JP2006309088A - Highly precise measurement method of microscope focusing position - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly precise measurement method of a sample position with which a focusing position and the sample position are measured with high precision and in a wide range even at the time of fluorescent image observation without a loss with respect to a biotic example not resistant to light irradiation, etc. and a sample with low reflectivity. <P>SOLUTION: When light for position measurement is passed through an area with high number of aperture including the entire reflection area of an edge part of an objective lens, intense reflecting light is obtained even in the sample with low reflectivity in a transparent sample, etc. by the entire reflecting light from a boundary surface of the sample and reflecting light from a large illumination angle. When the boundary surface of the sample changes in the height direction by Δz, the reflecting light is regarded to appear from a position shifted by Δx to be provided by Δx=2Δz×tanθ in the horizontal direction in the boundary surface of the sample and larger variation of Δx is obtained even to variation of Δz by increasing an angle of incidence θ. Highly precise measurement of the sample position is performed without affecting the florescent image observation by introducing position measurement light on an optical path of a microscope image by using a dichroic mirror for fluorescent in common with that for position measurement. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a sample focusing position high-precision measurement method for an optical microscope or a measuring device using light, and more particularly to a microscope capable of observing a fluorescent image and a sample focusing position high-precision measurement method suitable for position measurement.

Conventionally, in autofocus and shape observation, as shown in FIG. 4, a sample reflected or scattered as a sample is irradiated with light through an objective lens, and the reflected light is photographed with a CCD camera through an imaging lens. . The method used in a microscope as a method of knowing the depth (height) position of a sample is as shown in FIG. 4 in which half of the illumination light is shielded, light is incident from one side of the objective lens, and reflected from the sample boundary surface. A method of detecting light with an optical sensor is used. In this method, an image of a sample boundary surface by reflected light is formed on an optical sensor, and a change in the height position of the sample is obtained by utilizing a method of blurring the asymmetric image of reflected light on the sensor. If the illumination light to the sample is only on one side with respect to the optical axis, the reflected light will also be only on one side, so if the height of the sample changes, the image of the reflected light on the sensor will be blurred only on one side, so The position is also shifted to one side, and when the height of the sample is changed in the opposite direction, the blurring direction becomes the opposite side. Such a thing is often seen in patent literature (see, for example, Patent Literature 1 and Patent Literature 2).
JP-A-8-234093 JP 2005-62515 A

  In the conventional method, as shown in FIG. 4, a half mirror or a dichroic mirror is separately used for the measurement light only for introducing the measurement light into the optical path in the microscope tube. When observing feeble light as in fluorescence observation, even a microscopic observation, even a slight loss of fluorescence leads to image degradation. Even if a dichroic mirror is used, when the fluorescence passes through an extra dichroic mirror for measurement, a fluorescence loss of about 5 to 10% is generated at the minimum.

  In addition, the conventional method can measure the position with high accuracy in the case of a highly reflective sample such as a metal or a semiconductor, but it is micrometer in the case of a low reflectance sample such as a biological sample or a glass surface. Only a degree of accuracy could be obtained.

  Also, in the conventional method, as shown in FIG. 4, measurement light is focused on a sample boundary surface in a dot shape by an objective lens, which is not good for a biological specimen that is weak to light irradiation. When the image is out of focus, the image is suddenly blurred from the focused light, so the focus position is clear, but the measurable range tends to be narrow. The method of halving the illumination light with a light-shielding plate has a wide illumination luminous flux, so the signal that depends on the illumination angle is averaged and weakened. In addition, the light is reflected off the object surface and at the center of the objective lens and ignored. The problem that an error that cannot be generated occurs.

  Normally, when the in-focus position of the specimen is finely adjusted, it is difficult to position the reference position of the sample at the target position with high accuracy by the mechanical draft of the apparatus or fluctuation due to temperature. It was impossible rather than a skill.

  Therefore, the present invention provides a loss of fluorescence even when observing a fluorescent image in a position measurement using a microscope or an objective lens, and does not damage a specimen that is weak to light irradiation, such as a biological sample, and is transparent. A sample that can measure the focal position and the sample position over a wide range with high accuracy even in a sample having a low reflectance such as a sample, and can accurately position the reference position of the sample at a target position by the operator. An object of the present invention is to provide a focus position high-precision measurement method.

  In order to achieve the above object, a sample focal position high-precision measurement method according to claim 1 of the present invention is a microscope capable of observing a fluorescent image and, in position measurement, for measuring the position of light having a wavelength not used for fluorescent image observation. The position measurement light is introduced from the light source onto the optical path of the microscope tube using a fluorescent dichroic mirror, and the sample is irradiated to measure the reflected light returning from the sample boundary surface through the objective lens. By forming an image on the optical sensor, it is possible to measure the in-focus position and the sample height position without loss of fluorescence even when observing the fluorescence image.

  As a result, by using light of a wavelength not used for fluorescence image observation for position measurement, neither fluorescence excitation nor fluorescence image is affected. Further, by using a fluorescent dichroic mirror that reflects fluorescence excitation light and position measurement light and transmits fluorescence, a dichroic mirror can be used in common for fluorescence and position measurement. Regarding the wavelength characteristics of the dichroic mirror, the transmittance of fluorescence and the reflectance of fluorescence excitation light are regarded as important, and the reflectance of position measurement light may be designed with lower priority. Therefore, the dichroic mirror has various fluorescent dyes. In contrast, it can be manufactured without deteriorating the fluorescence characteristics. Since the fluorescence dichroic mirror is used in common to introduce the position measurement light into the optical path in the microscope tube, there is no fluorescence transmission through the extra measurement dichroic mirror, etc. Does not occur.

  According to a second aspect of the present invention, a sample focusing position high-accuracy measuring method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of observing a fluorescent image and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used, so that it can be combined with high accuracy even for transparent samples and low reflectance samples. The focus position and sample height position can be measured without loss even in fluorescence image observation.

  Thereby, due to the difference in refractive index between the sample and the support substrate (cover glass), when the illumination light is incident at a larger angle than the critical angle, total reflection occurs at the sample boundary surface. In addition, the reflected light intensity suddenly increases as the critical angle is approached even below the critical angle (FIG. 5). By using the total reflected light and the reflected light from a large illumination angle, it is possible to obtain a reflected light having a high intensity even in a transparent sample or the like having a low reflectance, and the light reaching the position measurement optical sensor can be obtained. Since the intensity increases, the sample position and the in-focus position can be measured with high accuracy.

  Since the measurement light is not condensed in a spot shape at the sample boundary surface, it is possible to avoid a bad influence on a biological specimen that is weak to light irradiation. The illumination light illuminates the area corresponding to the image of the slit of the light source instead of a spot at the in-focus position, so even if the sample height position changes from the in-focus position, it can be measured without a sudden change in the illumination range. Suitable for wide range. Since the illumination light flux is not wide through a limited area at the edge of the objective lens, in addition to obtaining a large signal depending on the illumination angle, reflection from other than the object surface and at the center of the objective lens Problems such as reflection (reflection near d = 0 in FIG. 5) can be avoided. Since the illumination light and reflected light are separated from each other with the optical axis in between, the reflected light can be completely separated from the illumination light by a mirror such as a total reflection prism without any loss in front of the sensor, which is also advantageous for low reflectance samples. It is.

  According to a third aspect of the present invention, the in-focus position high-precision measurement method uses a non-coherent (incoherent) light source or a non-coherent and low-coherent light source as a light source in a microscope capable of observing a fluorescent image and position measurement. Using this coherent light source, the position measurement light from this light source is introduced into the optical path of the microscope tube using a fluorescent dichroic mirror, and the sample is irradiated, and the reflected light returning from the sample boundary surface through the objective lens is reflected. An image is formed on the position measurement optical sensor, and the in-focus position and sample position can be measured without loss even in fluorescence image observation with higher stability and accuracy.

  As a result, when laser light is used as illumination light for the light source, interference occurs, causing noise and drift in position measurement, resulting in poor measurement accuracy. In this case, when the sample focusing position high-precision measurement method according to claim 1 is performed using a non-coherent (incoherent) light source or a coherent light source that has been made non-coherent and low-coherent, a fluorescent image is obtained. For observation, it is possible to perform more stable and accurate position measurement without loss.

  According to a fourth aspect of the present invention, a sample focusing position high-precision measurement method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of observing a fluorescent image and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, the output from the optical sensor for position measurement is obtained as a function of the sample height position, the sample position is measured from the optical sensor, and is further arranged linearly A wide range of sample height positions can be measured using an optical sensor, and by using the total reflection light at the sample boundary surface and the reflection light from a large illumination angle, it is possible to obtain a low reflectance sample such as a transparent sample. Was the focus position and the sample position to be without loss widely measurable fluorescent image observation with high accuracy.

  As a result, the sample height position Δz can be obtained from the optical sensor output by obtaining the output from the position measuring optical sensor as a function of the change Δz in the sample height position. If the optical sensor position is placed at a position conjugate with the sample boundary surface when the observation boundary is focused on the sample boundary surface, that is, if the sensor position is placed at a position conjugate with the sample observation surface, the sample position is determined using the sample boundary surface as a reference point. Can be requested. Furthermore, if optical sensors arranged in a straight line are used, the measurement range of Δz can be expanded by using outputs from a plurality of sensors, and a wider range of sample height positions Δz can be measured.

  According to a fifth aspect of the present invention, a sample focusing position high-precision measurement method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of fluorescent image observation and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used, so that it can be combined with high accuracy even for transparent samples and low reflectance samples. The focal position and the sample position are measured, and the position measuring optical sensor position is moved from the position conjugate with the sample observation surface by moving the position measuring imaging lens or the position measuring optical sensor position in the optical axis direction. There is, by moving the imaging lens or the optical sensor located in a direction perpendicular to the optical axis, and enable the determination of the sample height position different positions on the sample as a reference position.

  Thereby, when observing a position different from the sample boundary surface, by moving the imaging lens or the position measurement optical sensor position in the optical axis direction and shifting the position measurement optical sensor from the position conjugate with the sample observation surface, That is, by placing the sensor position at a position conjugate with the sample observation surface when observing a position different from the boundary surface, or moving the imaging lens or the optical sensor position in a direction perpendicular to the optical axis. Thus, the sample position and the in-focus position can be obtained using an observation position different from the sample boundary surface as a reference point.

  According to a sixth aspect of the present invention, a sample focusing position high-precision measurement method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of observing a fluorescent image and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used. Measure illumination light such as thin-layer oblique illumination method and total reflection illumination method that measure focus position and sample position and combine with focus drive mechanism, auto focus, sample position control, and illumination through objective lens It can be applied and the.

  Thereby, by combining with a focus driving mechanism, automatic focusing (autofocus) and sample position control can be performed. In addition, in illumination methods such as the thin-layer oblique illumination method and total reflection illumination method that illuminate via the objective lens, it is essential for the illumination light control to accurately know and control the sample position. be able to.

  According to a seventh aspect of the present invention, a sample focusing position high-precision measurement method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of observing a fluorescent image and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used, so that it can be combined with high accuracy even for transparent samples and low reflectance samples. The focal position and the sample position are measured, and an intermediate lens group is provided between the objective lens and the position measurement imaging lens. The conjugate positional relationship between the position measurement optical sensor and the sample side depends on the presence or absence of the intermediate lens group. does not change Can be placed urchin said intermediate lens group.

  As a result, the imaging magnification on the sensor depends on the ratio of the focal lengths of the intermediate lens group, so that the imaging magnification can be freely changed by changing the lens configuration of the intermediate lens group. When the imaging magnification on the sensor is increased, the resolution is high, but the sample height position measurable range is narrowed. When the imaging magnification is lowered, the resolution is low but the measurable range is widened. Therefore, the resolution and the measurable range can be changed by changing the imaging magnification by changing the intermediate lens configuration.

  In addition, this makes it possible to replace the objective lens having different magnifications by changing the intermediate lens configuration without changing the position measuring light source and the position measuring imaging lens and the optical sensor side. It is possible to easily cope with a magnification objective lens. When an objective lens having a different magnification, that is, a different focal length is used, the optical path on the objective lens side changes, but the difference in the optical path on the objective lens side can be absorbed by changing the intermediate lens configuration. And the optical path on the sensor side can be kept the same.

  In addition, this makes it possible to prevent the sample position measurable range from being narrowed when the distance between the objective lens and the position measuring imaging lens becomes long. If the distance between the objective lens and the imaging lens for position measurement is increased, the field of view is narrowed as the optical path is far from the center of the optical axis, and as a result, the sample height position measurable range is narrowed. By inserting the intermediate lens group, it is possible to prevent the optical path from moving away from the center of the optical axis, thus preventing the problem that the measurable range becomes narrow due to the length of the distance between the objective lens and the imaging lens for position measurement. Can do.

  According to an eighth aspect of the present invention, there is provided a sample focusing position high-accuracy measurement method, in which a microscope capable of observing a fluorescent image and position measurement are performed by using a dichroic mirror for fluorescence from a light source. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used, so that it can be combined with high accuracy even for transparent samples and low reflectance samples. The focal position and the sample position are measured, and an intermediate lens group is provided between the objective lens and the position-measuring imaging lens. By moving some of the intermediate lens groups, different heights on the sample are obtained. Based on the position It made it possible to determine the sample height position as the position.

  With this configuration, an intermediate lens group is provided between the objective lens and the position measurement imaging lens so that the conjugate positional relationship between the position measurement optical sensor and the sample side does not change depending on the presence or absence of the intermediate lens group. By moving the position of a part of the intermediate lens group, a position different from the sample boundary surface can be obtained without changing the position measuring light source and the position measuring imaging lens and the optical sensor side. It is possible to obtain a different height position on the sample as a focus position as a reference point. Changing the height position of sample observation changes the optical path on the objective lens side, which limits the range in which the in-focus position can be measured, but the difference in the optical path on the objective lens side is absorbed by the movement of the intermediate lens. Since the optical path on the imaging lens and the sensor side can be kept the same, it is possible to perform in-focus position measurement over a wide range with respect to the height position of sample observation.

  According to a ninth aspect of the present invention, a sample focusing position high-precision measurement method uses a fluorescence dichroic mirror from a light source for position measurement light in a microscope capable of observing a fluorescent image and position measurement. Introduced on the road, passes the position measurement light to the high numerical aperture area of the edge of the objective lens, irradiates the sample with a large illumination angle including the total reflection area, and returns the reflected light from the sample boundary surface through the objective lens Is imaged on the optical sensor for position measurement, and the total reflected light at the sample boundary surface and the reflected light from a large illumination angle are used, so that it can be combined with high accuracy even for transparent samples and low reflectance samples. The focus position and sample position are measured, and the position measurement intermediate lens or position measurement imaging lens is moved in conjunction with the designated focus position. The output from the position measurement optical sensor corresponds to the target focus position. As a value, by performing focusing fit between the objective lens and the specimen position, to allow the focusing position measurement over a wide range relates height.

  As a result, the position measurement intermediate lens or the position measurement imaging lens is moved in conjunction with the designated height position for focusing, and the output from the position measurement optical sensor becomes a value corresponding to the target focus height position. As described above, by performing focusing by matching the distance between the objective lens and the sample, it is possible to measure the focus position in a wide range with respect to the height position. In the method according to claim 8, the movement of the intermediate lens is performed for changing the focus position reference point and is not necessarily linked to the focusing. However, in the method according to claim 9, the intermediate lens or the imaging is performed. The feature is that the lens position is always linked to the designated height position for focusing.

  In this way, even if the sample observation position changes at a height different from the sample boundary surface, the position measurement sensor and the sample observation surface are always kept in the same conjugate positional relationship, and the optical path of the position measurement light Therefore, it is possible to avoid the optical path obstruction due to the large fluctuation of the optical path, and it is possible to measure the focus position in a wider range with respect to the height position of the sample observation.

  As described above, the sample in-focus position high-precision measurement method of the present invention is not damaged even for specimens that are vulnerable to light irradiation or the like, such as biological samples, in position measurement using a microscope or an objective lens, The in-focus position and the sample position can be measured with a high accuracy of 100 nm or more even in a transparent sample or the like with a low reflectance. Further, by combining with a focus driving mechanism, automatic focusing (autofocus) and sample position control can be performed. Also, when observing fluorescent images, light with a wavelength different from the wavelength used for fluorescence observation is used for position measurement, and fluorescence is not transmitted through extra dichroic mirrors for measurement, resulting in loss of fluorescence. Absent.

  The present invention uses light of a wavelength not used for fluorescent image observation for position measurement, introduces the position measurement light from a light source onto the optical path of a microscope tube using a fluorescent dichroic mirror, and an objective lens The position measurement light is passed through only the high numerical aperture area, and the reflected light returning from the sample boundary through the objective lens is imaged on the position measurement optical sensor. Then, using the reflected light from a large illumination angle including the total reflected light on the sample boundary surface, the in-focus position and the sample position are measured with high accuracy and stability without loss even in fluorescence image observation.

Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is an explanatory diagram of the basic principle of high-accuracy measurement of the specimen in-focus position according to the present invention.
In FIG. 1, attention is paid to the relationship between incident light and reflected light from a light source. When the sample boundary surface changes by Δz in the height direction, the reflected light is considered to come out from a position shifted by Δx in the horizontal direction within the sample boundary surface. This Δx is measured by a sample position measuring optical sensor. At this time, the following equation (1) is established between Δz and Δx.

Δx = 2Δz × tan θ (1)
Therefore, by increasing the incident angle θ, it is possible to obtain a larger change amount of Δx for the same change amount of Δz. That is, Δz can be obtained with higher accuracy. Therefore, it is better to pass the incident light from the light source through the high numerical aperture region at the edge of the objective lens. For this reason, it is possible to measure the in-focus position and the sample position with high accuracy of 100 nm or more even in a transparent sample or the like with a low reflectance.

  In addition, when total reflection is used, reflected light with sufficient intensity can be obtained even with weak illumination light, and the amount of light transmitted to the sample side is greatly reduced. However, the in-focus position and the sample position can be measured without causing damage. Therefore, it is necessary to irradiate the sample with a large illumination angle including the total reflection region by passing the incident light from the light source through the high numerical aperture region at the edge portion of the objective lens.

FIG. 5 shows the dependence of the reflected light intensity from the sample boundary surface on the light incident position when an aqueous solution (refractive index: 1.33) is used as the sample and optical glass (refractive index: 1.52) is used as the cover glass. Showing sex results. The upper diagram in FIG. 5 shows the actually measured value of the reflected light intensity at the position measuring sensor 9, and the reflected light becomes stronger when the distance d from the central axis of the incident light to the objective lens is larger than 1.5 mm. Recognize. The lower figure shows the theoretical calculated values of the reflectance corresponding to the upper figure, and is in good agreement with the actually measured values. In this apparatus, since light having a diameter of about 1 mm is used as incident light, the rising of reflected light intensity is observed when the incident position d is slightly smaller than the theoretical value.

  FIG. 5 shows that the reflected light intensity is remarkably strong in the total reflection region exceeding the critical angle, and there is a region where the reflected light is strong before the critical angle even if total reflection does not occur. In the focus position high-precision measurement method of the present invention, the total reflected light and the reflected light from a large illumination angle in a region where the incident position d is large, that is, the incident angle at the boundary surface is large and the reflected light intensity is large. Used.

  Note that reflected light is seen near the origin where the incident position d in the upper diagram of FIG. 5 is 0.3 mm or less. This is mostly due to reflection from the vicinity of the center of the objective lens or from the sample boundary surface, and is measured by the conventional method. Gives a non-negligible error. In the present invention, this region where the incident position d is small is not used for position measurement and is not affected at all by this problem.

  With reference to FIG. 2, the operation of the configuration for realizing the sample focus position high-accuracy measurement method of the present description to which this measurement principle is applied will be described. FIG. 2 shows an example in which a near-infrared narrow-directional LED or a low-coherence infrared light-emitting element is used as a light source, and a two-division photodiode is used as a position measurement sensor.

    In FIG. 2, 1 is a sample to be observed, 2 is a light source and slit which is a near-infrared narrow directional LED, 3 is a condenser lens for illumination, 4 is a common dichroic mirror for fluorescence and position measurement, 5 is an objective lens, 6 is an immersion liquid, 7 is a cover glass, 8 is an imaging lens, 9 is a photosensor for position measurement using a two-part photodiode, 10 is a dichroic mirror for position measurement, and 11 is a mirror. .

  The objective lens is a high numerical aperture 100 × oil immersion objective lens whose numerical aperture exceeds the refractive index of the sample. The sample 1 is disposed on a cover glass (glass substrate) 7 placed on a stage (not shown) of the microscope main body located above the objective lens 5 with an immersion liquid 6 interposed. In this case, the sample 1 is positioned at the focal position of the objective lens 5 through the immersion liquid 6 and the cover glass (glass substrate) 7. The objective lens can be used without being limited to 100 times or an immersion liquid type, and a larger numerical aperture provides a more accurate result. Although FIG. 2 is drawn with an inverted microscope, the present invention can be applied to both an upright type and an inverted type.

  The illumination light that has passed through the slit from the illumination light source for position measurement passes through the condenser lens 3 for illumination, is reflected by the mirror 11, is reflected by the dichroic mirror 10, and is the same optical path as the illumination light for observation such as fluorescence excitation light. The sample is bent at a right angle by the dichroic mirror 4 and introduced into the optical path in the microscope tube, and the light passes through the high numerical aperture region of the edge portion of the objective lens 5 at a large illumination angle including the total reflection region. 1 is irradiated. The mirror 11 is for separating the illumination light and the reflected light for position measurement. The mirror 11 is not disposed after the condenser lens 3 but is disposed in front of the imaging lens, and is used by bending the reflected light side. Is also possible.

  The reflected light returning from the surface of the sample 1 through the objective lens 5 is bent at a right angle by the dichroic mirror 4 and imaged on the position measuring optical sensor 9 through the imaging lens 8. The imaging lens 8 is arranged so that the sample 1 and the position measuring optical sensor 9 are in a conjugate position. If the condensing lens 3 is arranged so that the slit 2 of the light source, the sample 1, and the position measuring optical sensor 9 are in a conjugate position, a better measurement result can be obtained. Here, based on (1) described with reference to FIG. 1, it is possible to obtain a larger change amount of the lateral deviation Δx in the sample boundary surface with respect to the change amount Δz in the height direction of the sample boundary surface, Δz can be obtained with higher accuracy. In this way, by using the totally reflected light on the surface of the sample 1 and the reflected light from a large illumination angle, the in-focus position and the sample position can be measured with high accuracy even in a sample having a low reflectance such as a transparent sample. can do.

  Moreover, when laser light is used for the light source 1 as illumination light, interference occurs, causing noise and drift in position measurement, resulting in poor measurement accuracy. In this case, using the in-coherent light source or the incoherent light source that has been made incoherent and low incoherent to perform the above-mentioned sample focus position high-precision measurement method, the position is more stable and accurate. Measurement can be performed.

  Further, by obtaining the output from the position measuring optical sensor as a function of the change Δz of the sample position, the sample position Δz can be obtained from the optical sensor output. If the optical sensor position is placed at a position conjugate with the sample boundary surface when the observation focus is set on the sample boundary surface, the sample position can be obtained with reference to the sample boundary surface. By using an optical sensor arranged in a straight line such as a linear multi-division photodiode or photodiode array as a sensor, the function can be expanded in the Δz direction by connecting outputs from a plurality of sensors. It is possible to measure the range. The measurable range of Δz by this method is a measure of the visual field of the objective lens with respect to Δx that is related to Δz in equation (1).

  As shown in the control flow of FIG. 3, by using the position measurement photosensor 2-division photodiode, the output of the 2-division photodiode of the position measurement photosensor 9 is differentially amplified by the in-focus position measurement device 14, and a sample is obtained. Used as a position signal. In a linear optical sensor such as a multi-division photodiode, a sample position is calculated after amplification of a plurality of optical sensor outputs by the focusing position measuring instrument 14 and used as a sample position signal. This is used for the control signal of the focus driving mechanism 16 incorporated in the microscope 15 capable of fluorescence observation using the thin layer oblique illumination method and the total reflection illumination method, and the automatic focusing is performed, the sample position is controlled, and the objective lens is passed. Thus, the present invention can be applied to illumination light control such as a thin layer oblique illumination method and a total reflection illumination method for performing illumination.

  When the boundary surface of the sample 1 is observed, the position measuring optical sensor 9 is placed at a position conjugate with the sample boundary surface at the time of focusing. When observing a position different from the boundary surface, the position measurement optical sensor 9 is placed at a position conjugate with the sample boundary surface at the observation position, or a position conjugate with the position where the position measurement optical center intersects the optical axis. In this case, position measurement can be performed based on the observation position. That is, the position of the imaging lens 8 or the optical sensor 9 is moved in the optical axis direction, and the position of the optical sensor 9 is shifted from a position conjugate with the sample surface. Alternatively, the reference position can be changed by moving the imaging lens or the optical sensor position in a direction perpendicular to the optical axis. When observing a position different from the sample boundary surface, the optical path of the position measurement light changes, so that a good result can be obtained by adjusting the angle of the light from the position measurement light source. Thereby, even in a biological sample or a transparent sample with a low reflectance, the in-focus position and the sample position can be measured with high accuracy of 100 nm or more.

  Further, as in the example shown in FIG. 7, when the condenser lens 3 on the light source side and the imaging lens 8 are made common and configured by one imaging lens 8, the position of the imaging lens 8 is changed. Thus, the position of the optical sensor 9 is optically shifted from the position conjugate with the sample observation surface, and the sample position and the in-focus position can be obtained using a position different from the sample boundary surface as a reference point.

  Further, in the case of using the intermediate lens group, the conjugate positional relationship between the position measuring optical sensor and the sample side is not changed depending on the presence or absence of the intermediate lens group. As an example in the case of an infinitely corrected optical microscope, as shown in FIG. 8, the light emitted from the observation position of the sample becomes parallel light after passing through the objective lens 5, but after being imaged by the intermediate lens 12, The intermediate lens 13 returns to parallel light.

  When the focal lengths of the objective lens, the intermediate lens 12 and the intermediate lens 13 are fobj, f12 and f13, the width of the parallel light downstream of the intermediate lens 13, that is, the illumination light and the reflection on the imaging lens 8 side of the intermediate lens 13 are reflected. The light interval is proportional to fobj × f13 / f12. Therefore, even when an objective lens having a different magnification, that is, an objective lens having a different focal length foj is used, if the combination of f12 and f13 is selected so that foj × f13 / f12 is the same, as shown in FIG. The difference in the optical path on the side can be absorbed by the intermediate lens group, so that the optical path on the light source 2 side and on the imaging lens 8 side and the optical sensor 9 side can remain the same.

  Further, when a position different from the sample boundary surface is used as a reference point and a different height position on the sample is obtained as an in-focus position, the optical sensor 9 is optically moved by moving a part of the intermediate lens group. In particular, the position can be shifted from the position conjugate with the sample observation surface and the light source 2 side, the imaging lens 8 and the optical sensor 9 side can be changed without any change. The operation will be described with reference to the example of FIG. 10. By changing the sample height position, the position of the image by the intermediate lens 12, that is, the position where the light intersects between the intermediate lens 12 and the intermediate lens 13 is shifted. If one of the intermediate lenses is moved by the same distance as the amount of change in the image position and the distance between the intermediate lens 12 and the intermediate lens 13 is changed, the light source 2 and the imaging lens 8 side than the intermediate lens 13 The optical path is the same as before the change in the sample height position.

  When the sample height position is changed, the optical path on the objective lens side changes, which limits the range in which the in-focus position can be measured, but the difference in the optical path on the objective lens side is absorbed by the movement of the intermediate lens. Since the optical path of the reflected light can be kept symmetrical with respect to the optical axis, the focus position can be measured in a wide range with respect to the height position of the sample observation.

  In addition, if the intermediate lens for position measurement or the imaging lens for position measurement is moved in conjunction with the specified height position for focusing, the focus position can be measured in a wider range with respect to the height position for sample observation. Is possible. In the case of the example of FIG. 10, the distance between the intermediate lens 12 and the intermediate lens 13 is changed in conjunction with the height position in the case of the example of FIG. The relationship between the height position of the sample observation and the position of the intermediate lens or the imaging lens when the output from the position measuring optical sensor is set to 0 or a constant value is obtained in advance.

  As shown in the control flow of FIG. 11, in response to designation of the observation height position by the focusing ring of the microscope or the control software, the height position of the intermediate lens or the imaging lens is determined according to the relationship obtained in advance. Move in conjunction with the position corresponding to the height of observation. The focus drive mechanism of the microscope is fed back and focused so that the output from the position measuring optical sensor becomes 0 or a constant value. In this way, even if the sample observation position changes at a height different from the sample boundary surface, the optical sensor for position measurement and the sample boundary surface are always kept in the same conjugate positional relationship, and the optical path is always set to the optical axis. Therefore, it is possible to minimize the fluctuation of the optical path, so that the optical path obstruction due to the large fluctuation of the optical path can be avoided, and the focus position can be measured in a wider range with respect to the height position.

The focus position measurement depends on the magnification of the image on the position measurement optical sensor. If the imaging magnification is high, the resolution is high, but the measurable height position range is narrow. If the focal length of the imaging lens 8 is image, the imaging magnification is
Imaging magnification = (image / fobj) × (f12 / f13) (2)
Given in. Therefore, the focal lengths of the objective lens, intermediate lens, and imaging lens are selected and used according to the required resolution and measurement range.

  The size of the illumination range of the position measurement light is related to the resolution and the measurable height position range and is defined by the slit 2 of the light source. Assuming that the diameter of the slit 2 of the light source is Rslit and the focal length of the condenser lens 3 is f3, the diameter of the slit image in the sample 1, that is, the diameter Rillum of the illumination range of the position measurement light is Rslit × (fobj / f3) X (f12 / f13), and the diameter of the slit image at the sensor 9, that is, the diameter Rimage of the position measurement optical image is given by Rillum × (image / fobj) × (f13 / f12).

Therefore, the diameter Rimage of the position measurement optical image at the sensor 9 is
Rage = Rslit × (image / f3) (3)
It is. Since there is actually diffraction and image blur, the diameter of the slit image given by these equations is not strict. The diameter of the slit 2 of the light source and the focal length f3 of the condenser lens 3 are selected and used according to the required resolution and measurement range.

  In addition, near-infrared light that is weak in light irradiation damage to living biological samples is used as a light source for position measurement, and there is almost no damage to living biological samples that are vulnerable to light irradiation. You can know the position. The apparatus of the present invention can be used not only for various optical microscopes but also for scanning probe microscopes or measuring devices using light.

  The sample focusing position high-accuracy measuring method according to the first embodiment of the present invention includes a low-coherence high-intensity infrared light-emitting element having a wavelength of 820 to 850 nm as the light source 2 and a right-angle prism as the mirror 3 in the apparatus shown in FIG. The position measuring dichroic mirror 10 reflects near infrared light having a wavelength of 800 nm or more and transmits visible light, the objective lens 5 is an oil immersion 100 × objective lens having a numerical aperture of 1.45, and the imaging lens 8 has a focal length of 30 mm. The lens and the position measuring sensor 9 are composed of two-divided Si photodiodes. As the dichroic mirror 4, a fluorescent observation dichroic mirror that reflects near-infrared light having a wavelength of 800 nm or more is used for each fluorescent dye. For example, as the dichroic mirror 4 for observing GFP, which is a green fluorescent protein, a mirror that reflects fluorescence excitation light of 488 nm, transmits visible light of 500 nm to 700 nm, and reflects near infrared light of 800 nm or more is used.

  FIG. 6 shows the accuracy of the measurement result by the sample focusing position high-precision measurement method of the present invention and the measurement result of the stability with respect to the sample temperature. When cold water was added to the sample, the sample temperature suddenly cooled to 34.5 ° C and then recovered to the 36 ° C level. Along with this, the sample height position was measured up to 5 micrometers due to temperature drift. Changed. However, with this apparatus, the height position of the sample can be measured with high accuracy within ± 30 nm (0.03 micrometers) at any time except for the moment immediately after cooling, and the in-focus position can be maintained. .

  The sample focusing position high-accuracy measuring method of the second embodiment of the present invention uses the lens having a focal length of 15 mm as the imaging lens 8 in the apparatus shown in FIG. . When the focal length of the imaging lens 8 is 15 mm, the magnification of the image on the position measuring sensor 9 is halved compared to the case where the focal length is 30 mm. As a result, the accuracy of in-focus position measurement is reduced to about half, but the range of height positions that can be measured is widened.

  The sample focusing position high-accuracy measuring method of the third embodiment of the present invention is the same as that of the first embodiment except that a linear 8-divided photodiode is used as the position measuring sensor 9 in the apparatus shown in FIG. It is. When observing different height positions on the sample, by arranging the divided photodiodes in the direction in which the reflected light changes, the height position can be measured in a wider range. The range in which Δz can be measured by this method is a measure of the field of view of the objective lens with respect to Δx given by Δx = 2Δz × tan θ in equation (1).

  The sample focus position high-accuracy measuring method of the fourth embodiment of the present invention is constituted by the apparatus shown in FIG. The imaging lens 8 of the first embodiment (FIG. 2) and the condenser lens 3 on the light source side are made common and constituted by one imaging lens 8. The optical paths on both the light source 2 side and the position measuring sensor 9 side are arranged at angles different from the right angle. However, when the height position of the sample is changed, the change in the optical path becomes symmetric with respect to the optical axis, and the height position The measurement range is widened. By changing the imaging lens 8 in the optical axis direction, the position measuring optical sensor position can be shifted from a position conjugate with the sample observation surface, and a different height position on the sample can be obtained as the in-focus position. .

  The sample focusing position high-accuracy measuring method of the fifth embodiment of the present invention is composed of the apparatus shown in FIGS. An intermediate lens 12 and an intermediate lens 13 are added to the apparatus of the first embodiment (FIG. 2). The light emitted from the observation position of the sample becomes parallel light after passing through the objective lens, but after being imaged by the intermediate lens 12, the light is returned to parallel light by the intermediate lens 13.

  In the case of using different objective lenses, it is possible to easily cope with other optical systems without making any changes only by changing the combination of the intermediate lenses. When a 100 × objective lens is used as in the apparatus shown in FIG. 8, a lens with a focal length of 30 mm is used as the intermediate lens 12 and a lens with a focal length of 30 mm is used as the intermediate lens 13.

  When a 60 × objective lens is used as in the apparatus shown in FIG. 9, a lens having a focal length of 30 mm is used as the intermediate lens 12 and a focal length of 30 × 60/100 = 18 mm is used as the intermediate lens 13. The focal lengths of the objective lens, the intermediate lens 12, and the intermediate lens 13 are fobj, f14, and f15, and other combinations are possible as long as fobj × f13 / f12 are the same.

  In this way, the difference in the optical path due to the difference in the objective lens can be absorbed by the intermediate lens group, and the measurement is performed while the light source 2 side, the imaging lens 8 and the position measurement sensor 9 side are exactly the same. be able to.

  The sample focusing position high-accuracy measuring method of the sixth embodiment of the present invention is constituted by the apparatus shown in FIG. An intermediate lens 12 and an intermediate lens 13 are added to the apparatus of the first embodiment (FIG. 2). The light emitted from the observation position of the sample becomes parallel light after passing through the objective lens, but after being imaged by the intermediate lens 12, the light is returned to parallel light by the intermediate lens 13.

  When observing different height positions on the sample, by changing the distance between the intermediate lenses 12 and 15, the position measuring optical sensor position can be shifted from a position conjugate with the sample observation surface. Different height positions can be obtained as in-focus positions. Although the position of the intermediate lens 13 is changed in FIG. 10, the position of the intermediate lens 12 may be changed. When the height position of the sample observation is changed, the optical path on the objective lens side changes, and this limits the range in which the focus position can be measured, but the difference in the optical path on the objective lens side is absorbed by the movement of the intermediate lens, and the light source 2 Since the optical path on the side and the imaging lens 8 and the position measuring sensor 9 side can be kept the same, the focus position can be measured in a wider range with respect to the height position of the sample observation.

The sample focusing position high-accuracy measuring method of the seventh embodiment of the present invention includes the apparatus shown in FIG. 10 of the sixth embodiment and the focusing mechanism shown in FIG.
The position of the intermediate lens 12 or the intermediate lens 13 is changed in conjunction with the designation of the observation height position. The relationship between the observation height position and the position of the intermediate lens 12 or the intermediate lens 13 when the output from the position measuring sensor 9 is set to 0 or a constant value is obtained in advance. As shown in the control flow of FIG. 11, the intermediate lens 12 or the intermediate lens 13 is subjected to intermediate / image formation according to a predetermined relationship with respect to the observation height position designation 17 by the focusing ring of the microscope or the control software. The lens driving mechanism 18 moves in conjunction with the corresponding position of the observation height position. The output from the position measuring optical sensor 9 is fed back to the focus driving mechanism 16 of the microscope so that the output becomes 0 or a constant value, and is brought into focus.

  When observing different positions in the sample depth direction with a microscope, the distance between the sample and the objective lens is changed. Therefore, considering the height position of sample observation and the distance between the sample and the objective lens is a method that has been consistently performed in the microscopic method. However, with this conventional method, the distance between the sample and the objective lens is deviated between the intended value and the actual value due to thermal or mechanical factors. As a result, the sample is observed at the position.

  On the other hand, in the method of this embodiment, the change of the height position of the sample observation is made to respond by changing the position of the position measuring intermediate lens or the imaging lens. Thereafter, focusing is performed by changing the distance between the sample and the objective lens so that the output from the position measuring sensor becomes a value corresponding to the target focal position.

  In this way, the position measurement sensor and the sample observation surface are always kept in the same conjugate positional relationship, and the optical path variation of the position measurement light can always be minimized. Obstacles can be avoided, and the focus position can be measured in a wider range with respect to the height position of sample observation. As a result, it is possible to perform observation within a wider range of height positions without deviation from the designated observation height position.

  The present invention can be applied to both an upright microscope and an inverted microscope. In addition to various optical microscopes, the apparatus of the present invention can be used for a scanning probe microscope or a measuring device using light.

Explanatory drawing of the basic principle of the sample focus position high precision measurement of this invention. The structure which implement | achieves the sample focus position highly accurate measurement method of this invention. Control flow of the focus drive mechanism. The schematic diagram which measures the depth (height) direction position of the conventional sample. Data on the light incident position dependency of the reflected light intensity from the sample boundary surface to the objective lens. Data on the accuracy of the sample focus position high-precision measurement method. The structure which implement | achieves the sample focus position highly accurate measuring method of Example 4. FIG. The structure which implement | achieves the sample focus position highly accurate measuring method of Example 5. FIG. The structure which implement | achieves the sample focus position highly accurate measuring method of Example 5. FIG. The structure which implement | achieves the sample focus position highly accurate measuring method of Example 6. FIG. 10 is a control flow of the focusing mechanism described in the seventh embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample to be observed 2 Light source and slit 3 Condensing lens for illumination 4 Dichroic mirror 5 Objective lens 6 Immersion liquid 7 Cover glass 8 Imaging lens 9 Optical sensor for position measurement 10 Dichroic mirror for position measurement 11 Mirror 12 Intermediate Lens 13 Intermediate lens 14 In-focus position measuring instrument 15 Microscope 16 Focus drive mechanism 17 Observation height position designation by focusing ring / control software 18 Intermediate / imaging lens drive mechanism

Claims (9)

  In a microscope and position measurement capable of observing a fluorescent image, light of a wavelength not used for fluorescent image observation is used for position measurement, and the light for position measurement is used as a light source on the optical path of the microscope tube using a dichroic mirror for fluorescence. Incorporating into the method, a sample focus position high-precision measurement method that enables measurement of the focus position and the sample height position without loss of fluorescence even when a fluorescent image is observed.   In a microscope and position measurement capable of observing a fluorescent image, light is passed from the light source to the high numerical aperture area at the edge of the objective lens, the sample is irradiated at a large illumination angle including the total reflection area, and the objective lens is passed through the sample boundary surface. The reflected light that returns is imaged on the position measurement optical sensor, and by using the total reflected light on the sample boundary surface and the reflected light from a large illumination angle, even in a transparent sample or the like with a low reflectance sample 2. The sample focusing position high-accuracy measuring method according to claim 1, wherein the in-focus position and the sample height position can be measured with high accuracy without loss even in fluorescence image observation.   3. The position measurement is performed more stably and with high accuracy by using a non-coherent (incoherent) light source or a coherent light source made incoherent and low-coherent as the light source. The method for measuring the in-focus position of the sample as described in 1.   By obtaining the output from the optical sensor for position measurement as a function of the sample height position, the sample position is measured from the optical sensor, and a wider range of samples are obtained using the linearly arranged optical sensor. 3. The method for measuring the in-focus position of the sample according to claim 2, wherein the height position can be measured.   The position measuring optical sensor position is moved in the optical axis direction to shift the position measuring optical sensor position from a position conjugate with the sample observation surface, or the imaging lens or the optical sensor position. The sample focus position high-accuracy measuring method according to claim 2, wherein the sample height position is obtained by moving the lens in a direction perpendicular to the optical axis, using different height positions on the sample as a reference position.   By combining with a focus drive mechanism, it can be applied to illumination light control such as autofocusing, sample position control, thin-layer oblique illumination method or total reflection illumination method that performs illumination via an objective lens The sample focusing position high-precision measurement method according to claim 2.   An intermediate lens group is provided between the objective lens and the position measurement imaging lens, and the intermediate lens group can be arranged without changing the conjugate relationship between the sample and the position measurement sensor. The method for accurately measuring the in-focus position of the sample according to claim 2.   There is an intermediate lens group between the objective lens and the imaging lens for position measurement, and by moving the position of a part of the intermediate lens group, the sample height is set with a different height position on the sample as a reference position. 3. The method for measuring a focused position of a sample according to claim 2, wherein the position can be obtained. The position measurement intermediate lens or the position measurement imaging lens is moved in conjunction with the specified height position for focusing so that the output from the position measurement optical sensor becomes a value corresponding to the target focus height position. 3. The method for measuring the in-focus position of the sample according to claim 2, wherein the in-focus position can be measured in a wide range with respect to the height position by performing focusing by adjusting the distance between the objective lens and the sample. .

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