JP2007041510A - Observation apparatus and observation system provided with the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope apparatus provided with high expandability, which has high NA though it has a wide observation range and can acquire a feeble light emitting signal at a high S/N ratio, and a microscope system provided with the same. <P>SOLUTION: The microscope apparatus has an infinity-corrected objective lens 2 and an imaging lens 3, and satisfies a conditional expression, 4.56≤D*NA'≤10, where D is the parfocal distance of the objective lens 2 and NA' is the numerical aperture of the imaging lens 3. It is desirable to make a distance from the mount position 5 of the objective lens 2 to the most object-side surface of the imaging lens 3 variable and to satisfy a conditional expression, 0.5FL<W<1.2FL, where W is the variable amount of the distance, and FL is the focal length of the imaging lens 3. It is more desirable to satisfy conditional expressions, 0.4<D/FL<1.2 and 1<D/Φd<2.75, where Φd is the outside diameter of a connection at the mount of the objective lens 2. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an observation apparatus capable of observing the inside of a living cell and the like, and an observation system including the observation apparatus.

  In the state-of-the-art research field, various methods for observing cells for a long time (several days to several weeks) have been developed for the purpose of elucidating the functions of living organisms, analyzing the behavior of proteins, and elucidating interactions. It is coming. As an observation method using an observation apparatus such as a microscope for observing a lesion in a living cell, a fluorescence observation method is often used. In fluorescence observation, a fluorescent substance such as a specific fluorescent protein is used as a luminescent label, and a biological sample such as a biological cell is dyed and then irradiated with excitation light to emit fluorescence. The presence / absence and position of a specific part in the biological sample is detected.

Conventional observation apparatuses for observing living cells and the like including fluorescence observation and optical systems used therefor include, for example, those described in Patent Documents 1 to 4 below.
Japanese Patent Laid-Open No. 5-113540 Japanese Patent Laid-Open No. 10-31162 JP-A-11-231224 JP 2001-21812 A

By the way, in fluorescence observation, when a stimulus is applied to a biological sample, such as irradiation with excitation light, the stimulus itself may adversely affect the active state of the cell. For this reason, there is a need for an observation system that can stimulate a luminescent label with as low a stimulus as possible (exciting light having a low intensity) and detect a weak luminescent signal that accompanies it with extremely high efficiency.
At the same time, it prevents you from losing sight of how the live cells move, and at the same time observes a wide area and detects information from many cells at once to improve processing speed and work efficiency. There is also a need for an observation system that can do this.
Further, there is a demand for an observation system that can be used in combination with other observation methods and has a flexible expandability and compatibility with conventional observation systems.
However, in the field of conventional observation devices as described in Patent Documents 1 to 4, there has been no observation device and observation system that satisfy these requirements.

  The present invention has been made in view of the above-described conventional problems, and has a high extensibility capable of acquiring a weak emission signal at a high S / N with a high NA while having a wide observation range. An object is to provide an observation apparatus and an observation system including the observation apparatus.

In order to achieve the above object, an observation apparatus according to the present invention is an observation apparatus having an infinity-corrected objective lens and an imaging lens, wherein the focal length of the objective lens is D and the numerical aperture of the imaging lens is When NA ′, the following conditional expression (1) is satisfied.
4.56 ≦ D · NA ′ ≦ 10 (1)

In the observation apparatus of the present invention, the distance from the barrel position of the objective lens to the most object side surface of the imaging lens is variable, and the variable amount is W, and the focal length of the imaging lens. Is preferably FL, the following conditional expression (2) is preferably satisfied.
0.5FL <W <1.2FL (2)

In the observation apparatus of the present invention, it is preferable that the following conditional expressions (3) and (4) are satisfied.
0.4 <D / FL <1.2 (3)
1 <D / Φd <2.75 (4)
However, D is the same focal length of the objective lens, FL is the focal length of the imaging lens, and Φd is the outer diameter of the joint portion on the body of the objective lens.

  In the observation apparatus of the present invention, it is preferable that the rear focal position of the objective lens is located on the object side with respect to the barrel position of the objective lens.

  In the observation apparatus of the present invention, at least two objective lenses can be exchanged, and the difference in distance from the body position to the rear focal position in all the exchangeable objective lenses is ± 15 mm. It is preferable that each interchangeable objective lens is configured so that it is within the range.

  In the observation apparatus of the present invention, at least two objective lenses can be exchanged with different confocal distances, and the objective lenses can be exchanged when they are exchanged with any objective lens having different confocal distances. And a barrel position adjusting means for changing the barrel position of the objective lens corresponding to the same focal length of the replaceable objective lenses so that the front focal positions of the objective lenses mounted in the same manner are substantially matched. Is preferred.

  In the observation apparatus of the present invention, it is preferable that at least two imaging lenses are provided.

  In the observation apparatus of the present invention, it is preferable that a fluorescence observation illumination optical system is provided between the objective lens and the imaging lens.

  In the observation apparatus of the present invention, it is preferable that the objective lens is an immersion objective lens or a solid immersion objective lens.

  An observation system according to the present invention includes any one of the above-described observation apparatuses according to the present invention and an imaging unit that captures an object image from the observation apparatus.

The observation system according to the present invention is an infinity-correction type in which the observation device according to the present invention is the first observation device and the objective lens used in the first observation device has a different focal length and emission NA. An observation device having a second objective lens and a second imaging lens having the same focal length as the imaging lens used in the first observation device is used as a second observation device, and the image formation in the first observation device. The numerical aperture of the lens is NA ′, the same focal length of the objective lens is D, the distance on the optical axis from the object plane to the rear focal position of the objective lens is fb, and the second observation apparatus uses the second observation device. When the numerical aperture of the imaging lens is NA2 ′, the same focal length of the second objective lens is D2, and the distance on the optical axis from the object plane to the rear focal position of the second objective lens is fb2, The objective lens in the first observation device; and The imaging lens satisfies the following conditional expressions (5) to (7) with respect to the second objective lens and the second imaging lens in the second observation apparatus: .
0.3 ≦ NA2 ′ / NA ′ ≦ 0.88 (5)
0.5 ≦ D2 / D ≦ 0.87 (6)
−15 <fb2−fb <15 (7)

  Further, in the observation apparatus of the present invention, at least one of bright field observation, fluorescence observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation is provided on the side opposite to the objective lens with the object interposed therebetween. It is preferable to provide a transmission illumination optical system configured to be able to observe the above.

  In the observation apparatus of the present invention, the transmission illumination optical system includes a light source and a condenser lens, and bright field observation, fluorescence observation, phase difference observation, differential interference observation, and a substantially pupil position of the condenser lens, Preferably, at least one of various optical elements capable of observing Hoffman modulation contrast is switchably provided.

  An observation system according to the present invention includes any one of the observation apparatuses according to the present invention including the transmission illumination optical system, an imaging unit that captures an object image from the observation apparatus, and an object obtained via the imaging unit. The image processing means for superimposing the transmitted illumination observation image and the light emission observation image from the object is provided.

  In the observation system of the present invention, it is preferable that the observation system further includes display means for displaying the range of the image region superimposed through the image processing means together with the superimposed image on a display device.

  According to the observation apparatus of the present invention and the observation system equipped with the observation apparatus, it has a high extensibility capable of acquiring a weak emission signal at a high S / N with a high NA while having a wide observation range. An observation apparatus and an observation system including the observation apparatus are obtained.

Japanese Patent Application No. 2005-86644 filed by the present applicant for the purpose of enabling the detection of faint luminescence emitted from a specific site such as a living cell, as an invention that has led to the observation device and the observation system of the present invention. There is a weak light imaging optical system described in the specification and a microscope apparatus including the same.
In the process of conceiving the invention of Japanese Patent Application No. 2005-86644, the applicant of the present invention has introduced a living body into which a luciferase gene has been introduced while changing the conditions of an imaging lens that constitutes an imaging optical system used in a conventional general fluorescence microscope. A test was conducted on whether or not the observation of luminescence of cells was possible. As described above, the imaging optical system of the conventional observation apparatus cannot detect weak light emission from a specific part such as a living cell.
In general, an imaging optical system of an observation apparatus includes an objective lens, an imaging lens, and imaging means.
In the conventional observation apparatus, the numerical aperture NA of the objective lens is limited to about 1.4 at the maximum. Even in the case of an immersion objective lens in which oil is filled between the specimen and the objective lens and the numerical aperture NA of the objective lens can be increased by the refractive index of the oil, the refractive index of the oil is limited to about 2. It is to be done.

  In order to capture a bright image of cells that emit light, it is necessary to increase the numerical aperture on the exit side (that is, the numerical aperture NA ′ of the imaging lens). However, the numerical aperture NA ′ of the imaging lens used in the imaging optical system of the conventional observation apparatus is about 0.05, and is about 0.07 at the maximum. Further, the focal length D of a general objective lens is about 40 to 65 mm.

  Therefore, the applicant of the present invention uses, as an imaging lens, a plurality of types of objective lenses having a numerical aperture larger than 0.05 for an objective lens having a numerical aperture NA of 1.4 and the same focal length D = 45 mm. It was investigated whether the numerical aperture NA ′ of the imaging lens should be large enough to observe the luminescent image. The number of pixels of the image sensor was 765 × 510 pixels and the pixel size was 9 × 9 μm.

  As a result of the above test, in order to observe the luminescent image, when the numerical aperture NA of the objective lens is 1.4 and the focal length is 45 mm, the combined imaging lens requires a numerical aperture NA ′ of at least 0.15 or more. It turns out that. When the numerical aperture NA 'of the imaging lens is 0.15, the magnification from the object to the image plane is about 10 times (1.4 / 0.15).

  In the invention of Japanese Patent Application No. 2005-86644, it is assumed that the numerical aperture NA ′ of the imaging lens is required to be at least 0.15 for the purpose of capturing as much as possible the weak light emission from a specific part of the cell. Based on the above test results, the configuration of the imaging optical system necessary for efficiently observing the light emission image is derived. For this reason, in the microscope apparatus of the invention of Japanese Patent Application No. 2005-86644, unlike the conventional observation apparatus, the observation range is limited to a part.

  However, unlike the invention of Japanese Patent Application No. 2005-86644, the present invention satisfies the requirement of imaging weak light as brightly as possible, and can observe a wide range as with conventional observation apparatuses such as a microscope. The object of the present invention is to provide an observation system, that is, an observation apparatus and an observation system that have a wide field of view and a high NA as compared with a conventional observation apparatus and can be compatible with the conventional observation apparatus. Therefore, in the present invention, the numerical aperture larger than the numerical aperture of the imaging lens used in the imaging optical system of the conventional observation apparatus and smaller than the numerical aperture of the imaging lens used in the invention of Japanese Patent Application No. 2005-86644. Based on the above test results, the configuration of the imaging optical system necessary for efficiently observing light is derived based on the premise that several imaging lenses are used.

In the observation apparatus of the present invention, an imaging lens having the same focal length as that of the imaging lens used in the imaging optical system of the conventional observation apparatus in order to achieve a high NA with a wide field of view as compared with the conventional observation apparatus. Is used, the numerical aperture NA ′ of the imaging lens is larger than the numerical aperture of the imaging lens used in the imaging optical system of the conventional observation apparatus. At the same time, correction of aberrations becomes difficult, so more lenses are required, and in order to place many lenses, the distance on the optical axis from the object plane to the position where the objective lens is mounted, that is, the same focal point. The distance D must be increased.
The imaging range on the imaging element surface is obtained by dividing the product of the numerical aperture NA of the objective lens and the observation range by the numerical aperture NA ′ of the imaging lens. Therefore, simply increasing the numerical aperture NA ′ of the imaging lens reduces the imaging range on the imaging element surface, making it impossible to effectively use the imaging range on the imaging element surface.
In order to increase the numerical aperture NA ′ of the imaging lens to about 0.08 to 0.09 while maintaining the magnification from the object to the image plane at the same level as in the conventional observation apparatus, the diameter of the light beam emitted from the objective lens Need to be larger.
In order to increase the diameter of the light beam emitted from the objective lens, it is necessary to increase the numerical aperture NA of the objective lens.

However, the observation apparatus of the present invention is configured to satisfy the following conditional expression (1).
4.56 ≦ D · NA ′ <10 (1)
Here, D is the focal length of the objective lens, and NA ′ is the numerical aperture of the imaging lens.
If the conditional expression (1) is satisfied, an objective lens including a plurality of lens groups that can sufficiently correct aberrations even when the numerical aperture of the objective lens is increased can be obtained.

On the other hand, if the lower limit value of the conditional expression (1) is not reached, the numerical aperture NA ′ of the imaging lens becomes small, and the numerical aperture NA of the objective lens cannot be increased, which is compared with the conventional observation apparatus. An observation device with a wide field of view and high NA cannot be obtained. Alternatively, the focal length of the objective lens becomes short, and a high aberration performance cannot be obtained by arranging a plurality of lens groups in the objective lens.
On the other hand, if the upper limit value of the conditional expression (1) is exceeded, the focal length becomes too long, and the transmittance decreases due to the vignetting of light within the objective lens and the absorption of light of the glass material.

In the observation apparatus of the present invention, the distance from the barrel position of the objective lens to the most object side surface of the imaging lens is variable, and the variable amount is W, and the focal length of the imaging lens is FL. In this case, it is preferable that the following conditional expression (2) is satisfied.
0.5FL <W <1.2FL (2)
In the present invention, the distance between the objective lens and the imaging lens is variable, and various observations are made by inserting and removing various optical units on the optical path through which the parallel light flux passes between the objective lens and the imaging lens. The purpose is to respond to the method. Conditional expression (2) defines such a variable range of distance.
If the conditional expression (2) is satisfied, a substantially telecentric configuration in which the incident angle on the imaging surface is about 0 to 5 degrees can be achieved, and a reduction in light amount due to shading inherent to an imaging element such as a CCD can be suppressed. It becomes possible. Further, a sufficient space for combining an intermediate lens unit such as a conventional fluorescent illumination system can be secured between the objective lens and the imaging lens, and the system performance as an observation apparatus is improved.

On the other hand, if the lower limit value of the conditional expression (2) is not reached, the space between the objective lens and the imaging lens becomes narrow, and the system performance as an observation apparatus deteriorates. In addition, light rays are incident obliquely on the image sensor, which is not preferable because the amount of light around the image sensor decreases due to the influence of shading by the image sensor.
Further, if the upper limit value of the conditional expression (2) is exceeded, it is possible to take a sufficient space between the objective lens and the imaging lens, but similarly to the case where the lower limit value of the conditional expression (2) is not reached, Light rays are incident obliquely on the image sensor, which is not preferable because the amount of light around the image sensor decreases due to the influence of shading by the image sensor. In addition, the outer diameter of the imaging lens is increased, and the entire imaging optical system is increased in size.

Furthermore, in the observation apparatus of the present invention, it is preferable that the following conditional expressions (3) and (4) are satisfied.
0.4 <D / FL <1.2 (3)
1 <D / Φd <2.75 (4)
However, D is the focal length of the objective lens, FL is the focal length of the imaging lens, and Φd is the outer diameter of the joint portion of the objective lens with the barrel.

The magnification from the object to the image plane is determined by the ratio of the focal length of the objective lens to the focal length FL of the imaging lens or the ratio of the numerical aperture NA of the objective lens to the numerical aperture NA ′ of the imaging lens.
Further, as described above, the imaging range on the imaging element surface is obtained by dividing the product of the numerical aperture NA of the objective lens and the observation range by the numerical aperture NA ′ of the imaging lens. Therefore, when the numerical aperture NA ′ of the imaging lens is increased, the imaging range on the imaging element surface is reduced, and the imaging range on the imaging element surface cannot be effectively used.

The conditional expressions (3) and (4) are conditional expressions for efficiently capturing weak light with a magnification suitable for observing the magnification from the object to the image plane.
If the above conditional expression (3) is satisfied, the magnification from the object to the image plane becomes an appropriate magnification comparable to the imaging optical system of the conventional observation apparatus, and the numerical aperture NA ′ is the image formation of the conventional observation apparatus. Suitable for combination with an imaging lens having a numerical aperture larger than 0.05 to 0.06 and smaller than a numerical aperture of 0.15 in the weak light imaging optical system of the invention of Japanese Patent Application No. 2005-86644. It becomes an objective lens with a large numerical aperture NA, and it is easy to ensure high aberration performance.
At the same time, if the above conditional expression (4) is satisfied, it is possible to increase the numerical aperture NA ′ of the imaging lens by sufficiently securing the effective diameter of the lens group near the barrel position of the objective lens, In addition, it is possible to minimize the loss of the peripheral light amount.

On the other hand, if the lower limit of conditional expression (3) is not reached, the focal length of the objective lens becomes too short, and an objective lens with good aberration correction and high NA cannot be constructed.
If the upper limit of conditional expression (3) is exceeded, the focal length of the objective lens becomes too long, and the transmittance decreases due to vignetting of light within the objective lens, absorption of light from the glass material, and the like. It is not preferable because the loss of the amount of peripheral light becomes large.

If the lower limit of conditional expression (4) is not reached, the focal length of the objective lens becomes too short, making it difficult to correct aberrations of the objective lens.
If the upper limit value of the conditional expression (4) is exceeded, the effective diameter of the lens near the barrel position of the objective lens becomes too small, and the numerical aperture NA ′ is an imaging lens in the imaging optical system of the conventional observation apparatus. When the imaging lens is larger than the numerical aperture 0.05 to 0.06 and smaller than the numerical aperture 0.15 of the imaging lens in the weak light imaging optical system of the invention of Japanese Patent Application No. 2005-86644, It becomes impossible to take an image with an appropriate magnification comparable to that of the imaging optical system of the conventional observation apparatus with a magnification from the object to the image plane.

In the observation apparatus of the present invention, it is preferable that the rear focal position of the objective lens is located closer to the object side than the body position of the objective lens.
With this configuration, a high aberration performance can be obtained by arranging a plurality of lens groups in the objective lens.

In the observation apparatus of the present invention, it is preferable that at least two objective lenses whose rear focal position is located on the object side with respect to the body position are replaceable.
If comprised in this way, an observation use can be expanded by using the objective lens of a different numerical aperture and a focal distance.
In this case, it is preferable to configure each of these replaceable objective lenses so that the difference in distance from the barrel position to the rear focal position is within ± 15 mm in all replaceable objective lenses. .
If comprised in this way, each objective lens can be comprised as an objective lens which can arrange | position a some lens group substantially similarly and can obtain a high aberration performance.

Further, in the observation apparatus of the present invention, at least two objective lenses are provided so as to be interchangeable with different focal lengths, and are exchanged and attached when any objective lens having a different focal length is exchanged. It is preferable to provide a barrel position adjusting means for changing the barrel position of the objective lens corresponding to the same focal length of each replaceable objective lens so that the front focal positions of the objective lenses substantially coincide.
If comprised in this way, it can have a certain degree of compatibility with the conventional observation apparatus and can expand an observation use, and also the effort which adjusts a focus when an objective lens is replaced | exchanged can be saved.
In the imaging optical systems of the observation device of the present invention and the conventional observation device, if the focal length of the imaging lens is made the same and the same observation magnification is maintained, the focal length of the objective lens also becomes the same. However, when the focal distance D is increased as in the observation apparatus of the present invention, the distance on the optical axis from the rear focal position of the objective lens to the barrel position of the objective lens inevitably increases. When viewed from the attachment position, the rear focal position is located on the object side in the objective lens.

In the observation apparatus of the present invention, it is preferable that at least two imaging lenses are provided.
If comprised in this way, the observation use can be expanded.
In that case, the imaging lens may be provided so as to be replaceable.
Alternatively, an optical path branching unit may be provided on the side opposite to the object of the objective lens, and an imaging lens and an imaging unit may be provided on each optical path branched via the optical path branching unit.
If comprised in this way, observation of the light emission etc. from which a wavelength differs can also be performed simultaneously.

In the observation apparatus of the present invention, an epi-illumination unit such as a fluorescence observation illumination may be provided between the objective lens and the imaging lens.
If comprised in this way, according to a use, for example, light emission observation and fluorescence observation can be switched and performed, and the observation use as an observation system can be expanded further.

In the observation apparatus of the present invention, the objective lens may be constituted by an immersion objective lens or a solid immersion objective lens.
With this configuration, the numerical aperture NA of the objective lens can be increased, and the numerical aperture NA ′ of the imaging lens can be increased.

  The observation system of the present invention includes any one of the above-described observation apparatuses of the present invention and an imaging unit that captures an object image from the observation apparatus.

Further, in the observation system of the present invention, the observation apparatus according to the present invention is the first observation apparatus, and the infinity correction type in which the focal length and the emission NA are different from those of the objective lens used in the first observation apparatus. An observation device having the second objective lens and a second imaging lens having the same focal length as the imaging lens used in the first observation device, the second observation device, and the connection in the first observation device. The numerical aperture of the image lens is NA ′, the same focal length of the objective lens is D, the distance on the optical axis from the object plane to the rear focal position of the objective lens is fb, and the second observation apparatus uses the second observation device. When the numerical aperture of the imaging lens is NA2 ′, the same focal length of the second objective lens is D2, and the distance on the optical axis from the object plane to the rear focal position of the second objective lens is fb2. The objective lens in the first observation apparatus And the imaging lens is configured to satisfy the following conditional expressions (5) to (7) with respect to the second objective lens and the second imaging lens in the second observation apparatus: ing.
0.3 ≦ NA2 ′ / NA ′ ≦ 0.88 (5)
0.5 ≦ D2 / D ≦ 0.87 (6)
−15 <fb2−fb <15 (7)

Conditional expressions (5) to (7) are the conventional focal lengths of 45 to 90 mm, the focal length of the imaging lens of 180 mm, and the numerical aperture of the imaging lens of 0.03 to 0.07 in the observation system of the present invention. The conditions for providing compatibility with the imaging optical system in this observation system are defined.
If conditional expressions (5) to (7) are satisfied as in the observation system of the present invention, compatibility with the imaging optical system in the conventional observation system can be provided.
The lower limit value of conditional expression (5) is that the imaging optical system in the conventional observation system in which the numerical aperture of the imaging lens is 0.03 and the numerical aperture of the imaging lens in the observation of the present invention is 0.09. This is a lower limit value for compatibility with the imaging optical system in the system.
The upper limit value of conditional expression (5) is that the imaging optical system in the conventional observation system in which the numerical aperture of the imaging lens is 0.07, and the numerical aperture of the imaging lens in the observation of the present invention is 0.08. This is an upper limit value for providing compatibility with the imaging optical system in the system.

The lower limit value of conditional expression (6) is that the imaging optical system in the conventional observation system having a focal length of 45 mm and the imaging optical system in the observation system of the present invention in which the focal length satisfies ISO standard 90 mm. This is the lower limit for compatibility.
The upper limit value of the conditional expression (6) is compatible with the imaging optical system in the observation system of the present invention in which the focal length is 75 mm and the imaging optical system in the conventional observation system having the same focal length of 65 mm. It is an upper limit value for giving the nature.

When the phase difference observation ring slit and differential interference observation prism are commonly used in the conventional observation system and the observation system of the present invention, the rear focal position of the objective lens from the object plane is almost the same between both systems. You need to be in position.
Conditional expression (7) defines the allowable amount of misalignment.

Further, in the observation apparatus of the present invention, at least one of bright field observation, fluorescence observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation is provided on the side opposite to the objective lens with the object interposed therebetween. It is preferable to provide a transmission illumination optical system configured to be able to observe the above.
In the observation apparatus of the present invention, the transmission illumination optical system includes a light source such as a halogen light source, an LED light source, and a fiber light source, and a condenser lens. Bright field observation and fluorescence are provided at a substantially pupil position of the condenser lens. Preferably, at least one of various optical elements that enables observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation is switchably provided.

Illumination methods for performing fluorescence observation include epi-illumination and transmission illumination. Here, as in the observation apparatus of the present invention, if the fluorescence observation is performed by transmitted illumination, it is not necessary to arrange an illumination system between the objective lens and the imaging lens. Can be made compact. In addition, the aberration performance of the imaging lens having a large numerical aperture NA ′ and the telecentricity on the image side (imaging device side) of the imaging lens are improved.
Further, when the observation object is transparent, if the observation object is configured to be visualized with transmission illumination as in the observation apparatus of the present invention, transmission fluorescence observation, phase difference observation, differential interference observation, oblique illumination, and Hoffman By combining an observation technique such as modulation contrast observation with light emission observation, the position and shape of the observation object can be specified, and a light emission image can be acquired at the same time.
Note that an LED light source is preferable as the light source used in the above-described observation of various transmitted illuminations and epifluorescence observation. When the LED light source is used, the illumination light intensity can be adjusted electrically with low power consumption, and the fluorescence observation can be switched at high speed.

An observation system according to the present invention includes any one of the observation apparatuses according to the present invention including the above-described transmission illumination optical system, an imaging unit that captures an object image from the observation device, and an object obtained through the imaging unit. The image processing means for superimposing the transmitted illumination observation image and the light emission observation image from the object is provided.
If comprised in this way, the light emission image observation combined with the transmission observation image can be realized, and the light emission image can be acquired by specifying the position, shape, etc. of the observation object.
Note that the light emission image has a weak amount of light. On the other hand, the transmitted observation image by the transmitted illumination has a higher light quantity than the emission observation image. Therefore, in order to observe the emission observation image in combination with the transmission observation image, the brightness of the emission image and the transmission observation image captured by the imaging unit is adjusted so that these two images are superimposed. .

In the observation system of the present invention, it is preferable that the observation system further includes display means for displaying the range of the image region superimposed through the image processing means together with the superimposed image on a display device.
With this configuration, the superimposed observation region becomes clear, and the light emission observation position on the observation object can be easily grasped.

(First embodiment)
FIG. 1 is a block diagram illustrating a schematic configuration of an entire observation system including an observation apparatus according to a first embodiment of the present invention, and FIG. 2 is a principle explanatory diagram of an imaging optical system in the observation system illustrated in FIG.
The observation system according to the first embodiment includes an observation device, an imaging unit 4 and an imaging information processing display unit 7.
The observation apparatus includes an objective lens 2 and an imaging lens 3.
The objective lens 2, the imaging lens 3, and the imaging means 4 constitute the imaging optical system 1.
In FIG. 1, 5 indicates a position where the objective lens 2 is mounted, and 6 indicates an object such as a biological sample. In FIG. 2, O indicates the object plane, and I indicates the position of the imaging plane.
The objective lens 2 is configured as an infinity correction type objective lens, and converts light from the object 6 into substantially parallel light.
The imaging lens 3 focuses the parallel light flux from the objective lens 2 and forms an object image on the imaging surface of the imaging means 4.
The image pickup means 4 has an image pickup element such as a CCD or a CMOS.
The imaging information processing display means 7 is constituted by a personal computer, for example, and is an imaging control means (not shown) for controlling the imaging movement difference of the imaging means 4 and an image recording means for recording image information taken by the imaging means 4. (Not shown), measuring means (not shown) for converting the image information captured by the imaging means 4 into numerical information and measuring, image information captured by the imaging means 4, and numerical values measured by the measuring means Display means (not shown) for displaying information is provided.

The observation apparatus is configured to satisfy the following conditional expression (1), where D is the focal length of the objective lens 2 and NA ′ is the numerical aperture of the imaging lens 3.
4.56 ≦ D · NA ′ ≦ 10 (1)
In the observation apparatus, the distance from the barrel position 5 of the objective lens 2 to the most object-side surface of the imaging lens 3 is variable. When the variable amount is W and the focal length of the imaging lens 3 is FL, the following conditional expression (2) is satisfied.
0.5FL <W <1.2FL (2)

The observation apparatus is configured to satisfy the following conditional expressions (3) and (4) when the outer diameter of the joint portion of the objective lens 2 with the body is Φd.
0.4 <D / FL <1.2 (3)
1 <D / Φd <2.75 (4)

  Further, the objective lens 2 is configured such that the rear focal position Fb is located on the object side with respect to the body position 5 of the objective lens 2.

  In the observation system including the observation apparatus according to the first embodiment configured as described above, the light from the object 6 is converted into parallel light via the objective lens 2, and the imaging means 4 is connected via the imaging lens 3. The image is formed on the imaging surface and imaged through the imaging means 4. The observation image picked up by the image pickup means 4 is sent to the image pickup information processing display means 7. Image information sent to the imaging information processing display means 7 is recorded via an image recording means (not shown), converted into numerical information via a measuring means (not shown), measured, and displayed as a display means ( Image information and measured numerical information are displayed via (not shown).

At this time, according to the observation system provided with the observation apparatus of the first embodiment, the observation apparatus satisfies the conditional expression (1), and thus includes a plurality of lens groups that can sufficiently correct aberrations even when the numerical aperture is increased. The objective lens 2 can be configured.
In addition, since the observation apparatus satisfies the conditional expression (2), the incident angle of the principal ray on the imaging surface can be set to a substantially telecentric configuration of about 0 degree to 5 degrees, and shading inherent to the imaging element such as a CCD is performed. It is possible to suppress a decrease in the amount of light. Further, a sufficient space for combining an intermediate lens unit such as a conventional fluorescent illumination system can be secured between the objective lens 2 and the imaging lens 3, and the system performance as an observation apparatus is improved.
In addition, since the observation device satisfies the conditional expression (3), the magnification from the object to the image plane is an appropriate magnification comparable to that of the conventional observation device (imaging optical system), and the numerical aperture NA ′ is conventionally The numerical aperture of the imaging lens is larger than 0.05 to 0.06 in the observation device (imaging optical system) and the numerical aperture of the imaging lens is 0 in the weak light imaging optical system of the invention of Japanese Patent Application No. 2005-86644. The objective lens 2 having a numerical aperture NA suitable for combination with the imaging lens 3 smaller than .15 is obtained, vignetting in the objective lens 2 is small, and high aberration performance is obtained.
Further, since the observation apparatus satisfies the conditional expression (4), the numerical aperture NA ′ of the imaging lens 3 is increased by sufficiently securing the effective diameter of the lens group in the vicinity of the barrel position 5 of the objective lens 2. And the loss of the peripheral light amount can be minimized.
In addition, since the rear focal position Fb of the objective lens 2 in the observation apparatus is located closer to the object side than the body position 5 of the objective lens 2, a plurality of lens groups are arranged in the objective lens 2 to achieve high aberration performance. Obtainable.
Therefore, according to the observation system including the observation device of the first embodiment, it is possible to acquire a weak emission signal with a high S / N with a high NA while having a wide observation range.

(Second Embodiment)
FIG. 3 is a block diagram showing a schematic configuration of the entire observation system including the observation apparatus according to the second embodiment of the present invention.
In the observation system according to the second embodiment, the observation apparatus includes two objective lenses 2 ₁ and 2 ₂ that are interchangeable. Further, all of the interchangeable objective lenses 2 ₁ and 2 ₂ are configured such that the difference ΔFb between the barrel position 5 and the rear focal positions Fb ₁ and Fb ₂ is within ± 15 mm.

According to the observation system including the observation device of the second embodiment configured as described above, the use of observation can be expanded by using objective lenses having different numerical apertures and focal lengths.
Further, since all the replaceable objective lenses 2 ₁ and 2 ₂ are configured such that the difference ΔFb between the barrel position 5 and the rear focal positions Fb ₁ and Fb ₂ is within ± 15 mm, respectively. The objective lenses 2 ₁ and 2 ₂ can be configured as an objective lens that can obtain a high aberration performance by arranging a plurality of lens groups.
Other configurations and operational effects are almost the same as those of the observation system including the observation apparatus of the first embodiment.

(Third embodiment)
FIG. 4 is a block diagram showing a schematic configuration of an entire observation system including an observation apparatus according to the third embodiment of the present invention.
In the observation system according to the third embodiment, the observation apparatus includes two objective lenses 2 ₁ ′ and 2 ₂ ′ that are different from each other such that the focal lengths are D ₁ and D ₂ . Also, interchangeable objective lens 2 ₁ ', 2 _2' corresponds to the focal length D _1, D _2, the objective lens 2 ₁ ', 2 _2' cylinder with position adjusting means 8 for changing the cylinder mounting position of Is provided so that the front focal position of each objective lens when it is replaced and attached to the objective lens 2 ₁ ′ or 2 ₂ ′ substantially coincides with the surface O of the object 6. It has become.

According to the observation system provided with the observation apparatus of the third embodiment configured as described above, it is possible to widen the observation application. Further, since the position adjusting means 8 is provided, it is possible to save the trouble of adjusting the focus when the objective lenses 2 ₁ ′ and 2 ₂ ′ are exchanged.
Other configurations and operational effects are almost the same as those of the observation system including the observation apparatus of the first embodiment.

(Fourth embodiment)
FIG. 5 is a block diagram showing a schematic configuration of the entire observation system including the observation apparatus according to the fourth embodiment of the present invention.
In the observation system of the fourth embodiment, a dichroic mirror or beam splitter as the optical path branching means 9 is provided on the side opposite to the object 6 of the objective lens 2, and on each optical path branched via the optical path branching means 9. Imaging lenses 3 ₁ and 3 ₂ and imaging means 4 ₁ and 4 ₂ are provided. In FIG. 5, reference numerals 10 ₁ and 10 ₂ denote spectral filters arranged on the respective optical paths branched through the optical path branching means 9.

According to the observation system provided with the observation apparatus of the fourth embodiment configured as described above, it is possible to simultaneously observe a plurality of types of weak light having different wavelengths.
Other configurations and operational effects are almost the same as those of the observation system including the observation apparatus of the first embodiment.

(Fifth embodiment)
FIG. 6 is a block diagram showing a schematic configuration of an entire observation system including an observation apparatus according to the fifth embodiment of the present invention.
In the observation system of the fifth embodiment, the observation apparatus includes two imaging lenses 3 ₁ and 3 ₂ that are exchangeable.
According to the observation system including the observation apparatus of the fifth embodiment configured as described above, for example, by changing the numerical aperture NA ′ of the imaging lenses 3 ₁ and 3 ₂ , the object 6 to the image plane can be changed. Observation applications can be expanded, such as observation with different magnifications.
Other configurations and operational effects are almost the same as those of the observation system including the observation apparatus of the first embodiment.

(Sixth embodiment)
FIG. 7 is a block diagram showing a schematic configuration of the entire observation system including the observation apparatus according to the sixth embodiment of the present invention.
In the observation system of the sixth embodiment, the observation apparatus includes a fluorescence observation illumination optical system 11 that is detachable between the objective lens 2 and the imaging lens 3.
The fluorescence observation illumination optical system 11 includes an LED light source 12, an illumination lens 13, an excitation filter 14, a dichroic mirror 15, and a barrier filter 16 that cuts light other than fluorescence.

According to the observation system including the observation apparatus of the sixth embodiment configured as described above, fluorescence observation can be performed when the fluorescence illumination optical system 11 is attached. That is, light from the LED light source 12 passes through the illumination lens 13, light having an excitation wavelength passes through the excitation filter 14, and irradiates the object 6 through the dichroic mirror 15 and the objective lens 2. Light from the object 6 passes through the objective lens 2, light other than excitation light passes through the dichroic mirror 15, light other than fluorescence is removed through the barrier filter 6, and only the fluorescence is imaged by the imaging unit 4. .
On the other hand, when the fluorescent illumination optical system 11 is removed, light from the object 6 can be observed as in the first embodiment.
The fluorescent illumination optical system 11 may be fixedly attached. In that case, the dichroic mirror 15 and the barrier filter 16 may be detachable. For example, when observing light emission, the dichroic mirror 15 and the barrier filter 16 may be removed.
Other configurations and operational effects are almost the same as those of the observation system including the observation apparatus of the first embodiment.

(Seventh embodiment)
FIG. 8 is a schematic explanatory diagram of the entire observation system including the observation apparatus according to the seventh embodiment of the present invention.
In the observation system of the seventh embodiment, in addition to the configuration of the observation apparatus of any of the above embodiments, the objective lens 2 and the imaging lens 3 satisfy the following conditional expressions (5) to (7). It is configured.
0.3 ≦ NA2 ′ / NA ′ ≦ 0.88 (5)
0.5 ≦ D2 / D ≦ 0.87 (6)
−15 <fb2−fb <15 (7)
Where NA ′ is the numerical aperture of the imaging lens 3, D is the focal length of the objective lens 2, fb is the distance on the optical axis from the object plane to the rear focal position of the objective lens 2, and NA2 ′ is the second. The numerical aperture of the imaging lens 3 ₂ , D2 is the same focal length of the second objective lens 2 ₂ , and fb2 is the distance on the optical axis from the object plane to the rear focal position of the _second objective lens 2 ₂ .

The second observation apparatus is a conventional general microscope apparatus, and includes a second objective lens 2 ₂ and a second imaging lens 3 ₂ . The second imaging optical system 1 ₂ includes a second objective lens 2 ₂ , a second imaging lens 3 _2, and an imaging means 4 ₂ . In FIG. 7, 5 ₂ with barrel position, 6 ₂ object (biological sample), 7 ₂ is imaging information display means.
The second objective lens 2 ₂ is configured as an infinitely corrected objective lens having the same focal length and exit NA as the objective lens 2. The second imaging lens 3 ₂ has the same focal length as the imaging lens 3.

According to the observation system of the seventh embodiment, the objective lens 2 and the imaging lens 3 are interchanged with the second objective lens 2 ₂ and the second imaging lens 3 ₂ provided in the second observation apparatus. Can do. For this reason, according to the observation system of the seventh embodiment, compatibility with a conventional observation system can be provided, and high expandability can be obtained.

(Eighth embodiment)
FIG. 9 is a schematic explanatory diagram of an observation apparatus and an observation system including the observation apparatus according to an eighth embodiment of the present invention.
The observation system according to the eighth embodiment includes an observation apparatus, an imaging unit 4 that captures an object image from the observation apparatus, and an imaging information processing display unit 7 ′.
The observation apparatus has substantially the same configuration as that of any of the observation apparatuses in any of the observation systems according to the first to third embodiments, and a transmission illumination optical system on the side opposite to the objective lens 2 with the object 6 interposed therebetween. A system 21 is provided.

The transmission illumination optical system 21 includes an LED light source 22 and a condenser lens 23. Further, at least one of bright field observation, fluorescence observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation can be observed.
In FIG. 9, 24 is an aperture stop, and 25 is a field stop.
In the example of FIG. 9, an excitation filter 26 that transmits only a predetermined excitation wavelength and blocks other wavelengths is disposed between the LED light source 22 and the condenser lens 23, and has a predetermined fluorescence wavelength and emission wavelength. A barrier filter 27 that transmits only light and blocks other wavelengths is arranged between the objective lens 2 and the imaging lens 4 so that fluorescence observation can be performed by transmitted illumination. The filters 26 and 27 are arranged on the optical path when performing fluorescence observation, and are detachably provided so as to be removed from the optical path in observations other than fluorescence observation.

Further, the aperture stop 24 is detachably disposed at a substantially pupil position of the condenser lens 23 via a slider 30. In addition to the aperture stop 24, the slider 30 includes a phase difference observation ring slit 28, a differential interference observation prism 29, and a polarizing plate and a Hoffman modulation contrast observation slit aperture plate (not shown). Yes.
In the observation system according to the eighth embodiment, bright field observation (brightness stop 24) and fluorescence observation (brightness stop 24) are arranged at approximately the pupil position of the condenser lens via the slider 30 according to the observation application. Various optics enabling phase difference observation (phase difference observation ring slit 28), differential interference observation (differential interference observation prism 29), and Hoffman modulation contrast observation (polarizing plate and slit aperture plate for Hoffman modulation contrast observation) At least one of the elements can be switched.
In addition to the switching operation of the slider 30, the filters 26 and 27 are removed from the optical path during observations other than fluorescence observation. At the time of phase difference observation, the objective lens 2 is used for phase difference. Further, a Hoffman modulation contrast observation modulator is mounted in the vicinity of the objective lens 2 at the time of Hoffman modulation contrast observation.

  In addition to the functions of the imaging information processing display unit 7 in the observation systems of the first to seventh embodiments, the imaging information processing display unit 7 ′ includes a transmission illumination observation image of the object obtained through the imaging unit 4 and the object. Image processing means (not shown) for superimposing the emission observation image is further provided. Further, the imaging information processing display means 7 ′ displays the superimposed image area, for example, with its outline shape, circle or rectangular line, through the image processing means. In addition, display means (not shown) for displaying on the display device is provided.

According to the observation system provided with the observation apparatus of the eighth embodiment, it is not necessary to arrange an illumination system between the objective lens and the imaging lens, so that the space between the objective lens and the imaging lens can be made compact. Can do. In addition, the aberration performance of the imaging lens having a large numerical aperture NA ′ and the telecentricity on the image side (imaging device side) of the imaging lens are improved.
In addition, even when the observation object is transparent, observation images superposed by combining observation methods such as transmission fluorescence observation, phase difference observation, differential interference observation, oblique illumination and Hoffman modulation contrast observation with light emission observation The position and shape of the observation object can be specified, and a light emission image can be acquired at the same time.
Moreover, the range of the superimposed image area can be displayed on the display device together with the superimposed image, the superimposed observation area becomes clear, and the emission observation position on the observation object can be grasped. It becomes easy.
Other configurations and operational effects are substantially the same as those of the observation system including the observation apparatus according to the first to third embodiments.

  The observation apparatus and the observation system including the observation apparatus according to the present invention are useful in the fields of medicine and biology in which it is required to observe weak light with various observation techniques over a wide observation region.

It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 1st Embodiment of this invention. It is a principle explanatory view of an imaging optical system in the observation system shown in FIG. It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 2nd Embodiment of this invention. It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 3rd Embodiment of this invention. It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 4th Embodiment of this invention. It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 5th Embodiment of this invention. It is a block diagram which shows schematic structure of the whole observation system provided with the observation apparatus concerning 6th Embodiment of this invention. It is a schematic explanatory drawing of the whole observation system provided with the observation apparatus concerning 7th Embodiment of this invention. It is a schematic explanatory drawing of the observation apparatus concerning 8th Embodiment of this invention, and the whole observation system provided with the same.

Explanation of symbols

1, 1 ₂ imaging optical system 2, 2 ₁ , 2 ₂ , 2 ₁ ′, 2 ₂ ′ objective lens 3, 3 ₁ , 3 ₂ imaging lens 4, 4 ₁ , 4 ₂ imaging means 5, 5 ₂ position with body 6, 6 ₂ objects (biological samples)
7, 7 ₂ , 7 ′ Imaging information processing display means 8 Body position adjustment means 9 Optical path branching means 10 ₁ , 10 ₂ Spectroscopic filter 11 Fluorescence observation illumination optical system 12 LED light source 13 Illumination lens 14 Excitation filter 15 Dichroic mirror 16 Barrier filter 21 Transmission illumination optical system 22 LED light source 23 Condenser lens 24 Brightness stop 25 Field stop 26 Excitation filter 27 Barrier filter 28 Phase difference observation ring slit 29 Differential interference observation prism 30 Slider

Claims (15)

In an observation apparatus having an infinitely corrected objective lens and an imaging lens,
An observation apparatus satisfying the following conditional expression, where D is the focal length of the objective lens and NA ′ is the numerical aperture of the imaging lens.
4.56 ≦ D · NA ′ <10 The distance from the barrel position of the objective lens to the most object side surface of the imaging lens is variable,
2. The observation apparatus according to claim 1, wherein when the variable amount is W and the focal length of the imaging lens is FL, the following conditional expression is satisfied.
0.5FL <W <1.2FL The observation apparatus according to claim 2, wherein the following conditional expression is satisfied.
0.4 <D / FL <1.2
1 <D / Φd <2.75
However, D is the same focal length of the objective lens, FL is the focal length of the imaging lens, and Φd is the outer diameter of the joint portion on the body of the objective lens. The observation apparatus according to claim 3, wherein a rear focal position of the objective lens is located closer to an object side than a barrel position of the objective lens. At least two of the objective lenses are replaceable, and
5. Each replaceable objective lens is configured so that a difference in distance from the barrel position to the rear focal position is within ± 15 mm in all the replaceable objective lenses. The observation apparatus described in 1. The objective lens is provided so as to be exchangeable with at least two different focal lengths, and
Corresponding to the focal lengths of the interchangeable objective lenses so that the front focal positions of the objective lenses that have been replaced and replaced substantially coincide with each other when the objective lenses have different focal lengths. The observation apparatus according to claim 3, further comprising a body position adjusting unit that changes a body position of the objective lens. The observation apparatus according to claim 3, wherein at least two imaging lenses are provided. The observation apparatus according to claim 3, wherein a fluorescence observation illumination optical system is provided between the objective lens and the imaging lens. The observation apparatus according to claim 4, wherein the objective lens is an immersion objective lens or a solid immersion objective lens. An observation system comprising: the observation apparatus according to claim 1; and an imaging unit that captures an object image from the observation apparatus. 10. An infinitely corrected second objective lens having the same focal length and exit NA as the first observation apparatus and the objective lens used in the first observation apparatus. And an observation device having a second imaging lens having the same focal length as the imaging lens used in the first observation device, a second observation device, and a numerical aperture of the imaging lens in the first observation device. NA ′, D is the focal length of the objective lens, fb is the distance on the optical axis from the object plane to the back focal position of the objective lens, and the aperture of the second imaging lens in the second observation device When the number is NA2 ′, the same focal length of the second objective lens is D2, and the distance on the optical axis from the object plane to the rear focal position of the second objective lens is fb2,
The objective lens and the imaging lens in the first observation device satisfy the following conditional expression with respect to the second objective lens and the second imaging lens in the second observation device: The observation system according to claim 10.
0.3 ≦ NA2 ′ / NA ′ ≦ 0.88
0.5 ≦ D2 / D ≦ 0.87
−15 <fb2−fb <15 At least one of bright field observation, fluorescence observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation can be performed on the opposite side of the objective lens across the object. The observation apparatus according to claim 3, further comprising a transmission illumination optical system. The transmission illumination optical system has a light source and a condenser lens,
At least one of various optical elements that enable bright field observation, fluorescence observation, phase difference observation, differential interference observation, and Hoffman modulation contrast observation can be switched at a substantially pupil position of the condenser lens. The observation apparatus according to claim 12, characterized in that: An observation apparatus according to claim 12 or 13, and an imaging means for imaging an object image from the observation apparatus,
An observation system comprising image processing means for superimposing a transmitted illumination observation image of an object obtained through the imaging means and a light emission observation image from the object. 15. The observation system according to claim 14, further comprising display means for displaying a range of the image area superimposed through the image processing means together with the superimposed image on a display device. .

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