JP2012141452A - Automatic focus mechanism and microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To focus on a specimen with accuracy.SOLUTION: An automatic focus mechanism 10 includes: an illumination optical system 11 for collecting illumination light projected onto a specimen A with a cover glass 5 in a line shape; an objective lens 13 for projecting the illumination light collected in a line shape onto the specimen A and collecting reflection light from the cover glass 5; a detection unit 15 having an imaging surface 36 disposed obliquely with respect to an optical axis of the reflection light collected by the objective lens 13 and for photographing the reflection light and detecting image information; and a focus adjustment unit 17 for relatively moving the objective lens 13 and the specimen A with the cover glass 5 in the optical axis direction so that the highest position of light intensity in the image information detected by the detection unit 15 is positioned on the optical axis of the reflection light.

Description

  The present invention relates to an automatic focusing mechanism and a microscope apparatus.

  2. Description of the Related Art Conventionally, a virtual slide microscope apparatus is known that obtains a digital image of a specimen and displays it on a monitor by changing its magnification and observation position to simulate a microscope (see, for example, Patent Document 1). ). According to the virtual slide microscope apparatus, a plurality of images are acquired while moving the stage in a direction orthogonal to the optical axis of the objective lens, and these images are connected to each other to observe the microscope apparatus like a slide glass. A specimen larger than the range can be observed, but there is a problem that the image quality deteriorates due to the focus position of the objective lens being shifted due to the focus drift due to the environmental change around the microscope, the inclination of the specimen itself, or the like. The microscope apparatus described in Patent Document 1 acquires an image of a specimen by a camera arranged to be inclined with respect to the optical axis of the objective lens, and controls the focal position of the objective lens based on the analysis result of the image. .

International Publication No. 2005/114293

  However, since the microscope apparatus described in Patent Document 1 detects a position where the contrast of the specimen image is high as a focal position of the objective lens by image processing, the focal position of the objective lens is determined in a portion outside the transparent specimen or specimen. There is a problem that it cannot be detected.

  The present invention has been made in view of such circumstances, and it is an object of the present invention to provide an automatic focusing mechanism and a microscope apparatus that can accurately focus on a specimen.

In order to solve the above problems, the present invention employs the following means.
The present invention provides an illumination optical system for condensing illumination light applied to a specimen with a cover glass in a line shape, and irradiates the specimen with the illumination light condensed in the line shape, and reflects the light from the cover glass. An objective lens that condenses light and an imaging surface that is arranged to be inclined with respect to the optical axis of the reflected light collected by the objective lens, and detects image information by photographing the reflected light The objective lens and the sample with the cover glass are relative to each other in the optical axis direction so that the detection unit and the position with the highest light intensity in the image information detected by the detection unit are located on the optical axis. Provided is an automatic focusing mechanism including a focusing adjustment unit to be moved.

  According to the present invention, when illumination light is irradiated onto the specimen with the cover glass by the objective lens, the reflected light from the cover glass is photographed by the imaging surface of the detection unit. In this case, by disposing the imaging surface with respect to the optical axis of the reflected light from the cover glass irradiated with the linear illumination light, the distribution of the reflected light incident on the imaging surface is It will change along the slope.

  That is, in the in-focus state, the condensing position on the optical axis of the reflected light beam coincides with the imaging surface, and is shifted from the condensing position and enters the imaging surface as the distance from the optical axis increases. Accordingly, the light intensity in the image information is highest on the optical axis of the reflected light, and the light intensity decreases as the distance from the optical axis increases. Further, when the focal position of the objective lens fluctuates in the optical axis direction, the condensing position away from the optical axis in the reflected light beam coincides with the imaging surface. Therefore, the position with the highest light intensity in the image information is shifted from the optical axis of the reflected light.

  In the present invention, the focus adjustment unit relatively moves the objective lens and the sample with the cover glass in the optical axis direction so that the position with the highest light intensity in the image information coincides with the optical axis of the reflected light. Thus, it is possible to accurately focus on the specimen.

  The present invention provides an illumination optical system for condensing illumination light applied to a specimen with a cover glass in a line shape, and irradiates the specimen with the illumination light condensed in the line shape, and reflects the light from the cover glass. An objective lens that condenses light and an imaging surface that is arranged to be inclined with respect to the optical axis of the reflected light collected by the objective lens, and detects image information by photographing the reflected light The objective lens and the specimen with the cover glass are placed on the optical axis so that the detection unit and the position with the highest light intensity in the image information detected by the detection unit are separated from the optical axis by a predetermined distance. An automatic focusing mechanism is provided that includes a focusing adjustment unit that relatively moves in a direction.

  According to the present invention, the objective lens and the sample with the cover glass are moved in the optical axis direction so that the position where the light intensity in the image information is highest is separated from the optical axis by a predetermined distance by the operation of the focus adjustment unit. By moving the lens relative to each other, the in-focus position can be adjusted to a part of the sample located at a position away from the cover glass in the optical axis direction, such as a deep part of the sample.

  In the above invention, the imaging surface of the detection unit may be inclined around the minor axis of the light beam of the reflected light.

  With this configuration, since the optical path length of the reflected light incident on the imaging surface of the detection unit differs along the line direction of the light beam, if the focal position of the objective lens varies in the optical axis direction, the light in the image information The position with the highest intensity moves in the line direction of the reflected light beam. Therefore, it is possible to easily focus on the specimen only by matching the position of the highest light intensity in the image information with the optical axis of the reflected light in the line direction.

Moreover, in the said invention, it is good also as the inclination of the said imaging surface with respect to the optical axis of the said reflected light satisfy | filling the following formula | equation.
{(P × tan θ) / β ² } <{(1.2 × λ) / NA ² }
Where p: length of the light receiving element constituting the imaging surface (μm)
θ: inclination of the imaging surface with respect to the optical axis of the reflected light (rad)
β: Imaging magnification of the entire device (times)
NA: Numerical aperture of objective lens λ: Wavelength of light source (μm)

By making the resolution ((p × tan θ) / β ² (μm)) of the reflected light in the detection unit in the optical axis direction smaller than the focal depth ((1.2 × λ) / NA ² ) of the objective lens, The focal position of the lens can be adjusted with high accuracy.

Moreover, in the said invention, the said focus adjustment part is good also as setting the relative movement amount of the said objective lens and the said specimen with the following formula | equation.
(A × p × tan θ) / β ²
Here, a: the number of pixels from the center of the reflected light beam incident on the imaging surface to the center pixel on the imaging surface

  The actual focal position deviation amount on the specimen with the cover glass is the focal deviation amount (a × p × tan θ) of the reflected light on the imaging surface, which is 2 of the imaging magnification (β) of the entire apparatus. The value divided by the power. Therefore, with this configuration, the specimen can be focused with a small number of adjustments.

  In the above invention, an inclination adjusting unit may be provided that adjusts the inclination of the specimen with the cover glass so that the inclination of the imaging surface with respect to the specimen with the cover glass becomes a constant angle.

  When the inclination of the sample with the cover glass with respect to the imaging surface of the detection unit becomes small, the change in the distribution of reflected light incident on the imaging surface becomes small. That is, the difference in light intensity in the image information is reduced. On the other hand, when the inclination of the sample with the cover glass with respect to the imaging surface increases, the change in the distribution of reflected light incident on the image surface increases. That is, the difference in light intensity in the image information increases. Therefore, with this configuration, it is possible to acquire image information of a desired reflected light distribution by the operation of the tilt adjustment unit.

  The present invention provides an image of the specimen based on any one of the automatic focusing mechanisms described above, a stage on which the specimen is mounted and movable in the optical axis direction, and the image information acquired by the detection unit. A microscope apparatus including an image acquisition unit to acquire is provided.

  According to the present invention, it is possible to acquire an image of a specimen in a state of being accurately focused.

  The automatic focusing mechanism according to the present invention produces an effect that the specimen can be focused with high accuracy. In addition, according to the microscope apparatus of the present invention, there is an effect that an image of a specimen can be acquired in a state of being accurately focused.

(A) is a figure which shows a part of microscope apparatus which concerns on one Embodiment of this invention, (b) is the figure which looked at the microscope apparatus containing (a) from the direction with the power of a cylindrical lens. (A) shows a state in which the center of the reflected light beam in the line direction is incident on the imaging surface at the time of focusing, (b) shows a state in which one end side of the reflected light beam in the line direction is incident on the imaging surface, (C) is a figure which shows a mode that the other end side of the line direction in the light beam of reflected light injects into an imaging surface. It is a figure which shows the reflected light distribution on the imaging surface at the time of focusing. (A) shows a mode that illumination light condenses in the near side of a cover glass, (b) has shown a mode that reflected light is reflected from a cover glass at the time of (a). It is a figure which shows the reflected light distribution on an imaging surface in case the focus position of illumination light is in front of a cover glass. (A) When the focal position of the objective lens is on the near side of the cover glass, the center of the reflected light beam in the line direction is incident on the imaging surface, and (b) shows the line in the reflected light beam. FIG. 6C is a diagram illustrating a state in which one end side in the direction is incident on the imaging surface, and FIG. 8C is a diagram illustrating a state in which the other end side in the line direction in the reflected light flux is incident on the imaging surface. (A) shows a mode that illumination light condenses on the depth side of a cover glass, (b) has shown a mode that reflected light is reflected from the cover glass at the time of (a). It is a figure which shows the reflected light distribution on an imaging surface in case the focus position of illumination light exists in the depth side of a cover glass. (A) shows how the center in the line direction of the reflected light beam is incident on the imaging surface when the focal position of the objective lens is on the depth side of the cover glass, and (b) shows the reflected light beam in the same manner. FIG. 5C is a diagram illustrating a state in which one end side in the line direction is incident on the imaging surface, and FIG. 8C is a diagram illustrating a state in which the other end side in the line direction in the reflected light beam is incident on the imaging surface. It is a flowchart which shows the process of focusing on a sample by the automatic focusing mechanism which concerns on one Embodiment of this invention. It is a figure which shows the imaging surface comprised by two light receiving elements arrange | positioned along with the line direction of reflected light. (A) shows a mode that illumination light is condensed on the depth side of a cover glass, (b) is a figure which shows the reflected light distribution on the imaging surface in the case of (a). (A) shows a mode that illumination light is condensed on the surface of a cover glass, (b) is a figure which shows the reflected light distribution on the imaging surface in the case of (a). It is a figure which shows the reflected light distribution on an imaging surface when the inclination of the imaging surface with respect to the cover glass of the microscope apparatus which concerns on the 1st modification of one Embodiment of this invention is small. It is a figure which shows the reflected light distribution on an imaging surface when the inclination of the imaging surface with respect to the cover glass of the microscope apparatus which concerns on the 1st modification of one Embodiment of this invention is large. (A) is a figure which shows a part of microscope apparatus which concerns on the 2nd modification of one Embodiment of this invention, (b) looks at the microscope apparatus containing (a) from the direction with the power of a cylindrical lens. It is a figure. It is a figure which shows the reflected light distribution on the imaging surface at the time of focusing with the microscope apparatus of FIG. (A) shows a state in which the center of the reflected light beam in the line direction is incident on the imaging surface, and (b) shows a state in which one end side of the reflected light beam in the line direction is incident on the imaging surface. (C) is a figure which shows a mode that it similarly injects into the other end side imaging surface of the line direction in the light beam of reflected light. (A) shows a state in which the center in the line direction in the luminous flux of reflected light is incident on the imaging surface when the focal position of the objective lens is on the near side of the cover glass, and (b) shows the case of (a). It is a figure which shows the reflected light distribution on an imaging surface. (A) shows a state in which the center in the line direction in the luminous flux of reflected light is incident on the imaging surface when the focal position of the objective lens is on the depth side of the cover glass, and (b) shows the case of (a). It is a figure which shows the reflected light distribution on an imaging surface. It is a figure which shows the imaging surface comprised by two light receiving elements arrange | positioned along with the direction orthogonal to the line direction of reflected light. (A) shows a state in which the cover glass is tilted with respect to the optical axis of the illumination light, and (b) is a diagram showing a reflected light distribution on the imaging surface at (a).

An automatic focusing mechanism and a microscope apparatus according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 1A and 1B, the microscope apparatus 100 according to the present embodiment adjusts the focus of the specimen A, the stage 1 on which the specimen A is placed, the light source 3 that emits illumination light. The automatic focusing mechanism 10 to perform and the observation optical system 50 which acquires the image of the sample A are provided.
FIG. 1A is a view of a part of FIG. 1B viewed from a direction where the power of the cylindrical lens 23 is not present.

The specimen A is placed on the stage 1 and covered with a thin cover glass 5.
The stage 1 can be moved by the stage drive unit 2 in the direction of the optical axis of the illumination light and the direction orthogonal to the illumination light.
As the light source 3, for example, a mercury lamp, a halogen lamp, a tungsten lamp, a laser light source, an LED, or an LD can be used.

  The automatic focusing mechanism 10 illuminates the specimen A with illumination optical system 11 that condenses the illumination light emitted from the light source 3 in a line shape, and reflects from the cover glass 5. An objective lens 13 that collects light, a detection unit 15 that captures reflected light collected by the objective lens 13 and detects image information, and a focal point of the objective lens 13 based on the image information detected by the detection unit 15 And a focus adjustment unit 17 for adjusting the position.

  The illumination optical system 11 includes a collimating lens 21 that makes the illumination light emitted from the light source 3 substantially parallel light, and a cylindrical lens (linear light condensing element) 23 that condenses the illumination light that has become substantially parallel light in a line shape. And the slit 25 arranged at the condensing position of the cylindrical lens 23, and the illumination light passing through the slit 25 is projected in parallel to the entrance pupil position of the objective lens 13 so that the slit 25 and the specimen A surface are in a conjugate relationship. A relay lens 27, an excitation filter 29 that transmits only light in a predetermined wavelength band, and a dichroic mirror 31 that reflects the illumination light that has passed through the excitation filter 29 and the reflected light collected by the objective lens 13. Yes.

  The slit 25 allows only reflected light from the cover glass 5 to pass through for the return light returning from the specimen A and the cover glass 5. Thereby, the influence of light unnecessary for measurement by diffracted light or the like can be reduced, and the focal position of the objective lens 13 can be adjusted with high accuracy.

  The detector 15 reflects the illumination light emitted from the light source 3 and passed through the collimator lens 21, while transmitting the reflected light returning from the cover glass 5 through the dichroic mirror 31 in the reverse direction to the optical path, and a beam A cylindrical lens 35 that collects the reflected light that has passed through the splitter 33, and a photodetector 37 such as a CCD camera or a CMOS camera that detects image information by photographing the reflected light collected by the cylindrical lens 35; It has.

  The photodetector 37 is arranged at a position conjugate with the specimen A. The photodetector 37 includes an imaging surface 36 (see FIG. 3) including a plurality of (three or more) light receiving elements, and acquires a two-dimensional reflected light image based on the detected image information. ing. A substantially line-shaped reflected light is incident on the imaging surface 36 corresponding to the illumination light collected in a line on the specimen A. The imaging surface 36 is disposed to be inclined around the minor axis of the light beam with respect to the optical axis of the reflected light.

For example, the inclination of the imaging surface 36 with respect to the optical axis of the reflected light satisfies the following formula (1).
{(P × tan θ) / β ² } <{(1.2 × λ) / NA ² } (1)
Preferably, {(p × tanθ) / β 2} <{(1.2 × λ) / NA 2/5}
Here, p: length of the light receiving element constituting the imaging surface 36 (μm)
θ: inclination (rad) of the imaging surface 36 with respect to the optical axis of the reflected light
β: Imaging magnification of the entire automatic focusing mechanism 10 (times)
NA: Numerical aperture of objective lens 13 λ: Wavelength of light source 3 (μm)
When the inclination of the imaging surface 36 satisfies the above formula (1), that is, the resolution {(p × tan θ) / β ² } in the optical axis direction of the reflected light in the light detection unit 15 is set to the focal depth {( By making it smaller than 1.2 × λ) / NA ² }, the focal position of the objective lens 13 can be accurately adjusted.

  The focus adjustment unit 17 matches the sample A with the focal position of the objective lens 13 based on the piezo actuator 41 that moves the objective lens 13 in the optical axis direction and the reflected light image acquired by the photodetector 37. When the focus determination unit 43 determines whether or not the focus determination unit 43 determines that they do not match, the piezo actuator 41 is driven to adjust the position of the objective lens 13 in the optical axis direction. And a piezo drive unit 45.

  When the position with the highest light intensity in the image information is on the optical axis of the reflected light, for example, when the focal point of the reflected light is on the optical axis in the reflected light image, the focusing determination unit 43 When it is determined that the focal position of the lens 13 coincides with the sample A and the position with the highest light intensity in the image information is deviated from the optical axis of the reflected light, for example, the condensing point of the reflected light in the reflected light image. Is deviated from the optical axis, it is determined that the focal position of the objective lens 13 does not coincide with the specimen A.

The piezo drive unit 45 sets the movement amount L of the objective lens 13 in the optical axis direction by, for example, the following formula (2).
L = (a × p × tan θ) / β ² (2)
Here, a: the number of pixels from the center of the light beam of the reflected light incident on the imaging surface 36 (that is, the condensing position of the reflected light in the reflected light image) to the center light receiving element in the imaging surface 36 Actual at the sample position Is the value obtained by dividing the amount of focus shift (a × p × tan θ) in the optical axis direction on the imaging surface 36 by the square of the imaging magnification (β) of the entire automatic focusing mechanism 10. Therefore, by setting the amount of movement L of the objective lens 13 in the optical axis direction according to the above equation (2), the sample A can be focused with a small number of adjustments.

  The observation optical system 50 shares the objective lens 13 and the dichroic mirror 31 with the automatic focusing mechanism 10. The objective lens 13 collects fluorescence generated by irradiating the specimen A with the illumination light and exciting the fluorescent material. Further, the dichroic mirror 31 transmits the fluorescence condensed by the objective lens 13.

  In addition, the observation optical system 50 collects the fluorescence that has passed through the dichroic mirror 31 and forms an image, and captures the fluorescence imaged by the imaging lens 51 to obtain a fluorescence image of the specimen A. And an image acquisition device (image acquisition unit) 53 such as a CCD and an image acquisition control unit (not shown) for controlling image acquisition by the image pickup device 53.

In the imaging device 53, a substantially line-like fluorescent image corresponding to the illumination light condensed in a line on the specimen A is acquired.
The image acquisition control unit operates the stage driving unit 2 to move the stage 1 in a direction orthogonal to the line direction of the illumination light. Thereby, the illumination light is scanned two-dimensionally on the specimen A. The imaging device 53 is configured to store a line-like fluorescent image acquired at each condensing position of illumination light scanned two-dimensionally in association with the position of the stage 1. Thereby, a two-dimensional fluorescence image of the specimen A is acquired.

Next, operations of the automatic focusing mechanism 10 and the microscope apparatus 100 configured as described above will be described below.
First, focusing of the objective lens 13 will be described.
The specimen A is placed on the stage 1, the specimen A is covered with the cover glass 5, and illumination light is generated from the light source 3.

  Illumination light emitted from the light source 3 is made substantially parallel light by the collimating lens 21 and reflected by the beam splitter 33, and then condensed in a line by the cylindrical lens 23. The illumination light collected in a line shape passes through the slit 25 and is relayed in parallel to the entrance pupil position of the objective lens 13 via the relay lens 27, the excitation filter 29, and the dichroic mirror 31, and is sampled by the objective lens 13. A is irradiated.

  The reflected light reflected by the cover glass 5 is collected by the objective lens 13 and reflected by the dichroic mirror 31, and then passes through the beam splitter 33 via the excitation filter 29, the relay lens 27, the slit 25, and the cylindrical lens 23. The light is condensed by the cylindrical lens 35 and photographed by the photodetector 37.

  In this case, since the imaging surface 36 is disposed around the minor axis of the luminous flux with respect to the optical axis of the reflected light collected in a substantially line shape, the reflected light incident on the imaging surface 36 The optical path length varies along the line direction of the light beam. That is, the reflected light distribution on the imaging surface 36 changes along the inclination of the imaging surface 36.

  For example, when the focal position of the objective lens 13 is on the cover glass 5, as shown in FIG. 2A, the condensing position coincides with the imaging surface 36 at the center C in the line direction of the reflected light beam. To do. On the other hand, as shown in FIG. 2B, on the one end L side in the line direction in the light flux of the reflected light, the near side from the condensing position is incident on the imaging surface 36. In addition, as shown in FIG. 2C, the depth side of the light condensing position is incident on the imaging surface 36 on the other end R side in the line direction of the reflected light beam.

  Therefore, as shown in FIG. 3, the reflected light distribution on the imaging surface 36 is the most confined in the vicinity of the approximate center in the line direction, and gradually becomes wider from the middle to the both sides along the line direction. That is, in the reflected light image, the light intensity is highest on the optical axis of the reflected light, and the light intensity gradually decreases with distance from the optical axis. In FIG. 3, hatched portions indicate the distribution of reflected light. The same applies to FIGS. 5, 8, 12 (b), 13 (b), 14, 15, 17, 19 (b), 20 (b), and 22 (b). . Further, in FIG. 3, the symbol C indicates a position corresponding to the symbol C in FIG. 2A, the symbol L indicates a position corresponding to the symbol L in FIG. 2B, and the symbol R indicates the symbol C in FIG. The position corresponding to the symbol R is shown.

On the other hand, when the focal position of the objective lens 13 fluctuates in the optical axis direction, the most narrowed position of the reflected light distribution on the imaging surface 36 changes along the line direction of the reflected light flux.
For example, as shown in FIG. 4A, when the focal position of the objective lens 13 is on the near side of the cover glass 5, the reflected light distribution on the imaging surface 36 is reflected light as shown in FIG. This is the most narrowed shape at a position slightly shifted from the optical axis in the line direction (in FIG. 5, the sign L side). FIG. 4B shows a light flux of reflected light reflected from the cover glass 5.

  For example, as shown in FIG. 6A, at the center C in the line direction in the light flux of the reflected light, the depth side slightly enters the imaging surface 36 from the condensing position. Therefore, the reflected light distribution is slightly broadened. As shown in FIG. 6B, on the one end L side in the line direction in the light flux of the reflected light, a slightly nearer side than the condensing position is incident on the imaging surface 36. Therefore, the reflected light distribution is slightly broadened. As shown in FIG. 6C, on the other end R side in the line direction in the light flux of the reflected light, the depth side is further incident on the imaging surface 36 from the condensing position. Therefore, the reflected light distribution is further expanded. In FIG. 5, a symbol C indicates a position corresponding to the symbol C in FIG. 6A, a symbol L indicates a position corresponding to the symbol L in FIG. 6B, and a symbol R indicates a symbol in FIG. 6C. The position corresponding to R is shown.

  As shown in FIG. 7A, when the focal position of the objective lens 13 is on the depth side of the cover glass 5, the reflected light distribution on the imaging surface 36 is as shown in FIG. 13 is the most narrowed shape at a position shifted in the opposite direction to the case where the focal position of 13 is on the near side of the cover glass 5 (the sign R side in FIG. 8). FIG. 7B shows a light flux of reflected light reflected from the cover glass 5.

  For example, as shown in FIG. 9A, at the center C in the line direction in the light flux of reflected light, a slightly nearer side than the condensing position is incident on the imaging surface 36. Therefore, the reflected light distribution is slightly broadened. As shown in FIG. 9B, on the one end L side in the line direction in the light flux of the reflected light, the nearer side than the condensing position is incident on the imaging surface 36. Therefore, the reflected light distribution is further expanded. As shown in FIG. 9C, on the other end R side in the line direction in the light flux of the reflected light, the depth side slightly enters the imaging surface 36 from the condensing position. Therefore, the reflected light distribution is slightly broadened. In FIG. 8, the symbol C indicates the position corresponding to the symbol C in FIG. 9A, the symbol L indicates the position corresponding to the symbol L in FIG. 9B, and the symbol R indicates the symbol in FIG. 9C. The position corresponding to R is shown.

  As described above, the position where the reflected light distribution is most narrowed on the imaging surface 36, that is, the position where the light intensity is the highest in the reflected light image is the line of the reflected light beam according to the amount of deviation of the focal position of the objective lens 13. Change direction.

In the automatic focusing mechanism 10, focusing adjustment is performed according to the procedure shown in the flowchart of FIG. 10.
First, when the reflected light image is acquired by the detection unit 15 based on the reflected light from the cover glass 5 (step S1), the focusing determination unit 43 collects the reflected light condensing point (light) in the reflected light image. The position of the highest intensity) is detected (step S2).

  When the focusing determination unit 43 determines that the condensing point of the reflected light is on the left side of the optical axis in the reflected light image (step S3 “YES”), the piezo driving unit 45, for example, moves the objective lens 13 The piezo actuator 41 is controlled so as to move in the direction of approaching the stage 1. Thereby, the condensing point of the reflected light in the reflected light image is brought close to the optical axis.

  Subsequently, Steps S1 to S3 are repeated, and when the focus determination unit 43 determines that the condensing point of the reflected light is not on the left side of the optical axis but on the right side (Step S4 “YES”), the piezo drive is performed. For example, the piezo actuator 41 is controlled by the unit 45 so as to move the objective lens 13 away from the stage 1. Thereby, the condensing point of the reflected light in the reflected light image is brought close to the optical axis.

  Steps S1 to S4 are repeated, and when the focus determination unit 43 determines that the condensing point of the reflected light in the reflected light image is not on the left side or the right side of the optical axis (step S4 “NO”). Since the focal position of the objective lens 13 coincides with the sample A, the focusing adjustment is completed.

Next, a case where a fluorescent image of the specimen A is acquired will be described.
The stage 1 is moved in the direction orthogonal to the line direction of the illumination light by the operation of the stage drive unit 2 in a state where the focal position of the objective lens 13 is matched with the sample A by the automatic focusing mechanism 10, and the sample A The illumination light is scanned two-dimensionally above.

  The imaging device 53 acquires a line-like fluorescent image at each condensing position of the illumination light scanned two-dimensionally, and stores the acquired fluorescent image and the position of the stage 1 in association with each other. Thereby, a two-dimensional fluorescence image of the specimen A is acquired.

  As described above, according to the automatic focusing mechanism 10 and the microscope apparatus 100 according to the present embodiment, the imaging surface 36 of the photodetector 37 is inclined with respect to the optical axis of the reflected light collected in a line. And adjusting the position of the objective lens 13 in the optical axis direction so that the position of the condensing point of the reflected light in the reflected light image coincides with the optical axis of the reflected light by the focus adjustment unit 17. The specimen A can be accurately focused. In addition, an image of the specimen A can be acquired in a state of being accurately focused.

In the present embodiment, normal light observation (bright field, dark field, etc.) may be performed. In this case, a half mirror may be used instead of the beam splitter 33.
In the present embodiment, for example, the sample A may be subjected to Raman scattered light observation. In this case, a laser line filter (not shown) that efficiently transmits only the laser wavelength but does not transmit other wavelengths may be disposed between the light source 3 and the beam splitter 33. The laser light is not perfect monochromatic light, and the laser light contains weak noise in addition to the oscillation wavelength. For this reason, it is effective to remove the stray light of the laser beam in order to efficiently detect the weak Raman scattered light when performing the Raman measurement. By using the laser line filter, it is possible to improve the pure color of the laser and obtain Raman scattered light having a high S / N.

In the present embodiment, when laser light is used as illumination light, it is preferable to arrange a polarizing beam splitter and a λ / 4 wavelength plate in the optical path of the laser light and reflected light.
In this case, a polarizing beam splitter is arranged instead of the beam splitter 33, and a λ / 4 wavelength plate is arranged between the polarizing beam splitter 33 and the objective lens 13, for example, between the relay lens 27 and the dichroic mirror 31. And it is sufficient.

The laser beam reflected by the polarization beam splitter becomes linearly polarized light. However, when the laser beam passes through the quarter wavelength plate, it becomes circularly polarized light by the objective lens 13 and is focused on the specimen A. When this laser light is reflected by the cover glass 5, the rotation direction of the circularly polarized light is reversed, and when passing through the quarter wavelength plate, the reflected light has a polarization plane perpendicular to the incident laser light.
By irradiating the specimen A with circularly polarized laser light, the polarization characteristics of the specimen A can be suppressed and observed. Therefore, it is possible to obtain a high S / N signal in which a noise component is excluded from the detection signal (image information) detected by the photodetector 37, and the focus position of the objective lens 13 can be adjusted with high accuracy.

  In the present embodiment, the imaging surface 36 is configured by three or more light receiving elements. For example, as illustrated in FIG. 11, two imaging surfaces 36 are arranged side by side in the line direction of the light flux of reflected light. The imaging surface 36 may be configured by the light receiving elements S1 and S2. In this case, the focus adjustment unit 17 may control the focal position of the objective lens 13 so that the brightness of the light receiving elements S1 and S2 becomes bright, or the brightness of the light receiving element A1 and the light receiving element A2 may be controlled. The focal position of the objective lens 13 may be controlled so that the brightness is equal.

Further, in the present embodiment, the focus adjustment unit 17 causes the position with the highest light intensity in the image information detected by the detection unit 15 to be separated from the optical axis by a predetermined distance, that is, in the reflected light image. The position of the objective lens 13 in the optical axis direction may be adjusted by driving and controlling the piezo actuator 41 so that the focused point of the reflected light is separated from the optical axis by a predetermined distance.
By doing so, the focus adjusting unit 17 can adjust the focal position of the objective lens 13 to a part of the sample A that is located away from the cover glass 5 in the optical axis direction, such as a deep part of the sample A.

  For example, an observation body (not shown) composed of a specimen A (for example, a cell) and a culture solution is enclosed between a slide glass (not shown) and a cover glass 5 and is shown in FIGS. 12 (a) and 12 (b). As described above, a case where the inside of the specimen A is observed deeply will be described as an example. The distance between the position to be observed and the cover glass 5 is defined as an offset d (μm). This value is determined by the observer. FIGS. 13A and 13B show the focal position of the objective lens 13 when the offset d is not set.

In FIG. 12B, assuming that the central light receiving element on the imaging surface 36 is point O, the reflected light condensing position on the imaging surface 36 is point X, and the refractive index of the observation body is n, the reflected light on the imaging surface 36 is reflected. The position of the objective lens 13 in the optical axis direction is adjusted so that the number of pixels a from the condensing position X to the center light receiving element O satisfies the following formula (3).
a = β ² × (d / n) / (p × tan θ) (3)
The focus adjustment unit 17 adjusts the position of the objective lens 12 so that the center light receiving element O point on the imaging surface 36 and the focal point X point of the reflected light are shifted, thereby adjusting the focus position of the objective lens 13 to the cover glass. 5 away from the reflection position. Therefore, even when the position to be observed is away from the cover glass 5 as in the deep observation of the biological sample, the image acquisition of the specimen A and the focus adjustment can be performed simultaneously.

12 (a) and 12 (b), the offset is determined based on the surface of the cover glass 5 on the side of the observation body, but the thickness (t) and the refractive index (n_glass) of the cover glass 5 are known. In this case, the surface on the objective lens 13 side of the cover glass 5 may be used as the offset reference position.
In this case, the position of the objective lens 13 in the optical axis direction may be adjusted so as to satisfy the following expression (4).
a = β ² × (d / n + t / n_glass) / (p × tan θ) (4)
Since the interface between the cover glass 5 and the air has a higher reflectance than the interface between the cover glass 5 and the observation body, the surface of the cover glass 5 on the objective lens 13 side is used as an offset reference position. The contrast of the reflected light distribution on the surface 36 can be increased, and the focal position of the objective lens 13 can be adjusted with high accuracy.

This embodiment can be modified as follows.
For example, as a first modification, the stage drive unit (tilt adjustment unit) 2 tilts the specimen A and the cover glass 5 so that the tilt of the imaging surface 36 with respect to the cover glass 5 on the specimen A becomes a constant angle. It is good also as adjusting. In this case, the inclination of the stage 1 may be adjusted.

  When the inclination of the cover glass 5 with respect to the image pickup surface 36 is reduced, as shown in FIG. 14, the change in the reflected light distribution on the image pickup surface 36 is reduced, and the spread is spread even if it is separated from the center of the reflected light flux in the line direction. Get smaller. That is, the difference in light intensity in the reflected light image is reduced. On the other hand, when the inclination of the cover glass 5 with respect to the image pickup surface 36 increases, as shown in FIG. 15, the change in the reflected light distribution on the image pickup surface 36 increases, so that the reflected light spreads away from the vicinity of the center in the line direction. Becomes even larger. That is, the difference in light intensity in the reflected light image increases.

  Therefore, by adjusting the inclination of the stage 1 so that the inclination of the imaging surface 36 with respect to the cover glass 5 becomes a constant angle by the operation of the stage driving unit 2, a desired reflected light distribution, for example, the inclination of the imaging surface 36 is obtained. The image information of the reflected light distribution determined by can be acquired. For example, the inclination of the stage 1 may be adjusted every time the sample A is replaced, or may be adjusted during maintenance.

  As a second modification, for example, as shown in FIGS. 16A and 16B, the line direction in the luminous flux of reflected light on the optical axis between the cylindrical lens 35 and the photodetector 37 is shown. It is good also as arrange | positioning the knife edge 55 which interrupts | blocks half of this. FIG. 16A is a view of a part of FIG. 16B viewed from a direction in which the power of the cylindrical lens 23 is not present.

  In this case, as shown in FIG. 17, the reflected light distribution on the imaging surface 36 is such that the spread on the one end L side in the line direction and the spread on the other end R side are along each other along the direction orthogonal to the line direction. The reverse direction. FIG. 18A shows a state in which the center position in the line direction of the light flux of the reflected light is incident on the imaging surface 36 at the time of focusing, and FIG. 18B also shows that one end L side in the line direction is incident on the imaging surface 36. FIG. 18C also shows a state where one end R side in the line direction is incident on the imaging surface 36. FIG. 19A shows a reflected light beam incident on the imaging surface 36 when the focal position of the objective lens 13 is on the near side of the cover glass 5, and FIG. The reflected light distribution on the imaging surface 36 is shown. FIG. 20A shows a reflected light beam incident on the imaging surface 36 when the focal position of the objective lens 13 is on the depth side of the cover glass 5, and FIG. The reflected light distribution on the imaging surface 36 is shown.

  In this way, the reflected light distribution on the imaging surface 36 spreads in the opposite direction on one end L side and the other R side in the line direction along the direction orthogonal to the line direction of the light flux of the reflected light. The focal position of the objective lens 13 can be adjusted even if the two light receiving elements S1 and S2 are arranged side by side in a direction orthogonal to the line direction of the reflected light as in the imaging surface 36 shown in FIG.

  In this modification, when the cover glass 5 is inclined with respect to the optical axis of the illumination light as shown in FIG. 22A, for example, as shown in FIG. The light distribution has a shape extending in parallel along the line direction of the reflected light flux. Also in this case, as shown in the first modification, the stage driving unit 2 may adjust the tilt of the stage 1.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to the above-described one embodiment and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. Absent. Further, for example, in the above embodiment, the objective lens 13 is moved in the optical axis direction. However, the objective lens 13 and the specimen A may be moved relative to each other. For example, the stage 1 is moved in the optical axis direction. It is good also as adjusting the position of the sample A by moving to.

1 Stage 2 Stage drive unit (tilt adjustment unit)
DESCRIPTION OF SYMBOLS 5 Cover glass 10 Automatic focusing mechanism 11 Illumination optical system 13 Objective lens 15 Detection part 17 Focus adjustment part 36 Imaging surface 53 Imaging device (image acquisition part)
100 Microscope device A Specimen

Claims (7)

An illumination optical system for condensing illumination light irradiated on a specimen with a cover glass in a line;
An objective lens that irradiates the specimen with illumination light collected in a line shape and collects reflected light from the cover glass;
A detection unit that has an imaging surface disposed to be inclined with respect to the optical axis of the reflected light collected by the objective lens, and detects image information by photographing the reflected light;
Focusing that relatively moves the objective lens and the specimen with the cover glass in the optical axis direction so that the position with the highest light intensity in the image information detected by the detection unit is located on the optical axis. An automatic focusing mechanism including an adjustment unit. An illumination optical system for condensing illumination light irradiated on a specimen with a cover glass in a line;
An objective lens that irradiates the specimen with illumination light collected in a line shape and collects reflected light from the cover glass;
A detection unit that has an imaging surface disposed to be inclined with respect to the optical axis of the reflected light collected by the objective lens, and detects image information by photographing the reflected light;
The objective lens and the sample with the cover glass are relatively moved in the optical axis direction so that the position with the highest light intensity in the image information detected by the detection unit is separated from the optical axis by a predetermined distance. An automatic focusing mechanism including an in-focus adjustment unit.   3. The automatic focusing mechanism according to claim 1, wherein an imaging surface of the detection unit is tilted around a short axis of the light flux of the reflected light. The automatic focusing mechanism according to any one of claims 1 to 3, wherein an inclination of the imaging surface with respect to an optical axis of the reflected light satisfies the following expression.
{(P × tan θ) / β ² } <{(1.2 × λ) / NA ² }
Where p: length of the light receiving element constituting the imaging surface (μm)
θ: inclination of the imaging surface with respect to the optical axis of the reflected light (rad)
β: Imaging magnification of the entire device (times)
NA: Numerical aperture of objective lens λ: Wavelength of light source (μm) The automatic focusing mechanism according to any one of claims 1 to 4, wherein the focusing adjustment unit sets a relative movement amount between the objective lens and the sample according to the following expression.
(A × p × tan θ) / β ²
Here, a: the number of pixels from the center of the reflected light beam incident on the imaging surface to the center pixel on the imaging surface   The inclination adjustment part which adjusts the inclination of the sample with the cover glass so that the inclination of the imaging surface with respect to the sample with the cover glass becomes a constant angle. Automatic focusing mechanism. An automatic focusing mechanism according to any one of claims 1 to 6,
A stage on which the specimen is placed and movable in the optical axis direction;
A microscope apparatus comprising: an image acquisition unit that acquires an image of the specimen based on the image information acquired by the detection unit.

Images