle analysis device that can improve measurement accuracy.

[Summary of the invention] The gist of the present invention is to achieve the above objects.

The light beam irradiation surface of the flow cell includes an irradiation optical system that irradiates a light beam onto the sample particles flowing through the flow section in the flow cell, and a photometry optical system that measures the scattered light of the light beam scattered by the sample particles. The photometric optical system is characterized by being provided with focus detection means for projecting a photometric light beam onto the boundary surface and detecting the in-focus state of the photometric objective lens based on the positional relationship of the reflected image obtained by the photoelectric detector. This is a particle analysis device that performs

[Embodiments of the invention] The present invention will be explained in detail based on illustrated embodiments.

FIG. 1 is a configuration diagram of a particle analysis device, in which sample particles S pass through a flow section 2 in the center of a flow cell 1 perpendicular to the plane of the paper, and a laser light source 3 is arranged in a direction perpendicular to this flow. . On the optical axis 0 of the laser light emitted from the laser light source 3, an imaging lens 4 made up of two sets of cylindrical lenses orthogonally arranged on the laser light source 3 side with respect to the sample particles S is arranged. Further, on the optical axis 0 on the opposite side of the laser light source 3 with respect to the sample particles S, a light shielding plate 5, a condensing lens 6, and a photoelectric detector 7 are arranged in this order.

In addition, an autofocus unit (hereinafter referred to as AF unit) 9 including a photometric objective lens 8 and a condenser lens are arranged in a direction substantially perpendicular to the optical axis 0 of the laser beam and the center direction of the flow of the sample particles S. 10. Wavelength selection means 13.14.15 consisting of a diaphragm plate 11, a condensing lens 12, a gicroic mirror, etc. are arranged in sequence, and the wavelength selection means 13.14.15 installed diagonally with respect to the optical path reflect light. A barrier filter 16 is placed on the optical path in the direction in which the
Photoelectric detector 17, barrier filter 18/photoelectric detector 19
, barrier filter 20*photoelectric detector 21 are arranged, respectively. And these photoelectric detectors 17.19.
21 uses, for example, a photomultiplier capable of amplifying and detecting weak light.

Therefore, the laser light emitted from the laser light source 3 is shaped into an imaging beam with arbitrary long and short diameters by the imaging lens 4, which is made up of two sets of cylindrical lenses orthogonal to each other, and the sample particles S flowing through the flow section 2 is irradiated. Sample particle S
Among the scattered light irradiated and scattered, the forward scattered light is the irradiated light that has passed through the position where there is no sample particle S by the light shielding plate 5, and is focused on the photoelectric detector 7 via the condensing lens 6. The properties of the sample particles S are measured.

Further, the specimen particles S dyed with various fluorescent agents are focused as side scattered light onto the aperture plate 11 by the condenser lens 10 via the photometric objective lens 8 in the AF unit 9. By passing the side scattered light and fluorescence through this diaphragm plate 11 installed at a position conjugate to the sample particles S, a photometric signal with less noise can be obtained. The light beam after passing through the diaphragm plate 11 is made into a parallel light beam by a condenser lens 12, separated into side scattered light and fluorescence by a wavelength selection means 13 having appropriate spectral characteristics, and the side scattered light is passed through a barrier filter 16. and detected by the photoelectric detector 17, the sample particles S
Internal granularity can be observed.

On the other hand, the fluorescence passes through the wavelength selection means 13 and is separated into, for example, green fluorescence and red fluorescence by the wavelength selection means 14,
The green fluorescence passes through a barrier filter 18 to a photoelectric detector 19.
The red fluorescence is detected by the photoelectric detector 21 via the wavelength selection means 15 and the barrier filter 20, and the biochemical properties of the sample particles are observed.

Although a green/red dichroic mirror is used as the wavelength selection means 14 and 15 for selecting fluorescence, it is also possible to use wavelength selection means such as a spectroscopic prism or a diffraction grating that can separate wavelengths continuously. Alternatively, a beam diameter variable means such as a beam expander or a beam compressor may be inserted between the light source 3 and the imaging lens 4.

Here, we will discuss the second AF unit 9, which can increase the light collection efficiency of weak light and obtain an accurate in-focus state.
This will be explained with reference to FIGS. FIG. 2 is a configuration diagram of the AF unit 9 seen from the side, and FIG. 3 is a horizontal sectional view of the flow cell 1. A photometric objective lens 8 is installed in the center of the AF unit 9, a light source 22 is installed at the bottom to irradiate the flow cell l, and an aperture 2 is placed on the optical axis of the light source 22.
3. Convex lenses 24 are sequentially arranged. Further, a convex lens 25 is placed on the optical path of the light reflected by the boundary surface consisting of the front surface 2a and the rear surface 2b of the flow section 2 of the flow cell l.
The 1-segment elements 26 are sequentially arranged.

In this case, the objective lens 8 is arranged so that the focus is on the center of the flow section 2 and the condensed light beam from the flow section 2 becomes a parallel light beam by the objective lens 8. In this state, the AF unit A focusing optical system in 9 is arranged to detect the front surface 2a and rear surface 2b of the flow section 2. That is, the light beam passing through the aperture 23 is projected onto the front surface 2a and the rear surface 2b by the light source 22 via the convex lens 24, and the light beam is projected onto the front surface 2a and the rear surface 2b.
Two aperture images of the aperture 23 reflected by the a and rear surfaces 2b are respectively formed on the dividing element 26 via the convex lens 25.

Note that a two-split element is used as the splitting element 26, and the objective lens 8 is in focus in the state shown in FIG.
), the dividing element 26 is arranged so that an image of the same size M is formed on the two dividing planes of the dividing element 26, and the outputs from the two dividing planes are equal to each other. Also, Fig. 4(b)
, (c), in the splitting element 26, if the aperture image M shifts to one side and a difference occurs in the output of the two splitting planes, it can be seen that the objective lens 8 is not in focus.

In this way, when the outputs of the two dividing planes from the dividing element 26 are equal, the objective lens 8 is always focused on the center of the flow section 2, and a parallel light beam of the sample particle image is always obtained from the objective lens 8. Become. In addition, since the aperture plate 11 is arranged at the condensing position of the condenser lens 10, when focusing is achieved by moving the AF unit 9, the flow section 2 becomes in a conjugate relationship with the aperture plate 11, and the sample particles S The scattered light is accurately focused at the position of the aperture plate 11.

Here, since the objective lens 8 and the condensing lens 10 are combined so that a parallel light beam is formed between them, the position of the center axis of the flow cell 1 moves slightly on the optical axis when replacing the flow cell 1, etc. Even if you move the AF unit 9,
Focusing can be achieved simply by making the two outputs of the splitting element 26 equal. In addition, since the reflection at the flow section 2 is detected, even if the size of the flow cell 1, that is, the dimension from the surface to the flow section 2 changes slightly when replacing the flow cell 1, the focus adjustment will not be affected. .

In this way, in the embodiment, it is possible to focus easily and accurately, so the numerical aperture of the objective lens 8 is increased while maintaining accurate focus, the light collection efficiency of the optical system is improved, and the signal strength is increased. This means that it is possible to increase the Note that the wavelength of the focusing light source 22 is preferably separated from the wavelength of the laser light source 3 and the wavelength of fluorescence so that the measurement is not affected by scattered light within the flow cell 1. Preferably, a light source is used.

Further, a mechanism driven by the output signal of the dividing element 26 is provided, and an aperture image M of the aperture 23 is provided on two surfaces of the dividing element 26.
If the AF unit 9 is searched and moved on the optical axis by a drive mechanism until each of Improved operability. In addition, the AF unit 9
If the measurement start signal of the particle analyzer is emitted by the signal when the drive mechanism is stopped or the output signal from the splitting element 26 when in focus, the measurement start signal of the particle analyzer can be emitted when the objective lens 8 is not in focus. In addition, a means for displaying a focusing signal indicating that the aperture image M of the aperture 23 has been formed can be provided at a predetermined position of the dividing element 26, and the AF unit 9 can be manually activated. When operating, measurement may be started at the time this focusing signal is output.

Note that the arrangement of the focus detection optical system in the AF unit 9 is not limited to that in the embodiment; for example, the light source 22 may be arranged above the photometric objective lens 8; Although the case where the AF unit 9 is installed in the photometric optical system for light has been described, in the photometric optical system for forward scattered light, the AF unit is placed between the light shielding plate 5 and the condensing lens 6, and the same method is used. effect can be obtained. Of course, even better results can be obtained by installing such an AF unit in the side/front re-photometry optical system.

[Effects of the Invention] As explained above, the particle analyzer optical system according to the present invention easily and accurately adjusts the focus of the photometric objective lens by installing the focus detection means in the photometric optical system. This makes it possible to improve measurement accuracy and increase the numerical aperture of the objective lens, thereby improving fluorescence photometry intensity and enabling highly accurate analysis.

Furthermore, if desired, by providing a mechanism that drives the focus detection means, it becomes possible to perform fully automatic focus adjustment, and by providing a focus signal display mechanism, it is possible to easily perform focus adjustment manually. It is also possible to provide a mechanism that allows the device to move only when it is in focus, making the measurement even easier.

[Brief explanation of drawings]

The drawings show an embodiment of the particle analysis device according to the present invention, in which Fig. 1 is a configuration diagram of the optical system, Fig. 2 is a layout diagram of the optical system as seen from the side of the AF unit, and Fig. 3 is a cross-section of the flow cell. FIG. 4 is an explanatory diagram of the light image distribution on the split element surface. Reference numeral 1 is a flow cell, 2 is a flow section, 2a is a front surface, 2b is a rear surface, 3 is a laser light source, 4 is an imaging lens, 8 is an objective lens, 9J:l: AF unit, 10.12 is a condensing lens, 11 is an aperture plate, 13.14.15 is a wavelength selection means,
16.18.20 is barrier filter, 17.19.21
22 is a photodetector, 22 is a light source, 23 is an aperture, 24.25 is a convex lens, and 26 is a dividing element.

Claims (1)

[Scope of Claims] 1. An irradiation optical system that irradiates a light beam onto sample particles flowing through a flow section in a flow cell, and a photometric optical system that measures scattered light caused by the light beam being scattered by the sample particles; The photometric optical system includes a focus detection means for projecting a photometric light beam onto the boundary surface of the light beam irradiation surface of the distribution part and detecting the focused state of the photometric objective lens based on the positional relationship of the reflected image obtained by the photoelectric detector. A particle analysis device characterized by being installed inside. 2. The particle analysis device according to claim 1, wherein the light beam irradiated onto the two boundary surfaces of the light beam irradiation surface of the flow section is a parallel light beam. 3. The particle analysis device according to claim 2, wherein the photoelectric detector that detects two reflected lights from the flow section is a split type element. 4. The particle analysis apparatus according to claim 1, wherein the photometric objective lens and the focus detection means are arranged separately at predetermined positions within the same unit. 5. The particle analysis apparatus according to claim 4, wherein the unit is movable along the optical axis of the photometric optical system. 6. The particle analysis device according to claim 5, further comprising a mechanism for automatically searching and moving the unit until it reaches a focused state. 7. The particle analysis device according to claim 1, wherein a condenser lens is provided in the photometric optical system, and an aperture stop is installed at a position conjugate with the sample particle with respect to the objective lens and the condenser lens. 8. The particle analysis device according to claim 1, wherein the focus detection means uses light in a wavelength range outside the wavelength range used for photometry. 9. The particle analysis apparatus according to claim 1, further comprising focus state display means based on a signal from the focus detection means. 10. The particle analysis apparatus according to claim 1, wherein measurement can be performed only when the photometric objective lens is in focus.