JPH0660869B2 - Particle analyzer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a particle analyzer capable of determining a focused state of a photometric objective lens in a flow cytometer or the like.

[Prior Art] In a conventional particle analyzer used for a flow cytometer or the like, for example, 70 μm × 20 μm at the center of the flow cell is used.
The irradiation light is radiated to the specimen such as blood cells that are wrapped in the sheath liquid and pass through the circulation part with a minute rectangular cross section, and the resulting forward and side scattered light causes
It is possible to obtain particle properties such as size and refractive index. Also, for specimens that can be stained with a fluorescent agent,
By detecting the fluorescence of the sample from the side scattered light in a direction substantially perpendicular to the irradiation light, it is possible to obtain important information for analyzing the sample.

In order to perform accurate measurement with a flow cytometer, etc., the sample particles must be accurately focused by the objective lens for photometry so that spurious signals from objects other than the sample particles do not mix. I have to. Therefore, it is necessary to adjust the focus of the photometric objective lens, but in the conventional device, the operator manually performs the focus adjustment while flowing the standard sample before the measurement, and the operation is complicated. In addition, it is difficult to perform sufficiently accurate focus adjustment depending on the operator.

In addition, if the focal point moves during measurement, it cannot be confirmed, so it is not possible to determine whether a pseudo signal has been mixed during the measurement, and there is concern about the reliability of the data. .

Further, there is a drawback that the focus adjustment is required every time the nozzle, the flow cell or the like is exchanged, which makes the measurement troublesome. In addition, when performing fluorescence measurement, it is necessary to strengthen the weak fluorescence signal, but for that reason the photoelectric detector that detects fluorescence should be photomal.-Improve the luminous efficiency of the fluorescent agent-Power of the irradiation light source It has been considered to increase the light-collecting efficiency of the objective lens for photometry. Luminous efficiency of fluorescent agents has been actively researched at present, and the increase in the power of the irradiation light source can be considerably increased if the manufacturing cost is ignored. It will hurt the person and it is hard to say that it is a good method.

The improvement of the light-collecting efficiency of the photometric objective lens can be achieved by increasing the numerical aperture of the objective lens, but at the cost of this, there is an adverse effect that the depth of focus becomes shallow. If the depth of focus becomes shallower, even if the distance between the flow section and the objective lens moves slightly, not only the signal from the sample particle but also the signal from other objects will be mixed, and accurate measurement will be possible. I can't do it. As described above, the conventional apparatus has the drawbacks that the focus adjustment is complicated, sufficient fluorescence signal intensity cannot be obtained, and the analysis accuracy cannot be improved.

[Object of the Invention] An object of the present invention is to provide a focusing optical system for detecting a focused state of a photometric objective lens so that focus adjustment can be performed easily and accurately and sufficient fluorescence signal intensity is obtained. It is to provide a particle analysis device capable of improving measurement accuracy.

[Summary of the Invention] The gist of the present invention for achieving the above object is generated by irradiation optical means for irradiating a sample particle flowing through a flow section in a flow cell with a light beam, and irradiation of the sample particle with the light beam. A flow meter based on a detection output of the detecting means; a projection means for projecting a light flux onto a predetermined wall surface of the flow cell; a detecting means for detecting a reflected light from the wall surface; A particle analysis device, comprising: an adjusting unit that adjusts a focus with the photometric optical unit.

Embodiments of the Invention The present invention will be described in detail based on the illustrated embodiments.

FIG. 1 is a block diagram of a particle analyzer, in which a sample particle S passes through a flow passage 2 perpendicular to the paper surface of the center of a flow cell 1, and a laser light source 3 is arranged in a direction orthogonal to this flow. . On the light source O of the laser light L emitted from this laser light source 3, the laser light source 3
On the side, an imaging lens 4 formed by orthogonally arranging two sets of cylindrical lenses is arranged. Further, a light blocking plate 5, a condenser lens 6, and a photoelectric detector 7 are sequentially arranged on the optical axis O on the side opposite to the laser light source 3 with respect to the sample particle S.

Further, an autofocus unit (hereinafter referred to as an AF unit) 9 including a photometric objective lens 8 and a condenser lens 10 in a direction substantially orthogonal to the optical axis O of the laser light L and the center direction of the flow of the sample particles S, respectively. , The diaphragm plate 11, the condenser lens 12, the dichroic mirrors, and the like, and the wavelength selection means 13, 14, 15 are sequentially arranged, and are reflected by the wavelength selection means 13, 14, 15 obliquely arranged with respect to the optical path. The barrier filter 16 / photoelectric detector 17, the barrier filter 18 / photoelectric detector 19, the barrier filter 20 / photoelectric detector 21 are arranged on the optical path in the different directions. And these photoelectric detectors 17, 19, 21
For example, a photomul that can detect weak light by enhancing it is used.

Therefore, the laser light L emitted from the laser light source 3
Is formed into an image-forming beam having an arbitrary long diameter / short diameter by the image-forming lens 4 in which two sets of cylindrical lenses are orthogonalized,
The sample particles S flowing in the circulation unit 2 are irradiated. Of the scattered light that is irradiated and scattered on the sample particles S, the light that has passed through the position where the sample particles S are absent is removed from the forward scattered light by the light shielding plate 5, and is collected by the photoelectric detector 7 via the condenser lens 6. The light is emitted and the property of the sample particle S is measured.

The sample particles S dyed with various fluorescent agents are condensed as side scattered light on the diaphragm plate 11 by the condenser lens 10 via the photometric objective lens 8 in the AF unit 9. The side scattered light and the fluorescence can be passed through the diaphragm plate 11 installed at a position conjugate with the sample particle S to obtain a photometric signal with less noise. The light flux after passing through the diaphragm plate 11 is made into a parallel light flux by the condenser lens 12, and is split into side scattered light and fluorescence by the wavelength selection means 13 having appropriate spectral characteristics, and the side scattered light is barrier filter 16. And the sample particles S detected by the photoelectric detector 17.
Granularity inside can be observed. On the other hand, the fluorescence passes through the wavelength selection means 13, is separated into, for example, green fluorescence and red fluorescence by the wavelength selection means 14, the green fluorescence is detected by the photoelectric detector 19 through the barrier filter 18, and the red fluorescence is wavelength selected. The biochemical properties of the analyte particles are observed as detected by the photoelectric detector 21 via the means 15 and the barrier filter 20.

As the wavelength selection means 14 and 15 for selecting fluorescence, dichroic mirrors of two colors of green and red are used. For example, a wavelength selection means such as a spectral prism or a diffraction grating capable of continuously separating wavelengths is used. Good. Further, a beam diameter varying means such as a beam expander or a beam compressor may be inserted between the light source 3 and the imaging lens 4.

Here, regarding the AF unit 9 capable of increasing the collection efficiency of weak light and accurately obtaining the focused state,
This will be described with reference to FIGS. 2 is a side view of the AF unit 9, and FIG. 3 is a horizontal sectional view of the flow cell 1. An objective lens 8 for photometry is installed in the center of the AF unit 9, and a light source 22 is installed below so as to irradiate the flow cell 1 with light.
3 and the convex lens 24 are sequentially arranged. In addition, the convex lens 2 is provided on the optical path of the light reflected by the boundary surface formed by the front surface 2a and the rear surface 2b of the flow section 2 of the flow cell 1.
5, the dividing element 26 is sequentially arranged.

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

A two-division element is used as the division element 26, and since the objective lens 8 is in focus in the state of FIG. 2, two aperture images of the aperture 23 at this time are shown in FIG. 4 (a).
As shown in FIG.
The splitting element 26 is arranged so that the images are formed in the same manner and the outputs from the two split surfaces are equal to each other. In addition, Fig. 4 (b), (c)
As shown in FIG. 5, in the splitting element 26, when the aperture image M shifts to the side and a difference occurs in the output of the two splitting surfaces, it is understood that the objective lens 8 is out of focus.

Thus, when the outputs of the two split surfaces from the splitting element 26 are equal, the objective lens 8 is always focused on the center of the flow section 2, and the parallel light flux of the sample particle image is always obtained from the objective lens 8. Become. Further, since the diaphragm plate 11 is arranged at the condensing position of the condenser lens 10, when the AF unit 9 moves to focus, the flow section 2 has a conjugate relationship with the diaphragm plate 11 and scattering by the sample particles S. The light is accurately focused on the position of the diaphragm plate 11.

Here, since the objective lens 8 and the condenser lens 10 are combined so as to form a parallel light flux between them, the position of the central axis of the flow cell 1 slightly moves on the optical axis when the flow cell 1 or the like is replaced. Even so, by moving the AF unit 9,
Focusing can be achieved by simply equalizing the two outputs of the splitting element 26. Further, since the reflection in the flow section 2 is detected, even if the size of the flow cell 1, that is, the size from the surface to the flow section 2 is slightly different when the flow cell 1 is replaced, the focus adjustment is not affected.

As described above, in the embodiment, focusing can be performed easily and accurately. Therefore, the numerical aperture of the objective lens 8 is increased while the accurate focus is maintained, the condensing efficiency of the optical system is improved, and the signal strength is increased. Can be increased. The wavelength of the focusing light source 22 is preferably separated from the wavelength of the laser light source 3 or the wavelength of fluorescence so as not to affect the measurement due to scattered light in the flow cell 1 or the like. It is preferred to use a light source.

Further, a mechanism driven by the output signal of the splitting element 26 is provided, and the aperture image M of the aperture 23 is formed on the two surfaces of the splitting element 26.
If the AF unit 9 is searched and moved on the optical axis by the driving mechanism until the respective images are formed, and the driving mechanism is stopped by the focused signal, the in-focus state is automatically obtained, and further operation is performed. It improves the sex. Further, if the measurement start signal of the particle analyzer is issued by the signal of the state where the drive mechanism of the AF unit 9 is stopped or the output signal of the splitting element 26 at the time of focusing, the objective lens 8 is in focus. If not, inaccurate measurements can be avoided. Further, it is possible to provide a means for displaying a focusing signal indicating that the aperture image M of the aperture 23 has been formed at a predetermined position of the splitting element 26. When the AF unit 9 is manually operated, the focusing is performed. It suffices to start the measurement when the signal is output.

The arrangement of the focus detection optical system in the AF unit 9 is not limited to that of the embodiment, and the light source 22 may be arranged above the measurement objective lens 8, for example. Further, in the embodiment, the case where the AF unit 9 is installed in the photometric optical system for side scattered light has been described, but also in the photometric optical system for forward scattered light, the space between the light blocking plate 5 and the condenser lens 6 is described. The AF unit can be arranged in the same position to obtain the same effect. AF like this
It goes without saying that even better results can be obtained by installing the units in both the side and front photometric optical systems.

[Advantages of the Invention] As described above, the particle analysis apparatus according to the present invention includes a projection unit that projects a light beam onto a predetermined wall surface of a flow cell, a detection unit that detects reflected light from this wall surface, and a detection unit of this detection unit. By providing the adjusting means for adjusting the focus between the flow cell and the photometric optical means based on the output, the focus adjustment between the flow cell and the photometric optical means can be performed easily and accurately, which improves the measurement accuracy and improves the accuracy. Enables particle analysis.

[Brief description of drawings]

The drawings show one embodiment of the particle analysis apparatus according to the present invention. Fig. 1 is a configuration diagram of an optical system, Fig. 2 is an optical system layout diagram when the AF unit is viewed from the side, and Fig. 3 is a cross section of a flow cell. FIG. 4 and FIG. 4 are explanatory views of the light image distribution on the surface of the dividing element. 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, 9 is an AF unit, 10 and 12 are condenser lenses, 1
1 is a diaphragm plate, 13, 14 and 15 are wavelength selection means, 16,
Reference numerals 18 and 20 are barrier filters, 17, 19 and 21 are photoelectric detectors, 22 is a light source, 23 is an aperture, 24 and 25 are convex lenses, and 26 is a dividing element.

Claims (1)

[Claims] 1. A flow cell comprising: an irradiation optical means for irradiating a sample particle flowing through a flow section in the flow cell with a light beam; and a photometric optical means for measuring light generated by irradiation of the sample particle with the light beam. Projecting means for projecting a light beam on a predetermined wall surface, detecting means for detecting reflected light from the wall surface, and adjusting means for adjusting the focus of the flow cell and the photometric optical means based on the detection output of the detecting means. A particle analysis device having.