JPH0682173B2 - Transmission microscope imager - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a transmission microscope image pickup device, and in particular, a sample is two-dimensionally scanned with a light beam in the form of a minute spot and the transmitted light from the sample is projected onto an image sensor. The present invention relates to a transmission microscope image pickup device that forms a sample image by performing the above process.

(Prior Art) A transmission microscope image pickup device useful for biological observation has been widely put into practical use. The applicant of the present application has proposed in Japanese Patent Application No. 59-242419 a transmission microscope image pickup apparatus capable of obtaining optical information from a sample as an electric signal by using a laser light source and a linear image sensor. The transmission microscope image pickup device of the applicant of the present invention deflects a light beam projected from a laser light source in the main scanning direction and the sub-scanning direction by using two deflectors, and condenses the two-dimensionally deflected light beam into a condenser lens. The light beam that has passed through the sample is condensed by the objective lens, and a plurality of light receiving elements are minutely arrayed in the main scanning direction on the linear image sensor. The image is formed by projecting as a spot and receiving the transmitted light from the sample line by line. Therefore, this transmission microscope imaging device
Since the charge storage capacity of the linear image sensor is used, image distortion does not occur even if uneven scanning of the light beam occurs,
It has a great advantage that a high S / N ratio and a high resolution image signal can be obtained.

(Problems to be Solved by the Invention) In the transmission microscope image pickup apparatus described above, the sample is two-dimensionally scanned with the illumination light focused into a minute spot by the condenser lens, and the transmitted light from the sample is condensed by the objective lens. Therefore, the scanning point of the illumination light incident on the sample and the condensing point of the objective lens must be exactly matched. The relative displacement between the scanning point of the illumination light and the condensing point of the objective lens includes a displacement in the main scanning direction and a displacement in the sub-scanning direction orthogonal to this, but there is a displacement in the sub-scanning direction. When it occurs, the amount of light incident on the linear image sensor is significantly reduced, the amplitude of the image signal is reduced, the S / N ratio is degraded, and the resolution is also significantly reduced. In particular, when the objective lens or condenser lens is replaced, the lens magnification often differs slightly. In such cases, the magnification of the illumination optical system and the magnification of the observation optical system do not match each other, and as a result At the scanning point of the spot-shaped illumination light and the focal point of the objective lens, a relative position shift occurs in the sub-scanning direction, and the illumination light transmitted through the sample is not received by the objective lens, and the incident light amount on the linear image sensor is significantly As the image quality decreases, so does the resolution.

Therefore, the object of the present invention is to eliminate the above-mentioned drawbacks and to eliminate the relative positional deviation between the scanning point of the illumination light and the focal point of the objective lens due to the mismatch of the magnifications of the illumination optical system and the observation optical system. , The transmitted light from the sample can be accurately collected by the objective lens,
(EN) A transmission microscope image pickup device having a large amplitude and a high S / N ratio and capable of obtaining a high resolution image signal.

(Means for Solving the Problems) A transmission microscope image pickup device according to the present invention includes a light source that emits a light beam, and a device that deflects the light beam emitted from the light source in the main scanning direction and the sub-scanning direction orthogonal thereto. A condenser lens that projects the deflected light beam toward the sample, an objective lens that collects the transmitted light from the sample, and a plurality of elements arranged in the main scanning direction to receive the light beam from the objective lens. And a linear image sensor for outputting a photoelectric output signal, means for detecting a relative positional deviation in the sub-scanning direction between the scanning point of the illumination light incident on the sample from the condenser lens and the focal point of the objective lens, and detection And a means for correcting the magnification of the illumination optical system and / or the observation optical system on the basis of the amount of positional deviation.

(Operation) In the present invention, the deviation in the sub-scanning direction between the spot of the laser beam projected on the sample by the illumination optical system and the object point on the sample projected on the linear image sensor by the observation optical system. Since the detection optical system and the observation optical system are configured to correct the magnifications of the illumination optical system and / or the observation optical system according to the detected deviation so that the mutual magnifications match, the scanning point of the illumination light on the sample and the focusing of the objective lens It is possible to always match the points, and it is possible to accurately form an image of a bright and proven object point on the sample on the linear image sensor, increase the S / N of the photoelectric output signal, and improve the resolution. In addition, the operator does not have to worry about the adjustment of the magnification, and there is an advantage that the operator can concentrate on the speculum.

(Embodiment) FIG. 1 is a three-dimensional diagram showing the configuration of an embodiment of a microscope image pickup apparatus according to the present invention, in which many optical paths are relative to a horizontal plane.
It extends at a 45 ° angle or perpendicular to the horizontal. In this example, a transmission color microscope image pickup device will be described as an example. An Ar laser 1 that emits blue and green light beams and a He-Ne laser 2 that emits red light beams are used as light sources that emit light beams of three primary colors of blue, green, and red. The light beam emitted from the Ar laser 1 is inclined downward at an angle of 45 ° with respect to the horizontal plane, is incident on the first dichroic mirror 3, and becomes a blue component light with a wavelength of 488 nm and a green component light with a wavelength of 514.5 nm. To be separated. The blue light beam that has passed through the dichroic mirror 3 is expanded and collimated by the first expander 4, reflected by the right-angle prism 5 in a direction orthogonal to the horizontal plane, and further reflected by the right-angle prism 6 at an angle of 45 ° with respect to the horizontal plane. And is incident on the first acousto-optic element 7.
This first acousto-optic device 7 transmits a blue light beam to the X-axis of the sample surface.
It vibrates at high speed in the direction (direction orthogonal to the paper surface). The light beam deflected by the first acousto-optic device 7 is incident on the first non-parallel plane plate 8 which is the optical path correcting means. This non-parallel flat plate 8 is connected to a driving device (not shown), and is shown in FIG.
As shown in FIG. 5, the blue light beam is deflected in the Y direction orthogonal to the X direction by rotating about the optical axis in accordance with the deviation amount of the blue light beam from the regular optical path in the Y direction orthogonal to the X direction. Make a correction. As a result, even if the blue light beam deviates from the regular optical path in the Y direction due to variations in the laser emission angle and the like, the blue optical beam travels along the regular optical path by the optical path correction. The optical path-corrected blue light beam is reflected by the half mirror 9, transmitted through the half mirror 10, travels downward through an angle of 45 ° with respect to the horizontal plane via the relay lens 11, and is arranged in the horizontal mirror. Incident on. The vibrating mirror 12 is connected to a driving device (not shown) and rotates at a predetermined frequency to deflect the incident light beam in the Y direction orthogonal to the X direction of the sample.
The vibrating mirror 12 has a total reflection surface formed on both the front surface and the back surface thereof, and a light beam directed to the sample enters the front surface side and a light beam emitted from the sample enters the back surface side. The blue light beam reflected by the vibrating mirror 12 travels upward by an angle of 45 ° with respect to the horizontal plane, passes through a left / right reversing prism 13, and enters a relay lens 14 which functions as a magnification correcting means. The relay lens 14 is connected to driving means (not shown), and moves in the direction of arrow a or b along the optical axis according to the difference in magnification between an objective lens and a condenser lens, which will be described later, to perform magnification correction. The blue light beam that has passed through the relay lens 14 is reflected by the right-angle prism 15 in the vertical direction, is focused by the condenser lens 16 into a fine spot, and is incident on the sample 17. Therefore, the sample 17 is scanned in the X direction and the Y direction at a predetermined scanning frequency by the blue light beam focused into a minute spot, and the transmitted light from the sample 17 is condensed by the objective lens 18. The condenser lens 16 and the objective lens 18 are configured by lenses having the same magnification, and the scanning point of the light beam from the condenser lens 16 toward the sample 17 and the condensing point of the transmitted light from the sample 17 by the objective lens 18 coincide with each other. In this way, the condenser lens 16 and the objective lens 18 are arranged at positions conjugate with each other with the sample 17 interposed therebetween. The light beam condensed by the objective lens 18 is reflected upward by an angle of 45 ° with respect to the horizontal plane by the right-angled prism 19, passes through the relay lens 20, and is incident on the rear surface side of the vibrating mirror 12. The blue light beam reflected by the back surface of the vibrating mirror 12 enters the second dichroic mirror 22 via the relay lens 21. The second dichroic mirror 22 reflects only blue light and transmits light in other wavelength ranges. Therefore, the blue beam is reflected by the second dichroic mirror 22, passes through the imaging lens 23, and enters the plane-parallel plate 24. The plane-parallel plate 24 is rotated in the a or b direction to correct the optical path as shown in FIG. 3B. Further, the light beam is incident on the half mirror 25, and the reflected light is the displacement amount detector 26.
And the transmitted light is incident on the first linear image sensor 27. The first linear image sensor 27 is arranged at the image forming position of the image forming lens 23, and each light receiving element is arranged to receive the blue light beam from the sample 17 line by line in the main scanning direction (X direction). Are arranged one-dimensionally in a direction corresponding to, and the transmitted light from the sample 17 is sequentially received by each element to perform photoelectric conversion, and the charges accumulated in each element are sequentially read at a predetermined read frequency. Since the linear image sensor has a charge storage capability, each pixel of the sample 17 and each light receiving element of the linear image sensor 27 always have a 1: 1 relationship, and the scanning speed of the first acousto-optic element 7 is uneven. Even if the occurrence occurs, the amount of light received changes only slightly, and image distortion does not occur.

FIG. 4 is a diagram showing the configuration of the displacement amount detector 26. sample
Two photodetectors with the same shape in the direction corresponding to the Y direction of 17
50 and 51 are arranged so that the blue light beam from the sample 17 is incident on these first and second photodetectors 50 and 51. Then, the boundary line 1 between the first photodetector 50 and the second photodetector 51 is aligned with the center of the light receiving element of the linear image sensor 27. According to this structure, when the incident light on the linear image sensor 27 deviates in the Y direction, the two photodetectors of the displacement amount detector are detected.
Since the lights incident on 50 and 51 are displaced in the Y direction at the same time,
If the photoelectric output signals of the first photodetector 50 and the second photodetector 51 are supplied to the differential amplifier 52 to form a difference signal, the magnitude of this difference signal represents the deviation amount of the light beam, The direction polarity represents the shift direction. Therefore, this difference signal is transmitted to the first non-parallel plane plate 8 and / or the first non-parallel plane plate which is the optical path correcting means.
If the non-parallel flat plate 8 and / or the parallel flat plate 24 are driven according to the amount of displacement of the incident light with respect to the light receiving element of the first linear image sensor 27 in the Y direction, The optical path is automatically corrected for the direction,
The transmitted light from the sample can be accurately incident on each light receiving element of the linear image sensor 27.

Next, scanning of the green light beam will be described. The green light beam reflected by the first dichroic mirror 3 travels downward at an angle of 45 ° with respect to the horizontal plane and forms a right angle prism.
The light beam is reflected by the expander 29 to be expanded parallel light flux, is reflected by the right-angle prism 30 in the vertical direction, and is reflected by the right-angle prism 31 in the downward direction at an angle of 45 ° with respect to the horizontal plane.
Incident on. Then, the second acousto-optic element 32 vibrates at the same frequency as the first acousto-optic element 7 at a high speed,
The optical path is corrected by the non-parallel flat plate 33 and reflected by the half mirror 10 to enter the common optical path. Next, the light enters the vibrating mirror 12 through the relay lens 11 and is deflected in the Y direction. After that, the light travels in a common optical path, is focused by a condenser lens 16 into a fine spot, and is incident on a sample 17. Therefore, sample 17
The area scanned by the blue light beam will be scanned by the green light beam at the same scanning frequency. The green light beam that has passed through the sample 17 further travels in the common optical path, is reflected by the back surface of the vibrating mirror 17, passes through the second dichroic mirror 22, and enters the third dichroic mirror 34. The dichroic haller 34 reflects only the green light beam and transmits light in other wavelength ranges. Therefore, the green light beam is emitted from the third dichroic mirror 34.
Is reflected by the second linear image sensor 38 through the increasing lens 35 and the second parallel flat plate 36, and is then incident on the half mirror 37. Image signal is generated, the reflected light is incident on the second displacement amount detector 39, and the displacement amount of the incident light with respect to the second linear image sensor 38 in the Y direction is detected. These second
The configuration and operation of the linear image sensor 38 and the second displacement amount detector 39 of FIG.
Since it is the same as the displacement amount detector 26, detailed description thereof will be omitted.

Next, scanning of the red light beam will be described. The He-Ne laser 2 that emits the red light beam is arranged below the Ar laser 1 so as not to intersect the light beams emitted from the Ar laser 1. The red light beam emitted from the He-Ne laser 2 travels downward at an angle of 45 ° with respect to the horizontal plane, is converted into an expanded parallel light flux by the third expander 40, and is blue by the third acousto-optic element 41. And vibrates at the same frequency as the green light beam at a high speed, the optical path is corrected by the third non-parallel plane plate 42, enters the vibrating mirror 12 through the half mirrors 9 and 10 and the relay lens 11, and is deflected in the Y direction. To be done. In this way, if the vibrating mirror is shared for the three light beams of blue, green and red, the occurrence of a registration error in the Y direction can be effectively prevented. The red light beam reflected by the vibrating mirror 12 travels in a common optical path, is focused by the condenser lens 16 into a minute spot, and is incident on the sample 17. As a result, the same area of the sample 17 is scanned by the three light beams of blue, green and red at the same scanning frequency. The red light beam transmitted through the sample 17 further travels in the common optical path, is reflected on the back side of the vibrating mirror 12, and is reflected by the second and third diloic Millaus 22 and 34.
Through the increasing lens 43 and the third parallel plane plate 44, is reflected by the right-angle prism 45 and is incident on the half mirror 46, and the reflected light is incident on the third displacement amount detector 47 and is emitted from the regular optical path. The amount of displacement is detected, and the transmitted light is incident on the third linear image sensor 48 to create an image signal. Like this 3
If the light path is corrected by detecting the amount of displacement of each light beam from the regular light path for each light beam of a book, for example, each light beam from the sample will change even if the radiation angle of any laser light source changes. Can be automatically and accurately incident on each linear image sensor. Particularly in this example, by adjusting the first to third non-parallel plane plates 8, 32, 41 provided in the optical path on the illumination side, three light beams of blue, green and red are made to be one point on the sample 17. The image of the object point illuminated on the sample can be focused by adjusting the first to third parallel plane plates 24, 36 and 44 provided in the optical path on the observation side. , 38 and 48 can be accurately projected. In this way, it is possible to obtain a color image signal having a large amplitude, a high S / N, a high resolution, and no color shift.

FIG. 4A mainly shows a state in which the condenser lens 16 and the objective lens 17 have different magnifications and a positional deviation occurs between the scanning point of the light beam by the condenser lens 16 and the converging point of the objective lens 18. It is a schematic diagram cut by a plane orthogonal to the scanning direction,
FIG. 4B is a schematic diagram showing a state in which the scanning point and the converging point coincide with each other due to the magnification correction. As shown in FIG. 4A, the scanning point S of the light beam by the condenser lens 16 and the objective lens 18
When the focal point C of the sample 17 is displaced in the Y-axis direction of the sample 17, the illumination light transmitted through the sample 17 is not received by the objective lens 18, and as shown in FIG. The amount of light incident on the image sensor is reduced and the illumination efficiency is significantly reduced. Most of the causes of the positional deviation between the scanning point of the spot-shaped illumination light incident on the sample 17 and the condensing point of the objective lens 18 are due to the difference in the magnification of the illumination optical with respect to the sample 17 and the magnification of the observation optical system. To do. Therefore, in this example, the relay lens 14 arranged in the common optical path of the illumination optical system is used as a magnification correction means, and the relay lens 14 is moved in the direction of arrow a or b along the optical axis direction to increase the magnification of the illumination optical system. The observation light emitted from the sample 17 in accordance with the magnification of the observation optics is accurately incident on each linear image sensor 27, 38, 48.

Next, a drive control method of the relay lens 14 will be described. The positional shift amount between the scanning point and the condensing point is obtained from the amount of light incident on any of the linear image sensors. On the other hand, as shown in FIG. 5, the direction in which the relay lens 14 should be driven cannot be determined from the incident light amount of the linear image sensor. For this reason, in this example, the reference position of the relay lens 14 is determined, and the relay lens 14 is always driven in one direction to obtain a point at which the amount of incident light is maximized and adjustment is performed. When the adjustment is performed with the sample attached, the observation light modulated by the sample 17 is incident on the linear image sensor. Therefore, it is desirable to perform the adjustment without the sample 17 attached. As described above, if the relay lens is driven in the optical axis direction for adjustment based on the amount of light incident on the image sensor, it is possible to perform magnification correction with high accuracy with a simple configuration without the need for providing additional adjusting means.

The present invention is not limited to the above-described embodiments, but various modifications can be made. For example, in the above-described embodiment, the configuration is such that the relay lens of the illumination optical system is driven in the optical axis direction to correct the magnification, but the relay lens arranged in the observation optical system is driven, or the illumination optical system and the observation optical system are used. Both relay lenses may be driven.

Further, the magnification correction means is not limited to the relay lens, may drive an optical element other than the relay lens,
Further, a magnification correction means may be separately provided.

Further, in the above-mentioned embodiment, the linear image sensor for forming the image signal is used as the means for detecting the positional deviation between the scanning point of the illumination light and the condensing point of the objective lens. The photodetector may be provided as the positional deviation detecting means.

〔The invention's effect〕

As described above, according to the present invention, the illumination optical system and / or
Alternatively, since a means for correcting the magnification of the observation optical system is provided, even if the magnification of the illumination optical system and the magnification of the observation optical system do not match, the scanning point of the illumination light and the converging point of the objective lens can be accurately matched. Therefore, the transmitted light from the sample can be accurately incident on the image sensor.

As a result, it is possible to obtain an image signal having a large amplitude and a high S / N ratio, even when the lens is replaced.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of an embodiment of a transmission microscope imaging apparatus according to the present invention, FIGS. 2A and 2B are diagrams showing a configuration of an optical path correcting means, and FIG. 3 is a configuration of a displacement amount detector. 4A and 4B are diagrams showing the relationship between the scanning point of the illumination light and the focal point of the objective lens, and FIG. 5 is a graph showing the relationship between the shift amount and the incident light amount to the image sensor. Is. 1 …… Ar laser, 2 …… He-Ne laser 3,22,34 …… Dichroic mirror 4,29,40 …… Expander 5,6,15,19,30,31,45 …… Right angle prism 7, 32,41 …… Acousto-optic element 8,33,42 …… Non-parallel flat plate 9,10,25,37,46 …… Half mirror 11,14,20,21 …… Relay lens 12 …… Vibration mirror, 13 ...... Left / right reversing prism 16 …… Condenser lens, 17 …… Sample 18 …… Objective lens

Claims (2)

[Claims] 1. A light source for emitting a light beam, a means for deflecting the light beam emitted from the light source in a main scanning direction and a sub scanning direction orthogonal thereto, and a condenser for projecting the deflected light beam toward a sample. A lens, an objective lens that collects transmitted light from a sample, a plurality of elements arranged in the main scanning direction, a linear image sensor that receives a light beam from the objective lens and outputs a photoelectric output signal, and a condenser lens Means for detecting the relative positional deviation in the sub-scanning direction between the scanning point of the illumination light incident on the sample and the condensing point of the objective lens, and the illumination optical system and / or the observation optics based on the detected positional deviation amount. And a means for correcting the magnification of the transmission microscope image pickup device. 2. The structure according to claim 1, wherein a relay lens arranged in the illumination optical system and / or the observation optical system is configured to be driven in the optical axis direction to correct the magnification. Transmission microscope imaging device.